Video
New rattlesnake anti-venom saves man's lifeAntivenom quality control measures -
Defibrinogenation is the consequence of the consumption of clotting factors owing to the action of procoagulant enzymes in venoms, i. Therefore, the in vitro coagulant activity of venoms is likely to be a surrogate test for in vivo defibrinogenating effect.
Indeed, a relationship was shown between the ability of a polyspecific antivenom to neutralize in vitro coagulant and in vivo defibrinogenating activities of five viperid venoms Myotoxic activity of snake venoms is predominantly due to the direct action of PLA 2 s, and PLA 2 homologs, on the plasma membrane of muscle fibers 43 , However, no correlation between inhibition of PLA 2 activity and of myotoxicity is expected because in many venoms enzymatic phospholipid degradation is mostly due to non-toxic enzymes, as in the case of Bothrops asper which has an acidic PLA 2 with high enzymatic activity but being devoid of myotoxicity An alternative is the assessment of cytotoxicity on muscle cell lines, i.
Myotubes are good models of mature muscle fibers and are highly susceptible to myotoxic PLA 2 s The correlation between neutralization by antivenoms of in vivo myotoxicity and in vitro cytotoxicity on myotubes must be studied.
Likewise, the assessment of cytotoxicity in cell culture systems could become a surrogate assay for the analysis of dermonecrosis, a clinically significant effect of envenomings by spitting cobras in Africa and Asia 1 , The myogenic cell line C2C12 was used to assess cytotoxicity by venoms of five species of Naja sp.
from Africa and its neutralization by a polyspecific antivenom 60 , but whether this assay correlates with in vivo dermonecrosis remains to be investigated. A cell culture test using human keratinocytes was developed to study the cytotoxic action of Naja sp.
venoms and its neutralization by recombinant antibodies Since these venoms induce demonecrosis, this in vitro test could be of value to assess the neutralizing efficacy of antivenoms.
Cytotoxicity on kidney cell lines has been used in the analysis of nephrotoxic effects of venoms and toxins 62 and must be explored as a surrogate test for assessing antivenom efficacy, although venom-induced nephrotoxicity is of a multifactorial pathogenesis which also involves the effects of hemodynamic alterations Neuromuscular paralysis leading to respiratory arrest is one of the predominant effects of snakebite envenomings, particularly those caused by species of the family Elapidae, but also by some species of the family Viperidae 1 , It results from the action of a variety of neurotoxins at the neuromuscular junctions.
Post-synaptically acting polypeptides of the three finger toxins 3FTx family α-neurotoxins act by binding with high affinity to the cholinergic nicotinic receptor AChR at the motor end-plate of muscle fibers Neurotoxicity is also due to the action of PLA 2 s at the nerve terminal β-neurotoxins , by hydrolyzing phospholipids of the plasma membrane, inducing a calcium influx and the consequent alteration of the neurotransmitter exocytotic machinery Other types of neurotoxins include the dendrotoxins, present in mamba Dendroaspis sp venom, which are inhibitors of the voltage-dependent potassium channels Neurotoxins play a key role in the lethality of snake venoms.
Ex vivo neuromuscular preparations have been used by several groups to study the neurotoxic effect of venoms and isolated toxins. The most often used preparations are the chick biventer-cervicis and the mouse phrenic-diaphragm.
Once dissected out, these are placed in a bath containing a physiological solution, and muscle twitches are evoked by electrically stimulating the nerve Neurotoxicity is evidenced by the blockade of evoked muscle contractions.
This system has been used to assess the ability of antivenoms to neutralize the neuromuscular blocking effect [see, for example, Barfaraz and Harvey 68 ; Camargo et al. In the majority of these studies, the correlation with neutralization of lethality in vivo was not investigated, although it is likely that, owing to the relevance of neuromuscular paralysis in the overall toxicity of these venoms, such correlation is likely to occur.
Herrera et al. This system is also useful to assess the myotoxic effect of venoms These tests, however, require a specialized laboratory, and are therefore difficult to adapt to the routine quality control analysis of antivenoms. In addition, being ex vivo tests, they involve the use of animals.
An alternative to assess the inhibition of post-synaptically acting α-neurotoxins is an assay that quantifies the binding of these neurotoxins to purified AChR, such as those from the electric organ of fish, such as Torpedo californica Non-radioactive variations of this assay have been described, which have great potential for antivenom evaluation in vitro.
The basic set up of these procedures is based on the binding of purified AChR to α-neurotoxin bound to wells in microplates. After a washing step, antibodies against AChR are added, followed by conjugated secondary antibodies 73 , This procedure allows the detection of α-neurotoxins in venoms by a competition step whereby the venom is incubated with AChR before the addition to the α-neurotoxin coated plate These procedures have been adapted for the study of the ability of antivenoms to bind α-neurotoxins and thus to inhibit their binding to AChR An adaptation of this assay was used to assess its correlation with venom LD 50 of 20 elapid snake venoms, as well as the correlation of the neutralizing efficacy of an antivenom with the inhibition of AChR binding.
In both cases a significant correlation was found, especially in venoms containing a predominance of α-neurotoxins Owing to its simplicity and high-throughput nature, this assay could be adapted to antivenom development and quality control laboratories in the case of elapid neurotoxic venoms rich in α-neurotoxins Figure 2.
A potential limitation to the widespread implementation of these assays is the availability of purified AChR. This could be circumvented by the use of mimotopes and peptides derived from AChR which bind to α-neurotoxins 76 , 77 , as this will avoid the need to obtain the receptor from rays or eels.
Figure 2 In vitro assay for the assessment of the ability of antivenoms to bind to post-synaptically acting α-neurotoxins from snake venoms. A solution containing a fixed concentration of venom is incubated with various dilutions of antivenom. Then, antibodies both free and venom-bound are removed from free low molecular mass toxins including neurotoxins by ultrafiltration.
The filtrate containing these toxins is incubated with purified nicotinic acetylcholine receptor nAChR. Afterwards, the preparation is added to plate wells that had been coated with a purified α-neurotoxin. Upon incubation and washing, anti-nAChR antibodies are added, followed by washing and addition of conjugated anti-IgG antibodies.
After adding the corresponding substrate, the absorbance is recorded. The nAChR preparation, which is obtained from the electric organ of fish, could be substituted by synthetic peptides containing the binding site for α-neurotoxins. For details of this procedure, see Ratanabanangkoon et al.
For venoms in which β-neurotoxins predominate, a possible in vitro alternative would be the neutralization of PLA 2 enzymatic activity of the purified predominant neurotoxins.
Examples are taipoxin in Oxyuranus scutellatus 78 , β-bungarotoxins in Bungarus sp. In the case of venoms such as those of Bungarus sp. and Micrurus sp. In the cases of venoms, such as those of Dendroaspis sp, rich in other types of neurotoxins, i. dendrotoxins 80 , an as yet unexplored possibility would be the use of patch-clamp methods using oocytes expressing relevant receptors, such as voltage-dependent potassium channels 81 in the case of dendrotoxins.
These, however, require electrophysiology facilities which are not readily available in antivenom quality control laboratories. Venom solutions are applied to filter paper discs and then placed over the yolk sac membrane of shell-less eggs, followed by incubation at 37°C.
This model was initially proposed for the study of the hemorrhagic activity of viperid venoms and showed a good correlation with the in vivo intradermal rodent assay The model was then applied to the study of venom-induced lethality The death of the embryo was assessed by observing the cessation of heart beats, followed by the submergence of the yolk sac membrane into the yolk This model, however, cannot be applied for the study of neurotoxic venoms owing to the incipient development of neuromuscular junctions at this developmental stage in the chick embryo.
The model was also used for assessing its correlation with in vivo toxicity, i. lethality, in the analysis of neutralization of nine venoms by antivenoms A high correlation was found, suggesting the feasibility of using this system for evaluating antivenoms preclinical efficacy, except for neurotoxic venoms, for the reason indicated above.
The model is more economic than those performed in mice and is also more convenient from the 3Rs perspective. The application of -Omics technologies has had a high impact in the study of snake venoms, providing novel and relevant clues for understanding their evolution and composition in their ecological and medical contexts In particular, the field of proteomics as applied to venoms, i.
The baseline for antivenomic analysis is the proteomic characterization of venoms, with identification of the proteins and peptides after separation by reverse phase HPLC and one-dimension SDS-PAGE, and their quantification and classification in different protein families 86 , Hence, bound reactive and unbound non-reactive venom components are identified The percentage of non-reactive venom component is then estimated based on the comparison between the areas under the peak of bound and unbound fractions, allowing a quantitative assessment of immune reactivity.
Even though antivenomics is not a functional test in terms of neutralization of venom activities, it can shed valuable information for understanding the preclinical efficacy of antivenoms. Once the most relevant toxins in a venom are identified, the ability of antivenoms to recognize these components can be quantified through antivenomics, hence providing indirect evidence of efficacy of the antivenom.
Therefore, these guidelines recommend the use of antivenomics as a first screening test for the neutralizing ability of antivenoms, before moving to the in vivo tests 5.
As indicated above, the application of antivenomics to the analysis of the ability of antivenoms to recognize the most toxic components in a venom, as identified by the toxicity score, further potentiates the analytical power of this in vitro method.
Table 1 summarizes the information available on in vitro assays that have shown correlation with in vivo tests in the assessment of antivenom neutralizing ability. Table 1 Summary of the in vitro and ex vivo assays that have shown correlation with in vivo toxic activities of snake venoms in the assessment of the neutralizing ability of antivenoms.
Animal tests to assess venom toxicity and neutralization by antivenoms, particularly the mouse lethality assay, are associated with pain and distress, which may last for prolonged time intervals, as has been shown for crude venoms 94 , and purified myotoxic PLA 2 s 95 and hemorrhagic SVMPs The algogenic effect of venoms is due to the action of venom peptides and proteins that directly activate nociceptive pain sensing neural pathways, as well as by the action of endogenous inflammatory mediators released in tissues as a consequence of venom actions, which stimulate nociceptive receptors in neurons 94 , Despite the evident suffering induced in laboratory animals when assessing venom toxicity and neutralization by antivenoms, the scientific community in Toxinology, as well as antivenom manufacturers, have been slow at introducing interventions aimed at refining these tests with the use of analgesia.
One reason might be the possibility that analgesia affects the results of the tests, although this assumption has not received experimental support. Hence, it is time to consider the routine use of precautionary analgesia in these tests, along the lines indicated by the WHO 5.
The analgesics such as buprenorphine 98 , morphine and tramadol 99 , have been shown to be effective analgesics when used in experiments involving venoms that cause local tissue damage and death.
No differences in the extent of local hemorrhage, edema and myonecrosis induced by venom of Bothrops asper in mice were observed in mice pre-treated with morphine and tramadol, as compared to controls not receiving analgesia The analgesic effect of these drugs can be readily evaluated by using the Mouse Grimace Scale MGS and the mouse exploratory activity , which enable the quantification of pain.
It was shown that morphine and tramadol are effective in reducing pain in several models of envenoming by the venom of B. asper Likewise, the use of tramadol did not alter the results of the estimation of antivenom potency in the case of B.
asper venom and a polyspecific antivenom It is necessary to expand these observations to other venoms to assess whether similar results are obtained. In that case, the routine use of analgesia should be promoted in research and quality control laboratories.
The duration of the action of these analgesics in mice must be considered. It has been estimated that it is between 2 and 3 h for morphine , and up to 6 h for tramadol , whereas the action of buprenorphine in the rat lasts for 6—12 h Hence, in experiments to assess lethality and its neutralization, which usually last for 24 h, there is a need of subsequent administrations of the analgesic.
In the case of neurotoxic venoms, it is likely that opioid analgesics, such as the ones described, affect the outcome of the test.
In these cases, the use of milder analgesics, such as paracetamol, could be considered. The routine methods to estimate the LD 50 of venoms and the ED 50 of antivenoms usually last 24 or 48 h, depending on the route of injection 5 , 6.
Such prolonged time intervals involve much pain and distress in mice. Consequently, efforts are being carried out to make these tests less distressful. In this way, the range of doses to be used in a complete experiment, which usually works with five to seix mice per group, can be selected without having to sacrifice too many mice 5.
When the i. route is used in these tests, a 48 h observation period is established 5 , 6. However, our unpublished observations at Instituto Clodomiro Picado reveal that the number of mice dead at 24 h is the same as at 48 h, hence not justifying observations beyond 24 h.
A more drastic shift in the protocol to assess venom LD 50 and antivenom ED 50 uses a maximum observation period of 8 h [see, for example, Barber et al.
In this methodology, envenomed animals are observed at regular time intervals, e. Animals that are severely affected at any time interval, i. This modification of the classical methodology reduces the extent of animal suffering, although it may affect the precision of the results, as it has been observed that mice that appear moribund may then recover.
A balance needs to be made between the need to refine the lethality test and the need to ensure the robustness of the test for assessing antivenom efficacy.
This urges the development of studies to assess the correlation between the results of these improved protocols and those of classical protocols. There is an urgent need to develop in vitro assays that correlate with in vivo toxicity tests in the study of venoms and in the assessment of the neutralizing ability of antivenoms, along with the 3Rs paradigm Figure 3.
This goal must be strengthened by research funding agencies and agendas, regulatory agencies and diverse stakeholders related to antivenom development, manufacture and quality control.
This is a challenging task owing to the great complexity of the composition and mechanisms of action of venoms.
A research-based, case by case analysis is needed in order to determine which is the most appropriate in vitro assay for each venom-antivenom system, providing the highest correlation with in vivo toxic activities, particularly lethality. Figure 3 The 3Rs principles, as applied to the evaluation of the neutralizing ability of antivenoms.
The search for Replacement, Reduction and Refinement 3Rs should be actively pursued in the field of antivenom potency testing. Some examples of the implementation of these principles in antivenom testing are shown.
The best way to proceed along this line is to harness the growing body of information emerging from the study of venom toxicology and composition, which allows the identification of the most relevant toxic activities and toxins in each venom. This will facilitate the development of immunochemical or in vitro functional tests, enzymatic or otherwise, in substitution of animal-based assays.
In turn, this calls for a closer collaboration between researchers in the biochemistry and pharmacology of venoms and toxins with professionals and technicians in antivenom production and quality control laboratories.
Likewise, the regular use of analgesia in toxicity tests should be actively promoted in toxinological research and antivenom manufacture. It is expected that such initiatives will lead, in the short term, to a significant reduction in the number of animals used in research and antivenom development and potency evaluation, as well as in the suffering inflicted to those animals in the in vivo assays.
Inquiries on the sources of information used in this review can be directed to the corresponding author. JG prepared the first version of this manuscript.
MVa, AS, MH, MVi, GS, AS, CH, and GL revised and contributed to the content of the manuscript. All authors revised the final version of the work and agreed with its content. All authors contributed to the article and approved the submitted version.
This work was supported by Vicerrectoría de Investigación, Universidad de Costa Rica. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors thank our colleagues of the sections of antivenom production, quality control, and research from Instituto Clodomiro Picado, Universidad de Costa Rica, Juan J. Calvete Instituto de Biomedicina, Valencia, Spain , and Kavi Ratanabanangkoon Mahidol University, Thailand for fruitful discussions and collaborations on this topic.
Gutiérrez JM, Calvete JJ, Habib AG, Harrison RA, Williams DJ, Warrell DA. Snakebite envenoming. Nat Rev Dis Primers doi: PubMed Abstract CrossRef Full Text Google Scholar.
Chippaux JP. Snakebite envenomation turns again into a neglected tropical disease! J Venom Anim Toxins Incl Trop Dis Gutiérrez JM. Snakebite Envenomation as a Neglected Tropical Disease: New Impetus For Confronting an Old Scourge. In: Mackessy SP, editor.
Snake Venoms and Toxins , 2nd Edition. Boca Raton, Florida, USA: CRC Press Google Scholar. World Health Organization. Snakebite Envenoming. Strategy for Prevention and Control. Geneva: World Health Organization WHO Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins.
Gutiérrez JM, Solano G, Pla D, Herrera M, Segura A, Villalta M, et al. Assessing the preclinical efficacy of antivenoms: From the lethality neutralization assay to antivenomics. Toxicon — Russell W, Burch R. The Principles of Humane Experimental Technique. Xiong S, Huang C.
Synergistic strategies of predominant toxins in snake venoms. Toxicol Lett — Rucavado A, Nicolau C, Escalante T, Kim J, Herrera C, Gutiérrez J, et al. Toxins Basel CrossRef Full Text Google Scholar.
Bickler PE. Amplification of Snake Venom Toxicity by Endogenous Signaling Pathways. Calmette A. Les venins; les animaux venimeux et la sérothérapie antivenimeuse.
Paris: Masson et Cie Editeurs Vital Brazil O. Serumtherapia anti-ofídica. In: Brazil, V. Obra Científica Completa.
Niteroi: Instituto Vital Brazil Ahuja ML, Brooks AG. In vitro Test for Assay of Potency of Cobra Antivenene. Indian J Med Res — Christensen PA.
South African Snake Venoms and Antivenoms. Johannesburg: The South African Institute for Medical Research Theakston RDG, Lloyd-Jones MJ, Reid HA.
Micro-ELISA for detecting and assaying snake venom and venom-antibody. Lancet — Theakston RDG, Reid HA. Enzyme-linked Immunosorbent Assay ELISA in assessing antivenom potency.
Toxicon —5. Rungsiwongse J, Ratanabanangkoon K. Despite this, if immunoglobulin digestion is desired, another step to eliminate aggregates should be included 68, Purification processes have also been implicated in aggregate formation.
The second precipitation and resuspension steps that take place in the classic ammonium sulfate fractionation protocol for an IgG or F ab´ 2 product 10 , seem to increase the aggregates level when compared to caprylic acid purification, in which the immunoglobulins remain in a soluble form continuously 12, 22, 24, Another important cause of aggregate formation is long storage duration.
The final product will lose activity if stored several years due to antibody denaturation, which, in turn, gives rise to an increased aggregate level. Storage temperature of antivenoms in liquid form is probably an important factor in antibody denaturation and increasing aggregate formation.
Rojas et al. Furthermore, García et al. In , Pirquet and Schick studied the side effects caused by the administration of large quantities of a foreign serum containing antitoxins, a technique used mainly for the treatment of diphtheria and tetanus.
In particular, they stressed the fact that symptoms appeared more rapidly after a second exposure to the foreign serum than after the first administration. Their clinical descriptions included fever and rashes, and some reports of kidney damage with proteinuria, lymphadenopathy and joint symptoms Type III hypersensitivity is mediated by antigen-antibody complexes.
As a consequence of antivenom administration, the patient's immune system reacts by producing antibodies that attach to foreign antibodies antivenom , resulting in the formation of an immune complex. Such complexes can stimulate an acute inflammatory response that leads to complement activation and leukocyte infiltration, the so-called "serum sickness" syndrome.
This is a systemic late adverse reaction characterized by vasculitis, glomerulonephritis and arthritis due to intravascular formation and deposition of immune complexes that subsequently fix the complement and initiate the hypersensitivity reaction.
Patients may develop fever, lymphadenopathy, urticaria and arthritis The classic reaction, that occurs 7 to 15 days after the triggering injection, is known as the primary form of serum sickness. Similar manifestations that appear in a few days following the injection represent the accelerated form of serum sickness, which presumably occurs in subjects already sensitized.
To diminish the incidence of this reaction, it is important to reduce antivenom reactivity to the immune system. In this way, the solutions proposed above to attenuate type I anaphylactic reaction may be useful in this case as well. León et al. Nevertheless, the digestion process gives rise to an important activity loss through antibody denaturation so the amount of foreign protein present in a dose of F ab´ 2 antivenom should be larger than that in a whole IgG-based antivenom Therefore, by considering that the total amount of heterologous protein administered plays a more determinant role in the occurrence of serum sickness than the type of antibody preparation utilized, it is doubtful to establish a priori which molecule induces less response Alternatives including vaccination and the use of humanized antibodies or other neutralizing substances may be the best answer in a near future.
Antivenom contamination by endotoxins is the main cause of pyrogenic reactions in patients. Bacterial endotoxins consist of lipopolysaccharide, a major component of the outer cell membrane of gram-negative bacteria.
Endotoxins present strong biological effects on humans and other mammals when reach their bloodstream during bacterial infection or via intravenous application of a contaminated medicine.
Endotoxins are known to cause fever at very low doses and septic shock at higher doses The threshold level of endotoxin for intravenous applications is set to 5 endotoxin units EU per kilogram of body weight per hour.
The term EU describes the biological activity of an endotoxin Higher levels of endotoxins are related to bacterial infection or digestive tract injuries, but contamination at low concentrations can be found in pharmaceutical products.
Contamination of antivenom products with endotoxins take place if preventive measures are not followed during processing The presence of low doses of endotoxins in antivenoms generate an important increase of mild, early adverse reactions generally fever in patients 9.
To avoid endotoxins, the production laboratories must implement strict quality requirements in facilities, raw materials, process systems and equipment. Endotoxins are very stable molecules of varying size; their biologically active part can survive extremes of temperature and pH in comparison to proteins.
Temperatures from to °C and acids or alkalis of at least 0. Therefore, it represents a challenge to remove endotoxins from biological fluids including proteins. In addition to this, endotoxin shows a strong association with proteins, so steps that involve protein concentration also involve endotoxin concentration and steps that involve protein purification of other protein involve endotoxin elimination Thus, ammonium sulfate fractionation process tends to increase endotoxin level more than the caprylic acid purification of immunoglobulins in a production system, not only because of a higher endotoxin level in the raw materials and a longer process time, but also due to a specific concentration of endotoxins in the final precipitate, which corresponds to the IgG fraction Finally, if a product is accidentally contaminated and fails to pass the quality control, it should be discarded or reprocessed.
Decontamination is a costly alternative, so avoiding endotoxin contamination must be the preferred choice However, in unexpected cases, it is absolutely necessary to count with a decontamination procedure in order to save a given production batch that otherwise would be discarded. With this goal, several systems including ultrafiltration membranes and chromatography resins coupled to different ligands have shown good capacity to capture and remove endotoxins 73, 74, Unfortunately, the use of these systems involve variable yield losses, so this is another reason why they should be applied only to save occasional production batches but not as routine In the past century, antitoxic sera were widely used for diphtheria, tetanus and treatment of accidents with poisonous animals 10, , Nowadays, for tetanus and diphtheria treatments, the antitoxic sera have been replaced by vaccination, antibiotic therapy and human neutralizing antibodies, but for treating envenomation by snakes and others animals, heterologous antivenoms still remain the only effective solution.
Meanwhile the improvement in antivenom quality must focus on the increase of product purity and the reduction of aggregates, as well as on the implementation of good manufacturing practices GMP. Unfortunately the incorporation of refined purification techniques to antivenom production process and others commercials factors have carried on an important cost increase, thereby causing an strong antivenom shortage specially in the poorest countries 25, The best solution includes best quality antivenoms at an affordable cost 20, Open menu Brazil.
Journal of Venomous Animals and Toxins including Tropical Diseases. Submission of manuscripts About the journal Editorial Board Instructions to authors Contact.
Português Español. Open menu. table of contents « previous current next ». Text EN Text English. PDF Download PDF English. Toxins incl. snake antivenom; anaphylactic reaction; complement system activation; endotoxins.
REVIEW ARTICLE Snake antivenoms: adverse reactions and production technology Morais VM; Massaldi H Department of Biotechnological Development and Production, Hygiene Institute, School of Medicine, Universidad de la República, Montevideo, Uruguay Correspondence to Correspondence to: Victor Morais Departamento de Desarrollo Biotecnológico y Producción, Instituto de Higiene Av.
ABSTRACT Antivenoms have been widely used for more than a century for treating snakebites and other accidents with poisonous animals. CSA by the Fragment Fc of Heterologous Antibody In the past, it was presupposed that the presence of Fc fragments in antivenoms was the only, or the most important, cause of anaphylotoxic reactions CSA by Protein Aggregates The presence of protein aggregates can also provoke complement system activation.
CSA by Immune Complexes Type III Hypersensitivity In , Pirquet and Schick studied the side effects caused by the administration of large quantities of a foreign serum containing antitoxins, a technique used mainly for the treatment of diphtheria and tetanus.
PYROGENIC REACTIONS Antivenom contamination by endotoxins is the main cause of pyrogenic reactions in patients. Received: July 4, Accepted: August 20, Abstract published online: October 13, Full paper published online: February 28, Conflicts of interest: There is no conflict.
Chippaux JP, Goyffon M. Venoms, antivenoms and inmunotherapy. Theakston RDG, Warrell DA, Griffiths E. Report of a WHO workshop on the standardization and control of antivenoms.
Wilde H, Thipkong P, Sitprija V, Chaiyabutr N. Heterologous antisera and antivenins are essential biologicals: perspectives on a worldwide crisis. Ann Intern Med. Calmette A. Contribution a l'etude du venin des serpents. Immunisation des animaux et traitement de l' envenimation.
Ann Inst Pasteur. Contribution à l'ètude des venins, des toxins et des serums antitoxiques. Hawgood B. Doctor Albert Calmette founder of antivenomous serotherapy and of antituberculous BCG vaccination.
Clark RF, McKinney PE, Chase PB, Walter FG. Immediate and delayed allergic reactions to Crotalidae Polyvalent Immune Fab ovine antivenom. Ann Emerg Med. Dart RC, Mcnally J. Efficacy, safety, and use of snake antivenoms in the United States.
Otero-Patino R, Cardozo J, Higashi H, Nuñez V, Diaz A, Toro M, García M, Sierra A, García LF, Moreno A, Medina M, Castañeda N, Silva-Diaz JF, Murcia M, Cardenas SY, Dias da Silva WD.
A randomized, blinded, comparative trial of one pepsin-digested and two whole IgG antivenoms for Bothrops snake bites in Uraba, Colombia. The Regional Group of Antivenom Therapy Research REGATHER. Am J Trop Med Hyg.
Harms AJ. The purification of antitoxic plasmas by enzyme treatment and heat denaturation. Biochem J. Gutiérrez JM, Higashi HG, Wen FH, Burnouf T.
Strengthening antivenom production in Central and South American public laboratories: Report of a workshop.
Rojas G, Jiménez JM, Gutierrez JM. Caprylic acid fractionation of hyperinmune horse plasma: description of a single procedure for antivenom production.
Raweerith R, Ratanabanangkoon K. Fractionation of equine antivenom using caprylic acid precipitation in combination with cationic ion-exchange chromatography. J Immunol Meth. Pepin-Covatta S, Lutsch C, Grandgeorge M, Scherrmann JM. Immunoreactivity of a new generation of horse F ab 2 preparations against European viper venoms and the tetanus toxin.
Raw I, Guidolin R, Higashi H, Kelen E. Antivenins in Brazil: preparation. In: Tu AT editor. Handbook of natural toxins. New York: Marcel Dekker; Jadhav SS, Kapre SV. Antivenom producion in India. New York: Marcel Dekker; , p. Abbas AK, Lichtman AH, Pober JS editors. Cellular and molecular immunology.
Philadelphia: WB Sanders Co. Abbas AK, Litchman AH. Immediate Hypersensitivity. In: Abbas AK, Litchman AH editors. Philadelphia: Saunders Elsevier Science; Cruce JM, Lewis RE. Types I, II, III, and IV hypersensitivity.
In: Atlas of immunology. Florida: CRC Press; Morais V, Massaldi H. In developing countries, snakebite is regarded as an occupational health hazard for rural people and a socio-economic problem 18 , and therefore, PAV should be available at rural health centers. The lyophilized PAV is more suitable than liquid antivenom for long-term storage 2—3 years without requiring refrigeration at rural hospitals.
One of the key steps in the antivenom manufacturing process is the caprylic acid treatment of horse hyper-immunized serum to precipitate the non-IgG proteins The protein concentration of the aqueous solution of B1 and B2 of SL PAV was found to be 7.
In the present study, the albumin content of B2 is found within this limit however, this content is slightly higher in B1 Fig. Notably, ion-exchange chromatography of hyper-immunized serum after caprylic acid precipitation has been suggested to significantly reduce the contaminating proteins in PAV preparations However, adopting this process for antivenom manufacturing, which is not required according to the WHO guidelines, would increase the production cost of PAV and make it less affordable in the developing nations.
The gel filtration chromatogram of each batch of SL PAV was resolved into a major broad protein peak GF1 that eluted around fractions 42—68 mL Supplementary Fig. The percent protein content of GF1 in the two different batches of SL PAV was determined to be in the range from The SDS-PAGE analyses of both batches of SL PAV and their GF peak 1 under non-reduced and reduced conditions are shown in Fig.
Under the non-reduced conditions, the PAV samples and their GF peaks showed a smeared band at molecular mass ranging from to kDa Fig. Therefore, the pepsin digestion protocol should be properly followed and special attention should be given to avoid incomplete digestion of IgG molecules. SDS-PAGE analysis of the two batches of SL PAV and their gel filtration fractions under a non-reduced, and b reduced conditions.
The densitometry analysis of the SDS-PAGE protein bands of both batches of SL PAV and their respective GF peak GF1 suggested that they contained minor amounts of undigested IgG The SDS-PAGE analysis of the two batches of SL PAVs showed a minor amount of aggregate 4.
This result corroborates the characterization of SABU Indonesian antivenom that showed 7. The presence of incomplete or partially digested IgG in SL PAV was determined by enzyme-linked immunosorbent assay ELISA and western blot analyses using anti-Fc antibody. The ELISA and immunoblot analysis of the two batches of SL PAV showed This result is corroborated by the data from the densitometry analysis of SDS-PAGE bands of crude SL PAV Fig.
While partially digested IgG was present in the PAV in this study, further precision is recommended in this step to improve the product quality.
Determination of Fc content of IgG by Western blot analysis. Immunoblot analysis of the SL PAV B1, B2 and purified horse IgG was done by using anti horse IgG HRP conjugated Fc region-specific antibody. M represents protein molecular mass marker and B1 and B2 represent batch1 and batch 2 of PAV, respectively.
The primary antibody for both the analyses was raised against Fc region of IgG. The complement activation property of antivenom is primarily due to the Fc portion of the undigested IgG 14 , 33 , however, as already discussed removal of Fc portion of IgG by pepsin digestion does not guarantee the elimination of early anaphylactic reactions EARs induced by complement activation Noteworthy to mention, the WHO has not fixed any standard safe range for complement activation by antivenoms The complement activation shown by SL PAV in this study was assessed only by in vitro analysis and this should be confirmed and validated by in vivo pre-clinical studies or clinical case studies from antivenom-treated patients.
IgA and IgE molecules can be co-separated with the IgG molecule during precipitation of IgG from hyper-immune plasma and have no role in neutralizing the venom toxin 14 , Although no adverse effect of IgA has been reported, IgE contamination in antivenom preparations can induce a hypersensitivity reaction leading to anaphylactic shock in antivenom-treated patients The two batches of SL PAV were found to contain IgA Supplementary Fig.
S4 a—c but they were devoid of IgE contamination. The sterility assessment of SL PAV showed that both batches were free of microbial contamination Supplementary Fig.
During the processing of immunoglobulins of horse plasma, they may become contaminated with bacteria, and as a consequence, PAV can be contaminated with bacterial endotoxin Depending upon the potency and quality of product, the doses of antivenom ranges from 20 to mL and the maximum acceptable content of endotoxin in an antivenom preparation should be within a range of 2.
The endotoxin load in the two batches of SL PAVs was determined to be within the range of 1. S6 a,b. Cresol is used as preservative for long-term storage of antivenoms; however, the cresol content in antivenom should not exceed 0.
A high content of cresol in antivenoms has been reported to cause a hypersensitive reaction in patients RP-HPLC analysis showed that the cresol content in the two batches of SL PAV was within the range of 0.
S7 a—c , which is significantly below the limit of allowed preservative in an antivenom preparation. Thus, these results advocate for the quality and safety of the newly developed SL PAV.
The number of vials of PAV required to cure a snakebite patient depends on the severity of the envenomation amount of venom injected into the victim , efficacy of the antivenom, and sometimes repeated doses of PAV are administered over a period of 2—3 days. None of the tested mammalian cells showed cytotoxicity Supplementary Fig.
Furthermore, both batches of PAV were devoid of direct or indirect hemolytic activity against mammalian erythrocytes data not shown. A summary of the physicochemical properties and safety profiles of the two batches of SL PAV is shown in Table 1. For treating snakebite in SL, Indian PAV is mostly used, though its efficacy and neutralization potency towards SL snake venoms is of immense concern 13 , Further, Indian PAV does not contain antibody against HHV, even though the largest number of snake bites in SL are from this species 3 , 4.
Therefore, it would be essential to include HHV-specific antibodies in a PAV preparation for better protection against snakebite by this pit viper in SL.
This long-standing requirement of clinicians can be fulfilled by the newly developed SL PAV. The superiority of the newly developed country-specific SL PAV over Indian PAV was demonstrated by ELISA and Western blot analyses, two important laboratory techniques for quantifying the venom-antivenom cross-reactivity 43 , 44 , 45 , 46 , Although in vivo neutralization study of venom-induced lethality and toxicity is the gold standard for pre-clinical assessment of a PAV; however, prior to the in vivo pre-clinical assessment, the efficacy and potency of the antivenom can be assessed by determining the immunological cross-reactivity between venom and antivenom by ELISA and immuno-blotting techniques.
The in vitro neutralization of selected enzymatic activities and pharmacological properties of venom samples can also be evaluated.
The results of these tests can provide a clear understanding of the efficacy of antivenom. These assessments are highly recommended as they involve a minimal use of experimental animals and they are ethically acceptable 19 , 44 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , The result of the ELISA showed that both batches of SL PAV compared to Indian PAV demonstrated significantly higher immuno-recognition of the SL snake venom samples; PAVs demonstrated the highest and lowest immuno-recognition against the venom of E.
carinatus and B. caeruleus , respectively Fig. In addition, because Indian PAVs do not contain antibodies against HHV, their recognition towards HHV compared to that of SL PAV was extremely poor Fig. Though, a marginal recognition of H.
hypnale by Indian PAVs suggests that these venom toxins may share sequence similarities with the homologous toxins from Indian D. carinatus venoms as they all belong to the Viperidae family of snakes 6 , 44 , 45 , 49 , Several researchers have suggested that immunoblot analysis is an important in vitro laboratory test to determine the potency of PAV 45 , 46 , 48 , The results of immunoblot analysis were also in accordance with the ELISA results Fig.
S9 , S10 , S11 a,b. In this study, however, both batches of SL PAV compared to Indian PAV exhibited significantly higher immuno-recognition of low molecular mass proteins of SL snake venoms. Moreover, no batch to batch variations among B1 and B2 were seen in the immuno-recognition properties of country-specific SL PAV Fig.
Immunoblot cross-reactivity of SL venoms. a SDS-PAGE images of SL venoms. b — f Western blot images of SL venoms against two batches B1 and B2 of SL PAV, and Indian PAVs BSVL, PSVPL, and VINS. Same molecular marker M was used for all the blots. The Ponceau stained blot images are shown in supplementary Fig.
The full length unedited blot images are shown in supplementary Fig. Snake venom enzymes, particularly those from the Viperidae family of snakes, play a vital role in venom-induced toxicity and lethality 45 , 47 , 56 , Viperidae venoms are rich in enzymatic toxins like phospholipase A 2 PLA 2 , snake venom metalloprotease SVMP , and snake venom serine protease SVSP , which play an important role in the toxicity of venom 45 , 47 , 58 , 59 , 60 , 61 , 62 , Therefore, assessing the in vitro neutralization of catalytic activities and the pharmacological properties of these enzymes by commercial PAV is another important in vitro method for determining the efficacy of commercial antivenom.
The in vitro neutralization of some enzymatic activities and pharmacological properties of SL snake venoms by country-specific SL PAV, and Indian PAV was compared.
S12 a—e. Poor neutralization of PLA 2 activity is a major concern for venom-induced toxicity and lethality 64 , The enzymatic activity exhibited by HHV was better neutralized by SL PAV than by Indian PAVs since the latter do not contain antibodies against this venom.
In a nutshell, the in vitro laboratory tests provide convincing evidence for the significant improvement of the venom recognition property of the newly developed SL PAV, in comparison to Indian PAV.
Assessing the neutralization of in vivo lethality and toxicity of snake venoms by commercial PAV is the gold standard for determining their efficacy and it is a crucial step in the clinical assessment of PAV 14 , The in vivo venom neutralization potency of SL PAV is shown in Table 2.
Recently, the pre-clinical efficacy of a newly developed SL snake venom-specific PAV raised against SL snake venoms—DRV, ECV, HHV, and NNV by Instituto Clodomiro Picado ICP , Costa-Rica, has been assessed To the best of our knowledge, this PAV has not yet been marketed in SL.
Researchers have reported that B. ICP PAV does not contain B. caeruleus venom in the immunization mixture, therefore this PAV is devoid of antibodies against this medically important snake venom However, the paraspecific cross-neutralization of B.
caeruleus venom by antibodies against Elapdiae family of snake venom in ICP PAV may not be ruled out; nevertheless, it cannot ensure full protection to SL krait bite patients 69 , Consequently, it may be anticipated that owing to containing antibodies against B. caeruleus venom, SL PAV will provide better protection against krait envenomation.
In contrast, ICP PAV has a greater lethality neutralization potency against Viperidae venoms DRV, ECV, and HHV and a lower neutralization efficacy against NNV, compared to PSVPL SL PAV.
For the treatment against snakebite to be effective and convenient to the clinicians, the neutralization potency of PAV should preferably be adjusted according to the venom yield of snakes. It is further to be mentioned that the neutralization potency of the newly developed SL-specific PAV was based on the average venom yield of prevalent snake species with an intention to assist clinicians of SL who are well versed with the use and dose-regimen of Indian PAV.
Hospital management of snakebite with similar dosage of SL-specific PAV compared to Indian PAV which has been used for snakebite treatment by clinicians in SL for decades and they are well versed with the dosages would be expected to be easier and more clinically relevant than the currently used Indian PAV.
The published report on average venom yields of Sri Lankan species of snakes is not available, however, the same species of snakes in India have reported average venom yields of mg, In another study, the average venom yields have been reported as Further, the amount of venom that can be injected by envenomation into a patient would be expected to be proportional to the venom content yield of the species of snake.
Therefore, the neutralization potency of PAV against DRV, HHV, and NNV should be higher than that against ECV and BCV, because the venom yield per bite of these two snakes should therefore be lower than the former three species of snakes 73 , 74 , 75 , 76 , However, after the product SL PAV registration in SL further clinical trials may be undertaken for the effective clinical dose adjustment, if necessary.
Envenomation by the Elapidae family of snakes, such as the mambas Dendroaspis sp. induces a neurotoxic effect that is the primary cause of lethality; albeit, elapids also contain cytotoxins in their venom which lead to significant tissue necrosis 78 , In this case, the neutralization of lethality would be an ideal model for assessing the pre-clinical efficacy of antivenoms.
Nevertheless, the Viperidae snakes show a wide range of pathophysiological effects such as myonecrosis, dermonecrosis, hemorrhage, edema, coagulopathies, bleeding in various organs, hemodynamic disturbances, and renal disturbances 66 , 80 , These effects are cumulatively responsible for the venom-induced lethality.
Therefore, in the case of Viperidae venoms, pre-clinical evaluation of antivenoms by assessing the neutralization of lethality is not sufficient for an integrated evaluation of the antivenoms The WHO has recommended assessing the neutralization of other toxic activities with supplementary tests and the essential test of neutralization of lethality prior to marketing new antivenoms or introducing existing antivenoms into new geographical locales The in vivo neutralization of venom-induced toxicity by the newly developed SL PAV has been determined in mice.
Moreover, the toxicities of SL snake venoms are summarized in Supplementary Table S2. The tested pharmacological activities i. The neutralization potency of SL PAV towards hemorrhagic activity, necrotizing activity, and defibrinogenating activity was found to be higher against DRV, in comparison to ECV and HHV.
In contrast, the pro-coagulant activity displayed by HHV venom was better neutralized by SL PAV, compared to neutralization of the same activity against DRV and ECV Table 3. This may be due to the HHV showing much less pro-coagulant activity compared to the other two Viperidae snake venoms Supplementary Table S2.
In summary, grave concerns have been expressed by clinicians about using Indian PAVs for the treatment of snakebite in SL as it often shows only partial effectiveness in neutralizing the venom toxicity and it produces adverse clinical reactions in patients.
Consequently, because of the long-standing demand for a country-specific PAV for treating snakebite, the PAV was developed against the venoms of five most medically important snakes of SL.
The purity of the preparation and the safety of the newly developed SL PAV was found to be satisfactory and no significant variation was seen in the PAV composition or potency between two batches B1 and B2 of SL PAV; however, for an affirmative conclusion regarding batch-to-batch variation, future studies with more number of PAV batches are warranted.
However, following the good manufacturing practice GMP by the antivenoms manufacturers can result in production of different batches of PAV with uniform potency and composition Further, the immunological cross-reactivity studies and enzyme neutralization assay documented the superiority of SL PAV in comparison to Indian PAVs against SL venoms.
The pre-clinical study also provided convincing evidence for the neutralization of lethality and toxicity of SL snake venoms by SL PAV. Therefore, the findings of this study illustrate that the improved neutralization potency of PAV against SL snake venoms will greatly augment the hospital management of snake envenomation in SL.
Further pre-clinical and clinical studies are still warranted to understand the complex pharmacokinetics and pharmacodynamics, and the venom-antivenom interactions in vivo that can neutralize the venom-induced toxicity. The following five snake venoms pooled from 5 to 6 snakes from SL origin were used in this study: i Naja naja NNV , ii Daboia russelii DRV , iii Bungarus caeruleus BCV , iv Echis carinatus ECV , and v Hypnale hypnale HHV.
The venoms were collected from snakes of either sex of different age from various locations around Kandy city in SL as per prevalence of snakes in that area.
The Saw scaled vipers ECV were collected from North SL Jaffna. The collected venoms were stored lyophilized, and sent by University of Peradeniya SL who are collaborating with Premium Serums and Vaccines Pvt.
The equine PAV developed against SL snakes was obtained from Premium Serums and Vaccines Pvt. VINS , India Batch no. BSVL , India Batch no. A, expiry date: October Anti-goat erythrocyte polyclonal antibody raised in rabbit was obtained from Fitzgerald Industries International, USA.
The Pierce LAL Chromogenic Endotoxin Quantitation Kit was from Thermo Scientific, USA. The NHS-activated Sepharose 4 Fast Flow matrix was purchased from GE Healthcare. All other chemicals were of analytical grade and obtained from Sigma-Aldrich, USA.
Human embryonic kidney cells HEKT , murine hepatoma Hepa 1—6 cell line Hepa , and differentiated rat skeletal L6 myoblast cell lines were procured from ATCC American type culture collection , USA.
The texture, colour, and homogeneity of the PAV preparation were determined by visual inspection. The examination of textures like macroscopic collapse, color uniformity, cake shrinkage formation and adhesion properties of powder to the vials of PAVs was done by visual inspection and photographs of vials were also captured PAVs were dissolved in 10 mL of sterilized de-ionized water provided along with the antivenom and the turbidity of the solution was assessed by a turbidimeter model-CLD, Nephelometer, ELICO Ltd.
The 2 mL PAV solutions were transferred to pre-weighed microfuge tubes and after centrifugation of the tubes at 10, rpm for 10 min, the solutions were decanted, and the tubes were dried in vacuum and weighed again to determine the presence of the insoluble component, if any.
The pH of the aqueous PAV solution was determined using a digital pH meter Eutech Instruments, pH , USA. For determining the residual moisture content, a measured amount of PAV was heated at °C for 3 h in an oven and moisture content was determined by heat drying method The protein content of PAV was determined as described by Lowry et al.
Protein bands were visualized by Coomassie Brilliant Blue R staining. After scanning the gel, intensities of the protein bands were determined by ImageQuant TL 8. The percent of aggregate content band intensities above kDa in a particular batch of SL PAV was determined from the cumulative band intensity for that particular batch of PAV.
The procedures described in this section is adopted from our previous publications 19 , The tryptic fragments of PAV were reconstituted in 0.
Solvent A and B were 0. The peptides eluted from the HPLC column were then fed into a Nanomate Triversa Advion BioSciences, Ithaca, NY , equipped with an LC coupler and electrospray ionization ESI nanospray chip. The LC coupler connects the flow from the HPLC to the ESI chip, where the nano-ESI generated ions were transferred into an LTQ Orbitrap Discovery hybrid mass spectrometer Thermo Fisher Scientific, Bremen, Germany.
The ionization voltage was set to 1. mgf using ProteoWizard release version 3. The following search parameters were used; enzyme: trypsin, maximum missed cleavage sites: 2, precursor ion mass tolerance: 10 ppm, fragment ion tolerance: 0. The relative abundances of the identified proteins were calculated using Exponentially Modified Protein Abundance Index emPAI -based label-free quantification technique The MassSorter v3.
The incomplete pepsin digestion of IgG Fc content of IgG in the PAV preparation, if any, was determined by ELISA and Western blot analysis using HRP horseradish peroxidase -conjugated rabbit anti-horse IgG Fc specific antibody Sigma Aldrich, USA Absorbance was measured at nm in a microplate reader Multiskan GO, Thermo Scientific, USA.
A standard curve of the graded concentrations of purified horse IgG was prepared and Fc content was determined by ELISA under identical experimental conditions. The Fc content of test samples was compared to the standard curve of horse IgG.
For the immunoblot analysis, 20 µg of PAV protein in triplicate and horse IgG after separation by The image was photographed, scanned Epson image scanner, Epson America, Inc and the densitometry analysis of the developed blot was processed with ImageQuant TL 8.
Classical and alternative pathways of complement activation were determined by percentage of hemolysis induced by SL PAV batch 1 and batch 2 samples Human blood samples from healthy donor were collected without anticoagulant and allowed to clot at room temperature for 4 h. Goat blood obtained from slaughter house was collected in 3.
For determination of complement activation by classical pathway CP goat erythrocytes were washed with 1× PBS, pH 7. The antibody-sensitized goat erythrocytes were washed with 1× PBS pH 7. For alternative pathway AP of complement activation analysis, goat erythrocytes were washed with 1× PBS, pH 7.
Fifty micro liters of antibody sensitized goat erythrocytes for classical pathway or goat erythrocytes for alternative pathway were added to wells of a well microtiter plate containing 50 µL of human serum pre-incubated with 50 µl of SL PAV 1 mg at 37 °C.
The plate was centrifuged at rpm at 4 °C for 5 min Heraeus multifuge X1R, Thermo Scientific, USA and 50 µL of supernatant from each well was transferred to a new well plate containing µL of water.
The contents were mixed well by shaking, and the absorbance of mixture was measured at nm in a microplate reader. The occurrence of IgA and IgE in the SL PAVs, if any, was determined by Western blot analysis and ELISA of PAV against HRP-conjugated anti-horse IgA and anti-horse IgE antibodies, respectively dilution as described previously 19 , As a negative control, 20 µg for the Western blot analysis or ng for the ELISA of BSA was used.
The blots were scanned EPSON image scanner, Epson America, Inc. and their densitometry analysis was done by ImageQuant TL 8.
The sterility of the SL PAV was tested according to the WHO guidelines One mg of PAV solution in sterile water was incubated in trypticase soy broth and thioglycolate medium.
Control culture flasks were included for each medium. Flasks were incubated at 25 °C or at 35 °C for 14 days in trypticase soy broth and thioglycolate medium to test for fungal and bacterial cultures, respectively, using the appropriate controls.
Culture flasks were examined daily for bacterial or fungal growth by checking the optical density in a spectrophotometer at nm.
The level of endotoxin contamination in SL PAV, if any, was determined using a commercial diagnostic kit Pierce LAL Chromogenic Endotoxin Quantitation Kit, Thermo Scientific, USA Briefly, a standard curve of graded concentrations of E. coli endotoxin was plotted so that the endotoxin concentration in test sample can be determined as low as 0.
For endotoxin determination, 50 µL of SL PAV samples in triplicate was added to wells of microtiter plate and the plates were incubated at 37 °C for 5 min at dark. Then, 50 µL of Limulus amebocyte lysate LAL to each well was added, mixed gently on a plate shaker for 10 s followed by incubation at 37 °C for 10 min.
Thereafter µL of chromogenic substrate solution was added to each well and incubated for 6 min at 37 °C. From the standard curve, the concentration of endotoxin in PAV samples was determined. As a negative control, BSA was used. The use of a specified amount of preservative m -cresol in PAV preparations for their long-term storage was approved by the WHO in The m -cresol content was determined by reversed-phase ultra-high performance liquid chromatography RP-UHPLC of SL PAV on an Acclaim C 18 RP-UHPLC column 2.
The flow rate was 0. The isocratic programme for the mobile phase was optimized for 18 min The detection of m -cresol was observed at nm The percentage of m -cresol was determined against a standard curve of m -cresol run in UHPLC under identical experimental conditions.
Thereafter, cell viability was determined by the MTT-based method 91 and the result was expressed as PAV-induced cell death in percentage , if any, with respect to control PBS-treated cells H 2 O 2 was used as a cytotoxic agent positive control.
The immunological cross-reactivity of SL snake venoms against SL PAV and Indian PAV raised against venoms of the Big Four snakes of India was determined by ELISA and Western blot analysis 19 , 43 , 44 , Briefly, for ELISA ng of venom was coated for overnight at 4 °C in microtiter ELISA plate.
After washing the wells by using washing buffer phosphate buffer saline containing 0. For negative control, the venom samples were treated with naïve horse IgG and developed in parallel. After incubation with primary antibody, the excess antibodies were washed using washing buffer and incubated with anti-horse IgG HRP-conjugated secondary antibody produced in rabbit for 2 h at room temperature dilutions.
The reaction was stopped immediately by 2 M H 2 SO 4 and the absorbance was read at nm. For presenting the data, the absorbance values of PAV against venom samples was deducted from the absorbance of negative control. Immunoblotting experiments were performed as described previously by resolving the venom proteins in The SDS-PAGE protein bands were transferred to PVDF membrane in a semi-dry gel transfer system Amersham Bioscience, UK at 1.
The transfer efficiency was checked by Ponceau S staining of the membranes. The excess unbound antibodies were washed with TBS-T and incubated with anti-horse IgG ALP-conjugated secondary rabbit anti-horse antibodies for 1 h at room temperature.
Venom samples treated with horse naïve IgG served as negative control. Densitometry analysis of the blots was done using ImageQuant TL software 8. Briefly, venom 10 µg was pre-incubated with PAV µg in a predetermined ratio , protein: protein for 30 min at 37 °C followed by assaying the mixture for enzymatic activities and in vitro pharmacological properties of venom 44 , 45 , 51 , In vivo neutralization of lethality and other pharmacological effects of snake venom hemorrhagic activity, necrotizing activity, pro-coagulant activity, defibrinogenating activity, and myotoxicity of SL snake venoms by PAV raised against these snakes were evaluated in laboratory inbred Swiss albino mice males and females weighing between 18 and 20 g, age 3 to 4 weeks, following the WHO guidelines Ltd, Pune.
Dry food pellets Nutritive Life Sciences, Pune and purified filtered water were provided ad libitum. To determine the LD 50 , graded concentrations of venom from each species of snake in 5 mL of normal saline were injected intravenously into a group of five mice.
Animals were observed for 48 h and deaths during this period, if any, were recorded. The LD 50 was calculated by the Reed and Muench method by using the following formula.
The mixtures were incubated for 30 min at 37 °C, and then aliquots of a precise volume maximum 0. After injection, deaths were recorded at 48 h intravenous test and the results were analyzed using Reed and Muench method.
The ED 50 results were expressed as mg of venom neutralized by per mL of PAV and value was calculated using following formula After 3 h post injection, mice were euthanized using a carbon dioxide asphyxiation method.
The area of the injected skin was removed and the size of the hemorrhagic lesion was measured using calipers in two directions. The mean diameter of the hemorrhagic lesion for each venom dose was calculated and the mean lesion diameter was plotted against each venom dose to determine the minimum hemorrhagic dose MHD.
One unit of MHD is defined as the dose of venom that produces 10 mm diameter of skin hemorrhage.
The worldwide neglect of immunotherapeutic products for the treatment of snakebite has resulted Carbohydrates and Energy a Antivenom quality control measures paucity of msasures, safe and affordable therapy in many Quwlity World Anttivenom, particularly in Africa. NAtivenom Antivenom quality control measures high among Anfivenom most Healthy appetite control global health problems, Sculpting muscle definition thousands of Antivenom quality control measures measrues dying or becoming permanently maimed in developing countries each year because contrkl a lack of antivenom—a treatment that is widely available in most developed countries. This paper analyses the current status of antivenom production for sub-Saharan African countries and provides a snapshot of the global situation. A global survey of snake antivenom products was undertaken ininvolving 46 current and former antivenom manufacturers. Companies producing antivenom for use in sub-Saharan Africa were re-surveyed in and Variable potency and inappropriate marketing of some antivenoms mean that the number of effective treatments available may be as low as 2.Antivenoms from hyperimmune animal plasma are the only contrll pharmaceuticals against snakebites. The improvement mezsures downstream processing Body fat percentage and performance is of great interest, not only in terms of purity profile, but also from yield-to-cost perspective and rational measurse of plasma of animal origin.
We qualit on development of an efficient refinement strategy for F ab' 2 -based antivenom wuality. Process design was driven by the imperative to keep qualkty active principle constantly in solution Antivenim a precautionary measure to preserve stability of its conformation precipitation of active principle or its adsorption to chromatographic stationary Anrivenom has been completely avoided.
Final polishing was accomplished qualityy a combination Antovenom diafiltration and flow-through chromatography. This optimised jeasures looks mezsures promising for large-scale production of therapeutic antivenoms, since high yield of the active drug and fulfillment of the regulatory demand considering purity was achieved.
The recovery of the active substance was precisely determined Increase energy levels each purification step enabling accurate estimation of conyrol process cost-effectiveness.
Animal Antivenom quality control measures antivenoms constitute the most important therapy against snakebite envenoming. Nowadays this critical treatment has been ccontrol by severe shortage due controll low meazures of current productions, Antivenmo mostly affects developing countries as those suffering from contrlo morbidity and mortality rates.
Antivenoms' safety and efficacy in clinical setting are highly dependent on manufacturing procedure. Its design Antivenim be guided by the tendency to refine Antivenom quality control measures G from Seed packaging and labeling plasma qulaity in only a few easy, simple and efficient purification steps, qualitg antibody-based product of acceptable physicochemical features and good Maintains digestive balance of protective quailty.
Here, we developed a compact, feasible and economically viable refinement strategy for antivenom preparation which looks promising for large Sculpting muscle definition production as well.
Process design was driven by the imperative of keeping IgGs or F measurew 2 fragments constantly in Antivenoom in order Pomegranate Desserts preserve stability of auality conformations. In each of jeasures main steps—caprylic acid precipitation for removal of contaminants, pepsin digestion Antivenom quality control measures IgGs and chromatographic polishing of F ab' 2 active principle, optimal performance conditions were Belly fat burner strategies. Also, measues novel platform controo been supported with process efficiency data, so accurate estimation Allergy relief through air filtration the cost-effectiveness is enabled.
Sculpting muscle definition Kurtović T, Lang Balija M, Brgles M, Sviben D, Tunjić M, Cajner H, et al. PLoS Negl Trop Dis 13 Antivenpm : e Received: February 13, ; Accepted: May 1, ; Published: June 17, Copyright: © Kurtović et al.
This is an open contrll article quaoity under the terms of Antjvenom Creative Commons Attribution Licensewhich permits unrestricted use, distribution, and reproduction in any medium, provided the original Antivennom and source are credited.
Data Availability: All relevant contrl are within OMAD and food choices manuscript and its Supporting Qualiry files. The funders had Antiveenom role measyres study design, data collection and analysis, cojtrol to publish, or preparation of the manuscript.
Competing interests: Qualit authors have measres that Anttivenom competing interests Enhance physical speed. Antivenoms prepared from hyperimmune animal plasma, mostly Antienom or ovine, mfasures the only specific therapeutics for rapid counteracting post-snakebite pathophysiological manifestations.
Although there are various well established downstream processing strategies that have xontrol implemented into commercial scale production, optimisation of compact, high yielding and low-cost manufacturing procedures generating safe, efficacious and available immunotherapeutics is still of great interest.
Design of the ideal process should be guided by the tendency Ajtivenom refine immunoglobulin G from residual measuree proteins in only a few easy, contro and measurez purification steps, aiming Antivenom quality control measures good recovery of neutralising activity and regulatory acceptable physicochemical characteristics of the Sculpting muscle definition [ 1 ].
Quality of the final product depends Antivnom on immunisation scheme that should maximally boost humoral response, giving the highest possible titer qyality anti-venom antibodies. Many of so far Antivvenom strategies as the initial step employ salting-out procedure involving measuress or sodium sulphate [ 2 — 6 qualith that is associated with low purity profile of IgGs meashres well as ocntrol formation of aggregates [ 7 meqsures, 8 qualify.
Both shortcomings can contro prevailed by Antivenmo caprylic acid as an alternative xontrol agent [ 7910 ] which acts on the majority of plasma proteins without affecting IgG fraction by leaving it in measires and, consequently, Fresh leafy vegetables its contrlo and structural stability [ 1112 ].
Refinement principles employing caprylic acid have been successfully implemented into preparations of a whole series of highly efficacious equine or ovine IgG-based antivenoms [ 13 — 17 ]. They have also been proven beneficial for purification of F ab' 2 derivatives [ 1819 ] and monoclonal antibodies [ 20 ].
Following IgG extraction some antivenom manufacturers perform enzyme-mediated separation of the Fc portion of IgG, because it is not important for the neutralisation activity, while its removal contributes to reduction of foreign protein quantity in the product intended for use in humans.
It has been generally believed that the lack of the Fc fragment disables complement activation or inhibits the formation of immune complexes that are responsible for the onset of delayed hypersensitivity reactions [ 112122 ]. However, poor physicochemical features of the product, i.
turbidity, high content of IgG or contaminating protein aggregates, also exhibit detrimental impact, which was evidenced irrespective of the presence or absence of the Fc fragment [ 111222 ]. Thus, its role in adverse reactions still remains unclear.
Enzymatic cleavage can be performed either on unfractionated plasma [ 11823 ] or isolated IgGs [ 1 ] as well as simultaneously with removal of unwanted proteins by caprylic acid precipitation [ 19 ]. Both F ab' 2 or Fab antivenoms have been successfully and widely used in snakebite management for decades [ 1 ], with the former ones being considered more clinically efficacious due to their slower elimination rate creditable for long lasting action [ 24 ].
Such complexes may be removed by phagocytic cells, eliminating the toxins from relevant tissue locations. This mechanism does not operate in the case of Fab antibodies. The use of Fab fragments is often associated with recrudescence of envenomation signs, although their rapid distribution might represent desirable pharmacokinetic feature when dealing with venom toxins of comparable molecular weight.
Ion-exchange chromatography has been introduced into some refinement strategies as well, proving suitable for separation of F ab' 2 fragments from other plasma proteins under conditions preferring antibody adsorption on cation-exchange stationary phase material [ 18 ].
Additionally, it has been recognised also as a method of choice for the final polishing where an anion-exchange approach is favourably used [ 25 ]. Other chromatography techniques are also applicable, for example, purification of F ab' 2 fragments by means of affinity chromatography exclusively [ 26 ].
Our study aimed for integrating the most efficient segments of the existing technological knowledge from the field into a compact, feasible and economically viable purification strategy for preparation of equine plasma-derived antivenom based on F ab' 2 fragments.
At the same time, effort was put into the preservation of highest process yield and fulfillment of the regulatory requirements concerning final product purity and aggregate content. The aim was also to precisely quantify the recovery of the active drug in each process step to enable accurate estimation of the cost-effectiveness of the designed procedure.
A standard mouse diet Mucedola srl. Animal monitoring for signs of pain, suffering and distress associated with procedure was performed following severity assessment protocol.
Crude venom of V. ammodytes Vaa and two pools of Vaa -specific hyperimmune horse plasma HHP were provided by the Institute of Immunology Inc. Caprylic acid, o -phenylenediamine dihydrochloride OPDiodoacetamide IAAdithiothreitol DTTbovine serum albumin BSATween 20, thimerosal, 2- N-morpholino ethanesulphonic acid MES monohydrate and Tris base were from Sigma-Aldrich, USA.
Pepsin from porcine gastric mucosa, 0. Goat anti-horse F ab' 2 IgG conjugated with horseradish peroxidase HRP was from antibodies-onlineGermany.
All other chemicals used for preparation of buffers and solutions were from Kemika, Croatia. The bound antibodies were eluted with 20 mM citric acid, pH 2.
A highly purified IgG sample eIgG was used as standard in ELISA assay and as model substrate for preliminary optimisation of pepsin digestion. HHP was incubated at 56 °C for 1 h. After centrifugation at 3, × g for 40 min and discarding the pellet, caprylic acid was added to 0.
Precipitation was performed by vigorous stirring rpm at 23 °C for 1 h in thermomixer Eppendorf, Germanyfollowed by sample centrifugation 2, × g45 min. IgG-enriched supernatant was collected and filtered through a cellulose acetate filter with a pore size of 5 μm Sartorius, Germany.
Minimal caprylic acid concentration giving the highest IgG purity and preserving yield, as preliminary determined, was chosen as optimal for the precipitation. Preliminary optimisation of pepsin digestion was done using a model IgG substrate—highly pure IgG sample eIgG isolated from HHP by protein A based affinity chromatography.
Generally, substrate aliquots 2 mg mL -1 were pH adjusted using 0. Pepsin solution 5 mg mL -1 in 0. The final volume of reaction mixture, prepared in saline, was 1 mL.
Digestion was terminated at timed intervals with a 0. Since it was not possible to execute all runs simultaneously, their order was randomised to avoid systemic errors.
We used a regression function model covering linear contribution of each factor, but also non-linear for selected experimental area. The full factorial design was employed resulting in 4 experimental runs, each performed in triplicate 2 2 × 3.
The significance of the given factors was determined by means of ANOVA using Statistica All subsequent experiments were performed using real process IgG substrate—IgG fraction from the optimised caprylic acid fractionation step, and examining one variable at a time.
The common approach involved acidification to pH 3. Incubation was performed at 37 °C for 1. When optimal conditions were set, the procedure was scaled up fold. Samples from each experimental set were analysed by SDS-PAGE.
IgG-enriched supernatant following caprylic acid precipitation was diafiltrated into water or saline using Vivaspin device Sartorius, Germany with a kDa molecular weight cut-off MWCO polyethersulfone membrane. In each diafiltration step the buffer was exchanged by a factor of 8, ×.
Elution was performed with 1 M NaCl in the binding buffer. The absorbance was monitored at nm. After collecting the flow-through fraction, the bound components were eluted from the column material with binding buffer containing 1 M NaCl. The enzymatic activity of pepsin was measured spectrophotometrically on Multiskan Spectrum instrument Thermo Fischer Scientific, USA using haemoglobin as substrate.
Modified Ryle's protocol was followed [ 31 ]. Samples previously diafiltrated into 50 mM KCl, pH 2. Aliquots of 40 μL were incubated with μL of 2. Non-degraded substrate was precipitated by centrifugation at 2, × g for 10 min and absorbance of the supernatants was measured at nm.
Blanks were obtained by omitting samples from reaction mixtures. Staining was carried out with acidic Coomassie Brilliant Blue CBB R solution or, alternatively, with silver for detection of pepsin traces.
Isoelectric focusing, the first dimension of 2D gel electrophoresis, was performed in a ZOOM IPGRunner Mini-Cell Invitrogen, USA using immobilised pH gradient IPG strip 7 cm long, linear pH 3—10 Invitrogen, USA rehydrated with F ab' 2 sample μgaccording to the protocol provided by the manufacturer.
The following step voltage protocol was applied: V for 20 min, V for 15 min, V for 15 min and 2, V for min. Obtained spots served as starting material for mass spectrometry MS. Excised protein spots obtained by 2D gel electrophoresis of F ab' 2 sample were prepared for MS analysis by in-gel trypsin digestion, as follows.
Following reduction and alkylation gel pieces were washed with mM NH 4 HCO 3 and ACN, dried and rehydrated in 1—10 μL of porcine trypsin solution Roche, Germany 10 ng of trypsin per estimated 1 μg of protein for 45 min. Pooled extracts were purified by C 18 Zip-Tips Millipore, USAdried, dissolved again in 0.
Measurements were performed on an ultrafleXtreme Bruker, Germany in positive, reflectron ion mode. The instrument is equipped with SmartBeam laser nmand the applied acceleration voltage was 8 kV in the positive ion mode.
Obtained spectra were processed using FlexAnalysis 3. Following parameters were used: precursor ion mass tolerance ± ppm, product ion mass ± 1. Variable modifications such as N-acetylation, C-amidation, ammonia loss from N-terminal Cys, modification of N-terminal Gln to pyro-Glu, oxidation of Met, His or Trp and phosphorylation of Ser, Thr or Tyr were taken into account.
: Antivenom quality control measures| Introduction | Table 3 Neutralization of in vivo pharmacological activities of SL snake venoms by newly developed SL PAV. This is what led a group of Danish researchers to create a lateral-flow test capable of detecting species-specific envenoming long before symptoms set in. Google Scholar Englemann, W. The pre-clinical study also provided convincing evidence for the neutralization of lethality and toxicity of SL snake venoms by SL PAV. Englemann, W. |
| SYSTEMATIC REVIEW article | Escalante T, Ortiz N, Rucavado A, Sanchez EF, Richardson M, Fox JW, et al. R -value was used as a measure of the protective efficacy of each sample. Cheaper and better diagnostics would not only improve treatment, but also lower the cost of antivenom as well. Methodology of clinical studies dealing with the treatment of envenomation. Except for the in vitro coagulant activity, the rest of these assays involve the use of high numbers of mice, with the consequent suffering and distress inflicted in these animals because of the toxic action of venoms. |
| References | Gutierrez JM Animal Antisera Production; Quality, safety and efficacy problems, Instituto Clodomiro Picado, Universidad de Costa Rica. Process design was driven by the imperative of keeping IgGs or F ab' 2 fragments constantly in solution in order to preserve stability of their conformations. Clinical evidence from an authenticated case series. Snakebites are no new phenomenon. For treating snakebite in SL, Indian PAV is mostly used, though its efficacy and neutralization potency towards SL snake venoms is of immense concern 13 , |
| Antivenom Safety and Tolerance for the Strategy of Snake Envenomation Management | SpringerLink | Assessment of snake antivenom purity Antivenom quality control measures comparing physicochemical and immunochemical methods. Uqality A, Tanjoni Cotnrol, Antivenom quality control measures I, Moura-da-Silva Sculpting muscle definition, Furtado Mmeasures. This Hypoglycemia and autoimmune disorders is corroborated Anfivenom the data from the densitometry analysis of SDS-PAGE bands of crude SL PAV Fig. However, following the good manufacturing practice GMP by the antivenoms manufacturers can result in production of different batches of PAV with uniform potency and composition Google Scholar. An ELISA-based assay was developed for the quantification of gelatinase activity of viperid venoms. |
| Needs and Availability of Snake Antivenoms: Relevance and Application of International Guidelines | Safety of snake antivenom immunoglobulins: efficacy of viral inactivation in a complete downstream process. Biotechnol Prog. Google Scholar. Carneiro SM, Zablith MB, Kerchove CM, Moura-da-Silva AM, Quissell DO, Markus RP, Yamanouye N. Venom production in long-term primary culture of secretory cells of the Bothrops jararaca venom gland. Chippaux JP, Williams V, White J. Snake venom variability: methods of study. Chotwiwatthanakun C, Pratapaphon R, Akesowan S, Sriprapat S, Ratanabangkoon K. Production of potent polyvalent antivenom against three elapid venoms using a low dose, low volume, multi-site immunization protocol. Dichtelmüller H, Rudnick D, Kloft M. Inactivation of lipid enveloped viruses by octanoic acid treatment of immunoglobulin solution. Article PubMed Google Scholar. Duddu S, Dal MP. Effect of glass transition temperature on the stability of lyophilized formulations containing a chimeric therapeutic monoclonal antibody. Pharm Res. EMEA The European Agency for the Evaluation of Medicinal Products. Note for guidance on virus validation studies: the design, contribution and interpretation of studies validating the inactivation and removal of viruses. London: EMEA; Note for guidance on the production and quality control of animal immunoglobulins and immunosera for human use. Feige K, Ehrat FB, Kästner SB, Schwarzwald CC. Automated plasmapheresis compared with other plasma collection methods in the horse. J Vet Med A Physiol Pathol Clin Med. Gutiérrez JM, Avila C, Rojas G, Cerdas L. An alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica. Gutiérrez JM, Lomonte B, León G, Alape-Girón A, Flores-Díaz M, Sanz L, Angulo Y, Calvete JJ. Snake venomics and antivenomics: proteomic tools in the design and control of antivenoms for the treatment of snakebite envenoming. J Proteomics. Gutiérrez JM, Sanz L, Flores-Díaz M, Figueroa L, Madrigal M, Herrera M, Villalta M, León G, Estrada R, Borges A, Alape-Girón A, Calvete JJ. Impact of regional variation in Bothrops asper snake venom on the design of antivenoms: integrating antivenomics and neutralization approaches. J Proteome Res. Gutiérrez JM, León G, Lomonte B, Angulo Y. Antivenoms for snakebite envenomings. Inflamm Allergy Drug Targets. Gutiérrez JM, Solano G, Pla D, Herrera M, Segura A, Villalta M, Vargas M, Sanz L, Lomonte B, Calvete JJ, León G. Assessing the preclinical efficacy of antivenoms: from the lethality neutralization assay to antivenomics. ICH International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH; Kempf C, Stucki M, Boschetti N. Pathogen inactivation and removal procedures used in the production of intravenous immunoglobulins. Kim H, Nakai S. Simple separation of immunoglobulin from egg yolk by ultrafiltration. J Food Sci. Article CAS Google Scholar. Ko KY, Ahn DU. Preparation of immunoglobulin Y from egg yolk using ammonium sulfate precipitation and ion exchange chromatography. Poult Sci. Lazar A, Epstein E, Lustig S, Barnea A, Silberstein L, Reuveny S. Inactivation of West-Nile virus during peptic cleavage of horse plasma IgG. León G, Sánchez L, Hernández A, Villalta M, Herrera M, Segura A, Estrada R, Gutiérrez JM. Immune response towards snake venoms. Macedo SM, Lourenço EL, Borelli P, Fock RA, Ferreira Jr JM, Farsky SH. Effect of in vivo phenol or hydroquinone exposure on events related to neutrophil delivery during an inflammatory response. Meier J, Adler C, Hösle P, Cascio R. The influence of three different drying procedures on some enzymatic activities of three Viperidae snake venoms. Mem Inst Butantan. Niinistö K, Raekallio M, Sankari S. Storage of equine red blood cells as a concentrate. Vet J. Pikal MJ. Mechanism of protein stabilization during freeze-drying and storage: the relative importance of thermodynamic stabilization and glassy state relaxation dynamics. In: Rey L, May JC, editors. New York: Marcer Dekker Inc; Rial A, Morais V, Rossi S, Massaldi H. A new ELISA for determination of potency in snake antivenoms. Rojas G, Jiménez JM, Gutiérrez JM. Caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production. Sampaio SC, Rangel-Santos AC, Peres CM, Curi R, Cury Y. Inhibitory effect of phospholipase A2 isolated from Crotalus durissus terrificus venom on macrophage function. Sarciaux JM, Mansour S, Hageman MJ, Nail SL. Effects of buffer composition and processing conditions on aggregation of bovine IgG during freeze-drying. J Pharm Sci. Schersch K, Betz O, Garidel P, Muehlau S, Bassarab S, Winter G. Systematic investigation of the effect of lyophilizate collapse on pharmaceutically relevant proteins I: stability after freeze-drying. Segura Á, León G, Su C-Y, Gutiérrez J-M, Burnouf T. Segura Á, Herrera M, González E, Vargas M, Solano G, Gutiérrez JM, León G. Stability of equine IgG antivenoms obtained by caprylic acid precipitation: towards a liquid formulation stable at tropical room temperature. Segura A, Herrera M, Villalta M, Vargas M, Gutiérrez JM, León G. Assessment of snake antivenom purity by comparing physicochemical and immunochemical methods. Solano S, Segura Á, León G, Gutiérrez JM, Burnouf T. Low pH formulation of whole IgG antivenom: impact on quality, safety, neutralizing potency and viral inactivation. Teixeira C, Cury Y, Moreira V, Picolob G, Chaves F. Inflammation induced by Bothrops asper venom. Theakston RD, Warrell DA, Griffiths E. Report of a WHO workshop on the standardization and control of antivenoms. Wang W. Instability, stabilization and formulation of liquid protein pharmaceuticals. Int J Pharm. Wang W, Singh S, Zeng D, King K, Nema S. Antibody structure, instability and formulation. World Health Organization. Handbook for good clinical research practices GCP. Geneva: WHO; Guidelines for the production, control and regulation of snake antivenom immunoglobulins. Xie G, Timasheff N. Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured than for the native protein. Protein Sci. Article CAS PubMed Central PubMed Google Scholar. Zychar BC, Castro Jr NC, Marcelino JR, Gonçalves LR. Phenol used as a preservative in Bothrops antivenom induces impairment in leukocyte-endothelial interactions. Download references. Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. You can also search for this author in PubMed Google Scholar. Correspondence to Guillermo León. Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. Biomedicina de Valencia IBV-CSIC, Valencia, Spain. Reprints and permissions. Immediate and delayed allergic reactions to Crotalidae Polyvalent Immune Fab ovine antivenom. Ann Emerg Med. Dart RC, Mcnally J. Efficacy, safety, and use of snake antivenoms in the United States. Otero-Patino R, Cardozo J, Higashi H, Nuñez V, Diaz A, Toro M, García M, Sierra A, García LF, Moreno A, Medina M, Castañeda N, Silva-Diaz JF, Murcia M, Cardenas SY, Dias da Silva WD. A randomized, blinded, comparative trial of one pepsin-digested and two whole IgG antivenoms for Bothrops snake bites in Uraba, Colombia. The Regional Group of Antivenom Therapy Research REGATHER. Am J Trop Med Hyg. Harms AJ. The purification of antitoxic plasmas by enzyme treatment and heat denaturation. Biochem J. Gutiérrez JM, Higashi HG, Wen FH, Burnouf T. Strengthening antivenom production in Central and South American public laboratories: Report of a workshop. Rojas G, Jiménez JM, Gutierrez JM. Caprylic acid fractionation of hyperinmune horse plasma: description of a single procedure for antivenom production. Raweerith R, Ratanabanangkoon K. Fractionation of equine antivenom using caprylic acid precipitation in combination with cationic ion-exchange chromatography. J Immunol Meth. Pepin-Covatta S, Lutsch C, Grandgeorge M, Scherrmann JM. Immunoreactivity of a new generation of horse F ab 2 preparations against European viper venoms and the tetanus toxin. Raw I, Guidolin R, Higashi H, Kelen E. Antivenins in Brazil: preparation. In: Tu AT editor. Handbook of natural toxins. New York: Marcel Dekker; Jadhav SS, Kapre SV. Antivenom producion in India. New York: Marcel Dekker; , p. Abbas AK, Lichtman AH, Pober JS editors. Cellular and molecular immunology. Philadelphia: WB Sanders Co. Abbas AK, Litchman AH. Immediate Hypersensitivity. In: Abbas AK, Litchman AH editors. Philadelphia: Saunders Elsevier Science; Cruce JM, Lewis RE. Types I, II, III, and IV hypersensitivity. In: Atlas of immunology. Florida: CRC Press; Morais V, Massaldi H. Economic evaluation of snake antivenom production in the public system. J Venom Anim Toxins incl Trop Dis. Steinbuch M, Audran R. The isolation of IgG from mammalian sera with the aid of caprylic acid. Arch Biochem Biophys. Gutiérrez JM, Rojas E, Quesada L, León G, Nuñez J, Laing GD, Sasa M, Renjifo JM, Nasidi A, Warrell DA, Theakston RDG, Rojas G. Pan-African polyspecific antivenom produced by caprylic acid purification of horse IgG: an alternative to the antivenom crisis in Africa. Trans R Soc Trop Med Hyg. Saetang T, Treamwattana N, Suttijitpaisal P, Ratanabanangoon K. Quantitative comparison on the refinement of horse antivenom by salt fractionation and ion-exchange chromatography. J Chromatog B. Gutiérrez JM, Lomonte B, León G, Rucavado A, Chaves F, Angulo Y. Trends in snakebite envenomation therapy: Scientific, technological and Public Health considerations. Curr Pham Des. Krifi MN, El Ayeb M. Dellagi K. The improvement and standardization of antivenom production in developing countries: comparing antivenom quality, therapeutical efficiency, and cost. J Venom Anim Toxins. Chotwiwatthanakun C, Pratanaphon R, Akesowan S, Sriprapat S, Ratanabanangkoon K. Production of potent polyvalent antivenom against three elapid venoms using a low dose, low volume, multi-site immunization protocol. Sriprapat S, Aeksowan S, Sapsutthipas S, Chotwiwatthanakun C, Suttijitpaisal P, Pratanaphon R, Khow O, Sitprija V, Ratanabanangkoon K. The impact of a low dose, low volume, multi-site immunization on the production of therapeutic antivenoms in Thailand. Ferreira Junior RS, Nascimento N, Couto R, Alves JB, Meira DA, Barraviera B. Laboratory evaluation of young ovines inoculated with natural or 60Co-irradiated Crotalus durissus terrificus venom during hyperimmunization process. Oussedik-Oumehdi H, Laraba-Djebari F. Irradiated Cerastes cerastes venom as a novel tool for immunotherapy. Immunopharmacol Immunotoxicol. Jones RG, Corteling RL, Bhogal G, Landon J. A novel Fab based antivenom for the treatment of the mass bee attacks. Chippaux JP, Lang J, Amadi-Eddine S, Fagot P, Le Mener V. Short report: treatment of snake envenomation s by anew polyvalent antivenom composed for highly purifyied F ab' 2 Results of a clinical trial in northern Cameroon. Fernandez I, Takeara HA, Mota I. Isolation of IgG T from hyperimmune horse anti-snake venom serum: its protective ability. Fernandez I, Takeara HA, Santos CR, Cormont F, Latinne D, Bazin H, Mota I. Neutralization of bothropic and crotalic venom toxic activities by IgG T and IgGa subclases isolated from immune horse serum. Fernández I, Lima EX, Takehara HA, Moura-da-Silva AM, Tanjoni I, Gutierrez JM. Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica. Pope CG. The action of proteolyitic enzymes on the antitoxin and proteins in inmune sera: I True digestion of the proteins. Br J Exp Path. The action of proteolyitic enzymes on the antitoxin and proteins in inmune sera: II Heat denaturation after partial enzyme action. Sjostrom L, Al-Abdulla I, Rawat S, Smith D, Landon J. A comparation of ovine and equine antivenoms. Ariaratnam CA, Meyer WP, Perera G, Eddleston M, Kuleratne AM, Attapattu W, Sheriff R, Richards AM, Theakston RDG, Warrel DA. A new monospecific ovine Fab fragment antivenom for the treatment of envenoming by the Sri Lankan Russell's viper Daboia russelli russelli : a preliminary dose-finding and pharmacokinetics study. Harrison RA, Hasson SS, Harmsen M, Laing GD, Conrath K, Theakston RDG. Neutralization of venom-induced haemorrhage by IgG from camels and llamas immunised with viper venom and also by endogenous, non-IgG components in camelid sera. Herrera M, León G, Segura A, Meneses F, Lomonte B, Chippaux JP, Gutiérrez JM. Factors associated with adverse reactions induced by caprylic acid-fractionated whole IgG preparations: comparison between horse, sheep and camel IgGs. Hamrock DJ. Adverse events associated with intravenous immunoglobulin therapy. Int Immunopharm. Ferreira RN, Machado de Avila RA, Sanchez EF, Maria WS, Molina F, Granier C, Chávez-Olórtegui C. Antibodies against synthetic epitopes inhibit the enzymatic activity of mutalysin II, a metalloproteinase from bushmaster snake venom. Rafael A, Tanjoni I, Fernandes I, Moura-da-Silva AM, Furtado MF. An alternative method to access in vitro the hemorrhagic activity of snake venoms. Sánchez EE, García C, Pérez JC, de La Zerda SJ. The detection of hemorrhagic proteins in snake venoms using monoclonal antibodies against Virginia opossum Didelphis virginiana serum. Stábeli RG, Magalhães LM, Selistre-de-Araujo HS, Oliveira EB. Antibodies to a fragment of the Bothrops moojenii-amino acid oxidase cross-react with snake venom components unrelated to the parent protein. Tamarozzi MB, Soares SG, Marcussi S, Giglio JR, Barbosa JE. Expression of recombinant human antibody fragments capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom. Biochim Biophys Acta. Harrison RA, Wuster W, Theakston RGD. The conserved structure of snake venom toxins confers extensive immunological cross-reactivity to toxin-specific antibody. Tanjoni I, Butera D, Bento L, Della-Casa M, Marques R, Takehara H, Gutiérrez JM, Fernández I, Moura da Silva A. Colombini M, Fernandes I, Cardoso DF, Moura-da-Silva AM. Lachesis muta muta venom: immunological differences compared with Bothrops atrox venom and importance of specific antivenom therapy. Galán J, Sánchez E, Rodríguez-Acosta A, Pérez J. Neutralization of venoms from two Souther Pacific rattlesnakes Crotalus helleri with comercial antivenoms and endothermic animal sera. Lizano S, Domont G, Perales J. Natural phospholipase A2 myotoxin inhibitor proteins from snakes, mammals and plants. Peréz J, Sánchez E. Natural protease inhibitors to hemorrhagins in snake venoms and their potential use in medicine. Nuñez V, Otero R, Barona J, Fonnegra R, Jiménez S, Osorio R, Quintana J, Díaz A. Inhibition of the toxic effects of Lachesis muta, Crotalus durissus cumanensis and Micrurus mipartitus snake venoms by plant extracts. Pharm Biol. Mors W, Nacimento MC, Pereira BMR, Pereira NA. Plant natural products active against snake bite, the molecular approach. Fortes-Dias C. Endogenous inhibitors of snake venom phospholipases A2 in the blood plasma of snakes. Jurgilas P, Neves-Ferreira A, Domont G, Perales J. PO41, a snake venom metalloproteinase inhibitor isolated from Philander opossum serum. Neves-Ferreira A, Cardinale N, Rocha S, Perales J, Domont G. Isolation and caracterization of DM40 and DM43, two snake venom metalloproteinase inhibitors from Didelphis marsupialis serum. Surza L, Rocha G, Lomonte B, Neves-Ferreira A, Trugilho M, Junqueira-de-Azevedo I, Ho P, Domont G, Gutiérrez JM, Perales J. Functional analysis of DM64, an antimyotoxic protein with immunoglobulin-like structure from Didelphis marsupialis serum. Eur J Biochem. Thwin M, Gopalakrishnakone P. Snake envenomation and protective natural endogenous proteins: a mini review of the recent developments - Abbas AK, Lichtman AH, Pober JS. Mecanismos efectores de la inmunidad humoral. In: Abbas AK, Lichtman AH, Pober JS. Inmunología celular y molecular. Espana: Mc Graw Hill; Abbas AK, Lichtman AH. Effector mechanisms of humoral immunity. In: Schmitt W, Hacker N, Ehlers J. Philadelphia: Elsevier Science Saunders; García M, Monge M, León G, Lizano S, Segura E, Solano G, Rojas G, Gutiérrez JM.. Effect of preservatives on IgG aggregation, complement-activating effect and hypotensive activity of horse polyvalent anti-venom used in snakebite envenomation. Otero R, Gutiérrez JM, Rojas G, Nuñez V, Díaz A, Miranda E, Uribe AF, Silva JF, Ospina JG, Medina Y, Toro MF, García ME, León G, García M, Lizano S, de La Torre J, Márquez J, Mena Y, González N, Arenas LC, Puzón A, Blanco N, Sierra A, Espinal M, Arboleda M, Jiménez JC, Ramírez P, Díaz M, Guzmán MC, Barros J, Henao S, Ramírez A, Macea U, Lozano R. A randomized, blinded clinical trial of two antivenoms , prepared by caprylic acid or ammonium sulphate fractionation of IgG, in Bothrops and Porthidium snake bites Colombia: correlation between safety and biochemical characteristics of antivenoms. Otero R, León G, Gutiérrez JM, Rojas G, Toro MF, Barona J, Rodríguez V, Díaz A, Nuñez V, Quintana JC, Ayala S, Mosquera D, Conrado L, Fernández D, Arroyo Y, Paniagua C, López M, Ospina C, Alzate C, Fernández J, Meza J, Silva J, Ramírez P, Fabra P, Ramírez E, Córdoba E, Arrieta A, Warrell DA, Theakston RDG. Efficacy and safety of two whole IgG polyvalent antivenoms, refined by caprylic acid fractionation with or without â-propiolactone, in the treatment of Bothrops asper bites in Colombia. León G, Lomonte B, Gutiérrez JM. Anticomplementary activity of equine whole IgG antivenoms: comparison of three fractionation protocols. Effect of pepsin digestion on the antivenom activity of equine immunoglobulins. Jones RG, Landon J. Enhanced pepsin digestion: a novel process for purifying antibody f ab' 2 fragments in high yield for serum. J Immunol Methods. A protocol for enhanced pepsin digestion a step by step method for obtained pure antibody fragments in high yield from serum. Rojas G, Espinoza M, Lomonte B, Gutiérrez JM. Effect of storage temperature on the stability of the liquid polyvalent antivenom produced in Costa Rica. Silverstein AM. Clemens Freiherr von Pirquet: Explaining immune complex disease in Nat Immunol. León G, Monge M, Rojas E, Lomonte B, Gutierrez JM. Comparison between IgG and F ab' 2 polyvalent antivenoms: neutralization of systemic effects induced by Bothrops asper venom in mice, extravasation of muscle tissue, and potential for induction of adverse reactions. Silva de Freitas S, Machado RL, De Arruda EJ, Costapinto C, Alves Bueno S. J Memb Sci. Petsch D, Anspach FB. Endotoxin removal from protein solutions. J Biotechnol. Brunn GJ, Platt JL. The etiology of sepsis: turned inside out. Trends Mol Med. Erridge C, Bennett-Guerrero E, Poxton I. Structure and function of lipopolysaccharides. Microbes Infect. Fang H, Wei J, Yu Y. In vivo studies of endotoxin removal by lysine-cellulose adsorbents. Victor V, Rocha M, De La Fuente M. Immune cells: free radicals and antioxidants in sepsis. Int Inmunopharmacol. Machado RL, De Arruda EJ, Santana CC, Bueno SMA. Evaluation of a chitosan membrane for removal of endotoxin from human IgG solutions. Process Biochem. Massaldi H, Morais V. Evolution of endotoxin contamination during production of a therapeutic serum. PDA J Pharm Sci Technol. Anspach FB. |
0 thoughts on “Antivenom quality control measures”