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Potency testing of tetanus antitoxin must be performed in vivo, in a very painful, stressful and prone to high variability assay. It is, therefore, mandatory to find alternatives to this kind of potency assessment. Immunochemical tests as ELISA or ToBI test are already available but usually results in a poor correlation to the in vivo protection. Considering research and development of mono and oligoclonal antibodies against tetanus and the improvement of equine polyclonal antitoxin production and control, we developed an alternative instrumental test for tetanus antitoxin by using surface plasmon resonance. Tetanus antitoxin from hyperimmune equine sera (16 batches) were tested and the results indicated excellent concordance and correlation to the in vivo test (Lin's Ró=0.9). This innovative approach should now be improved in order to extend it to oligoclonal and monoclonal human antibodies aiming to replace mice for the potency assessment of tetanus antitoxin especially during research and development steps.

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BACKGROUND: Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab')2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. METHODS: Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p < 0.05. RESULTS: Horse F(ab')2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates. CONCLUSIONS: Anti-botulinum AB (bivalent), anti-diphtheric, antitetanic and anti-rabies horse F(ab')2 immunoglobulins probably contain acceptable levels of aggregates, although other improvements on the preparations must be carried out. Protein profile analysis and in vitro anticomplementary activity of F(ab')2 immunoglobulin preparations should be included as quality control steps, to ensure acceptable levels of aggregates, contaminants and whole IgG molecules on final products, reducing the chances of adverse reactions in patients.

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This review presents the main contributions to our knowledge regarding the development of antivenoms for therapeutic use in victims of venomous animal bites. We cover the progress of serum therapy since tetanus and diphtheria antitoxins in Germany and France until the current scenario of antivenom production worldwide. During these more than 120 years of antivenom development, many researchers contributed to establish what are nowadays the antivenoms used for therapeutic purpose. The history of antivenoms development is fascinating! This review aims to recognize all those who contributed to the establishment of new sera, new methodologies and saving lives: much more than Calmette and Vital Brazil.

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Background Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab)2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p 0.05. Results Horse F(ab)2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates...(AU)

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Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab′)2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p < 0.05. Results Horse F(ab′)2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates. Conclusions Anti-botulinum AB (bivalent), anti-diphtheric, antitetanic and anti-rabies horse F(ab′)2 immunoglobulins probably contain acceptable levels of aggregates, although other improvements on the preparations must be carried out. Protein profile analysis and in vitro anticomplementary activity of F(ab′)2 immunoglobulin preparations should be included as quality control steps, to ensure acceptable levels of aggregates, contaminants and whole IgG molecules on final products, reducing the chances of adverse reactions in patients.(AU)

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This review presents the main contributions to our knowledge regarding the development of antivenoms for therapeutic use in victims of venomous animal bites. We cover the progress of serum therapy since tetanus and diphtheria antitoxins in Germany and France until the current scenario of antivenom production worldwide. During these more than 120 years of antivenom development, many researchers contributed to establish what are nowadays the antivenoms used for therapeutic purpose. The history of antivenoms development is fascinating! This review aims to recognize all those who contributed to the establishment of new sera, new methodologies and saving lives: much more than Calmette and Vital Brazil.

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Background: Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab')2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods: Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p < 0.05. Results: Horse F(ab')2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates. Conclusions: Anti-botulinum AB (bivalent), anti-diphtheric, antitetanic and anti-rabies horse F(ab')(2) immunoglobulins probably contain acceptable levels of aggregates, although other improvements on the preparations must be carried out. Protein profile analysis and in vitro anticomplementary activity of F(ab')2 immunoglobulin preparations should be included as quality control steps, to ensure acceptable levels of aggregates, contaminants and whole IgG molecules on final products, reducing the chances of adverse reactions in patients.

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The assessment of the capacity of antivenoms to neutralize the lethal activity of snake venoms still relies on traditional rodent in vivo lethality assay. ED50 and LD50 assays require large quantities of venoms and antivenoms, and besides leading to animal suffering. Therefore, in vitro tests should be introduced for assessing antivenom neutralizing capacity in intermediary steps of antivenom production. This task is facilitated when one key lethal toxin is identified. A good example is crotoxin, a P-neurotoxin phospholipase A(2)-like toxin that presents anticoagulant activity in vitro and is responsible for the lethality of venoms of Crotalus durissus snakes. By using rotational thromboelastometry, we reported recently one sensitive coagulation assay for assessing relative potency of the anti-bothropic serum in neutralizing procoagulant activity of Bothrops jararaca venom upon recalcified factor-XII-deficient chicken plasma samples (CPS). In this study, we stablished conditions for determining relative potency of four batches of the anti-crotalic serum (ACS) (antagonist) in inactivating crotoxin anticoagulant activity in CPS (target) simultaneously treated with one classical activator of coagulation (agonists). The correlation coefficient (r) between values related the ACS potency in inactivating both in vitro crotoxin anticoagulant activity and the in vivo lethality of whole venom (ED50) was 0.94 (p value < 0.05). In conclusion, slowness in spontaneous thrombin/fibrin generation even after recalcification elicit time lapse sufficient for elaboration of one dose-response curve to pro-or anti-coagulant agonists in CPS. We propose this methodology as an alternative and sensitive assay for assessing antivenom neutralizing ability in plasma of immunized horses as well as for in-process quality control.

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Considering that the scarcity of venom represents a huge challenge for biochemical and functional studies of Micrurus species (coral snakes), in this report we describe for the first time the influence of pilocarpine administration prior to venom milking on the yield and protein composition of Micrurus corallinus venom. The administration of pilocarpine resulted in an increase of about 127% in the volume of venom milked, with similar protein content. Venoms showed similar protein bands distribution and intensity by SDS-PAGE and equivalents RP-HPLC profiles. Our proteomic analysis showed that venoms milked in the presence and absence of pilocarpine presented comparable protein profiles, in terms of protein composition and relative abundance. The toxins identified were assigned to 13 protein families and represent the most complete M. corallinus venom proteome described so far, in terms of number of protein families identified. Our data indicate that the administration of pilocarpine prior to venom milking increases the venom yield and does not change significantly the venom composition of M. corallinus. The employment of pilocarpine represents a useful approach to increase the yield of venom not only for Micrurus species, but also for other genera of snakes with limitations regarding the amount of venom available.

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Background Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab)2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p 0.05. Results Horse F(ab)2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates...

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Micrurus snakebites can cause death by muscle paralysis and respiratory arrest a few hours after envenomation. The specific treatment for these snake envenomations is the intravenous application of heterologous antivenom. In Brazil, this antivenom is produced from horses that are immunized with a mixture of Micrurus corallinus and Micrurus frontalis venoms, which are snakes that inhabit the south and southeastern regions of the country. Previously, we demonstrated that the coral antivenom, which is used in human therapy, was not able to neutralize several of the toxic venom effects from some Micrurus species that inhabit the country, as measured by in vitro and in vivo assays. The present study aimed to investigate the immunogenic properties of Micrurus spp. venoms, as well as the cross-reactivity and neutralization potential of experimental monovalent and polyvalent sera that were produced in different animal species. The present data showed that Micrurus venoms exhibited the same immunogenicity pattern in the three utilized animal species and that the specific antisera presented a large cross-reactivity when analyzed with ELISA and Western blot assays. Nonetheless, these positive results were not well correlated with the neutralizing potential of the antisera. Thus, the establishment of a new antigenic mixture to produce novel more efficient therapeutic Micrurus antivenom is not a simple task. Further studies, particularly with the Micrurus lemniscatus, Micrurus altirostris and Micrurus surinamensis venoms, are necessary to establish new strategies for the production of antivenoms with broad neutralizing activity for the treatment of accidents involving coral snakes throughout the country.

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BACKGROUND: Snake envenoming is a significant public health problem in underdeveloped and developing countries. In sub-Saharan Africa, it is estimated that 90,000-400,000 envenomations occur each year, resulting in 3,500-32,000 deaths. Envenomings are caused by snakes from the Viperidae (Bitis spp. and Echis spp.) and Elapidae (Naja spp. and Dendroaspis spp.) families. The African continent has been suffering from a severe antivenom crisis and current antivenom production is only sufficient to treat 25% of snakebite cases. Our aim is to develop high-quality antivenoms against the main snake species found in Mozambique. METHODS: Adult horses primed with the indicated venoms were divided into 5 groups (B. arietans; B. nasicornis + B. rhinoceros; N. melanoleuca; N. mossambica; N. annulifera + D. polylepis + D. angusticeps) and reimmunized two times for antivenom production. Blood was collected, and plasma was separated and subjected to antibody purification using caprylic acid. Plasmas and antivenoms were subject to titration, affinity determination, cross-recognition assays and in vivo venom lethality neutralization. A commercial anti-Crotalic antivenom was used for comparison. RESULTS: The purified antivenoms exhibited high titers against B. arietans, B. nasicornis and B. rhinoceros (5.18 x 106, 3.60 x 106 and 3.50 x 106 U-E/mL, respectively) and N. melanoleuca, N. mossambica and N. annulifera (7.41 x 106, 3.07 x 106 and 2.60 x 106 U-E/mL, respectively), but lower titers against the D. angusticeps and D. polylepis (1.87 x 106 and 1.67 x 106 U-E/mL). All the groups, except anti-N. melanoleuca, showed significant differences from the anti-Crotalic antivenom (7.55 x 106 U-E/mL). The affinity index of all the groups was high, ranging from 31% to 45%. Cross-recognition assays showed the recognition of proteins with similar molecular weight in the venoms and may indicate the possibility of paraspecific neutralization. The three monospecific antivenoms were able to provide in vivo protection. CONCLUSION: Our results indicate that the anti-Bitis and anti-Naja antivenoms developed would be useful for treating snakebite envenomations in Mozambique, although their effectiveness should to be increased. We propose instead the development of monospecific antivenoms, which would serve as the basis for two polyvalent antivenoms, the anti-Bitis and anti-Elapidae. Polyvalent antivenoms represent an increase in treatment quality, as they have a wider range of application and are easier to distribute and administer to snake envenoming victims.

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Envenomation by poisonous animals is a neglected condition according to the World Health Organization (WHO). Antivenoms are included in the WHO Essential Medicines List. It has been assumed that immunoglobulin G (IgG) antivenoms could activate the complement system through Fc and induce early adverse reactions (EARs). However, data in the literature indicate that F(ab')2 fragments can also activate the complement system. Herein, we show that several batches of IgG and F(ab')2 antivenoms from the Butantan, Vital Brazil, and Clodomiro Picado Institutes activated the complement classical pathway and induced the production of C3a; however, only those antivenoms from Clodomiro Picado generated C5a. Different protein profiles (IgG heavy chain, protein contaminants, and aggregates) were observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses. Our results show that various antivenoms from different producers are able to activate the classical pathway of the complement system and generate anaphylatoxins, and these findings suggest that factors, such as composition, contaminant proteins, and aggregates, may influence the anticomplementary activity of antivenoms in vitro. Therefore, there is a need to further improve antivenom production methods to reduce their anticomplementary activity and potential to cause EARs.

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Victims of massive bee attacks become extremely ill, presenting symptoms ranging from dizziness and headache to acute renal failure and multiple organ failure that can lead to death. Previous attempts to develop specific antivenom to treat these victims have been unsuccessful. We herein report a F(ab)(´)(2)-based antivenom raised in horse as a potential new treatment for victims of multiple bee stings. The final product contains high specific IgG titers and is effective in neutralizing toxic effects, such as hemolysis, cytotoxicity and myotoxicity. The assessment of neutralization was revised and hemolysis, the primary toxic effect of these stings, was fully neutralized in vivo for the first time.

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Envenomation by Loxosceles species (brown spider) can lead to local dermonecrosis and to serious systemic effects. The main toxic component in the venom of these spiders is sphingomyelinase D (SMase D) and various isoforms of this toxin are present in Loxosceles venoms. We have produced a new anti-loxoscelic serum by immunizing horses with recombinant SMase D. In the present study, we compared the neutralization efficacy of the new anti-loxoscelic serum and anti-arachnidic serum (the latter serum is used for therapy for loxoscelism in Brazil) against the toxic effects of venoms from spiders of the genus Loxosceles. Neutralization tests showed that anti-SMase D serum has a higher activity against toxic effects of L. intermedia and L. laeta venoms and similar or slightly weaker activity against toxic effects of L. gaucho than that of Arachnidic serum. These results demonstrate that recombinant SMase D can replace venom for anti-venom production and therapy.

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The effect of Bothrops antivenom on blocking the disturbances induced by Bothrops jararaca venom in leukocyte-endothelial interactions (LEI) at the microcirculation of the cremaster muscle in mice was evaluated using intravital microscopy. Our findings showed that an i.v. injection of Bothrops antivenom, per se, induced changes in LEI, similar to those induced by an s.c. injection of B. jararaca venom, and that Bothrops antivenom can also induce ephemeral symptoms, such as tremor and dyspnea in mice. These effects were mostly due to phenol used in Bothrops antivenom as a preservative, since animals injected i.v. with a phenol solution, but not with phenol-free Bothrops antivenom, presented those effects on LEI, and also tremor and dyspnea. In addition, phenol-free antivenom abrogated venom-induced changes in LEI parameters. The present data demonstrate that Bothrops antivenom contains antibodies that neutralize toxins of B. jararaca venom that impair LEI, and suggest that the phenol used as a preservative in it can originate some undesired effects.

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A reducao da ativacao do complemento atraves de uma alteracao do fragmento Fc das imunoglobulinas pela beta-propiolactona foi obtida em soros hiperimunes equinos antivirus rabico, venenos Bothrops e toxina difterica. Os resultados foram avaliados por teste de anafilaxia em cobaias, e comparados com aqueles obtidos com os mesmos soros purificados por precipitacao salina (sulfato de amonio), seguidos ou nao por digestao enzimatica com pepsina. Os niveis de pureza proteica foram para o soro antibotropico de 184,5 mg/g e 488,5 mg/g tratado pela beta-propiolactona e digeridos pela pepsina, respectivamente...(au)

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