Caprylic acid fractionation of hyperimmune horse plasma: Description of a simple procedure for antivenom production (original) (raw)
Related papers
Biomedical Research (Aligarh), 2018
Antivenoms used for treatment of envenomation caused by like snakes and scorpions, are mostly produced in equine (horses) or ovine (sheep and goat). In Iran, the production of polyspecific scorpion antivenom has been started since 70 y ago and purified with ammonium sulfate fractionation after pepsin digestion of Fc fragment from IgG. Since this conventional method is time consuming, expensive and the product has lower purity. In this study we used either caprilic acid (Octatonic acid) alone or with combination with ammonium sulfate for separation and removal of impurities and precipitation of specific F(ab´) 2 against scorpion venom. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) showed that purification with caprilic acid at a final concentration of 3.5% is optimal to obtain immunoglobulins essentially free of albumin. The second method of purification includes combination of 1.5% (v/v) caprilic acid with 20% saturation with ammonium sulfate. Both procedures are performed after peptic digestion of IgG (removal of Fc fragment) at 37°C for 4 h at pH=3.5. After peptic digestion pH is raised to 4.8 with 1.5 molar sodium hydroxide to stop enzyme digestion then in the first method 3.5-5% caprilic acid was added with strong agitation for one hour the impurities were removed with filter paper and the supernatant was dialyzed against CIS buffer. In the second method after adjusting pH=4.8 ammonium sulfate was added to 20% saturation then 1.5% caprilic acid was added with strong agitation for one hour then slurry is passed through filter paper the precipitated impurities discarded and supernatant was dialyzed.
Evaluation of four different immunogens for the production of snake antivenoms
Toxicon, 1992
Q. LI and C. L. OwNBY. Evaluation of four different immunogens for the production of snake antivenoms. Toxicon 30, 1319-1330, 1992 .-Four different immunogens were used to produce polyvalent antivenom in rabbits to the venoms of Bothrops atrox, Crotalus atrox, Crotalus adamanteus and Crotalus durissus terricus. The immunogens were: (1) unfractionated mixture of the four crude venoms, and three fractions of the mixture as follows, (2) HPLC gel filtration high (> 50,000) mol. wt fraction, (3) HPLC gel filtration medium (14,000-50,000) mol. wt fraction, and (4) HPLC gel filtration low (< 14,000) mol. wt fraction. The resultant immune sera were compared with commercial antivenom (Wyeth, polyvalent Crotalidae) for total IgG content, ELISA reactivities, patterns of Western blots and ability to neutralize lethal and local hemorrhagic activities of the four venoms. The results indicate that the rabbit antivenoms had significantly higher ELISA reactivity and blotting signals than Wyeth antivenom. However, neither ELISA nor Western blotting signals correlated with the ability of the antivenoms to neutralize the lethal or hemorrhagic activities of the venoms. The protective ability of the antivenoms varied considerably. In general, antivenoms generated by using fractionated venoms as immunogens exhibited greater protective ability than antivenom produced by using the mixture of four venoms as immunogen. Some of the antivenoms provided greater or comparable protective ability for certain venoms when compared to Wyeth antivenom. It appears that the use of certain venom fractions as immunogens is a promising alternative for production of effective antivenoms .
Tropical Journal of Pharmaceutical Research
To immunize antiserum of horse with Androctonus crassicauda scorpion venom in order to achieve an antivenom with higher purity by combined caprylic acid/ammonium sulfate. Methods: The optimum pH to terminate enzymatic digestion was evaluated. Purification was performed by various combinations of caprylic acid (0 to 2.5 mL %) and ammonium sulfate (0 to 20 g %) at 25, 30 and 37 ºC. The effects of three factors (caprylic acid, ammonium sulfate and temperature) were evaluated based on precipitation of non-immunoglobulin proteins. Antivenom purity was evaluated by determining the concentration of desired soluble protein and undesired albumin, as well as by turbidity and titration. Results: The results showed that the optimum pH for inhibition of enzyme activity and precipitation of impurities was 4.8. SDS-PAGE revealed that the highest impurity precipitation and lowest protein aggregation was occurred at the combination of 1.5 mL % caprylic acid and 10 g % ammonium sulfate at 37 ºC. Conclusion: The modified method of purification significantly decreases turbidity, albumin impurity concentration and processing time but increased antibody titer and purity of antivenom. Therefore, it is a potentially suitable method for purifying antivenom in commercial production.
Toxicon, 1988
alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica. Toxicon 26, 411-413, 1988 . -The ability of several batches of polyvalent antivenom to neutralize indirect hemolytic activity of Bothrops riper venom was studied using a sensitive plate test . All samples of antivenom tested effectively neutralized this activity . A highly significant correlation was observed between neutralization of indirect hemolytis and neutralization of lethal activity . This simple and sensitive in vitro test could be used to monitor antibody levels in horses immunized to produce polyvalent antivenom .
Toxicon, 1998
Olo rtegui. Neutralizing potency of horse antibothropic antivenom. Correlation between in vivo and in vitro methods. Toxicon 36, 1433±1439, 1998.Ð The correlation coecients between in vivo neutralization of lethal toxicity (ED 50 ), neutralization of the hemolytic activity (PLA 2 ) and levels of antibodies measured by ELISA, was investigated to test the potency of horse anti-bothropic antivenom. Twenty six horses were hyperimmunized with Bothrops venoms (B. alternatus, B. jararaca, B. jararacussu, B. neuwiedii and B. moojeni). To set up an indirect ELISA, for neutralization of PLA 2 activity and for determination of ED 50 in Swiss mice, the whole Bothrops jararaca venom (reference venom for assessing the bothropic antivenom potency in Brazil) was used. The toxic fraction (puri®ed from B. jararaca venom by Sephadex G-100 chromatography) was also used as antigen for ELISA. All antivenoms analyzed eectively neutralized the lethal activity in the range of 1.6 to 9.6 mg/ml of antivenom. The correlation coecient between ED 50 and ELISA antibody titers against the crude venom and toxic fraction was r = 0.65 (P < 0.001) and r = 0.85 (P < 0.0001), respectively. Correlation between ED 50 and neutralization of PLA 2 activity was r = 0.52 (P < 0.01), and the correlation between ELISA antibody titers and neutralization of PLA 2 activity was r = 0.58 (P < 0.002). Thus, the ELISA which measures only the antibody against the major toxic fraction of the B. jararaca venom should be most suitable for use as an in vitro assay of bothropic antivenom potency. #
Biologicals, 2015
There is a need to introduce innovations in the manufacture of snake antivenoms to increase the supply of these products worldwide. In this work, the fractionation of equine hyperimmune plasma with a new methodology that includes an aqueous two phase system (ATPS) as a primary purification step was compared with the traditional method of caprylic acid precipitation. Hyperimmune plasma from horses immunized with the venoms of three snakes from sub-Saharan Africa was used as starting material for the production of both formulations. After being adjusted to the same lethal neutralizing activity, both antivenoms were compared in terms of their immunoreactivity, neutralization of in vitro venom activities, physicochemical characteristics, and stability. Their performance in terms of yield and purity was also assessed. The neutralization profile of in vitro enzymatic activities and the immunoreactivity, analyzed by ELISA and antivenomic approaches, were very similar for both preparations. Likewise, they behaved similarly in stability studies. However, ATPS-fractionated antivenom showed improved physicochemical profile and immunochemical purity and yield, mainly owing to its lower protein content. Additionally, this methodology allowed the recovery of albumin as a byproduct. ATPS purification constitutes a promising technology for antivenom production and should be further evaluated at preclinical and clinical levels.
Journal of Venomous Animals and Toxins including Tropical Diseases, 2006
In this study we optimized an enzyme-linked immunosorbent assay (ELISA) to evaluate bothropic venom levels in biological samples. These samples were obtained by two distinct protocols. In the first one, Swiss mice were injected with 1 LD 50 of Bothrops jararaca (B. jararaca) venom and 15 minutes later, animals were treated with ovine antibothropic serum. Blood and spleen homogenate samples were obtained 6 hours after antiserum therapy. Ovine antibothropic serum significantly neutralized venom levels in serum and spleen. In the second protocol, BALB/c mice were injected with 1 LD 50 of bothropic venom by either intraperitoneal (IP) or intradermal (ID) route and venom levels were evaluated 1, 3 and 6 hours after, in blood, spleen homogenates and urine. Serum and splenic venom levels were significantly higher in animals envenomed by IP route comparing with animals envenomed by ID route. Higher venom levels were also detected in urine samples from animals envenomed by IP route. However, these differences were not statistically significant. These results demonstrated that the optimized ELISA was adequate to quantify venom levels in different biological samples. This assay could, therefore, substitute the in vivo neutralizing assay and also be useful to evaluate the severity of human and experimental envenomations.
Toxicon, 2012
Optimization of caprylic acid precipitation of equine plasma non-immunoglobulin proteins for antivenom preparation was achieved by regression analysis of the responses of three highly significant factors assayed by factorial design. The factors studied were caprylic acid concentration, plasma pH and temperature, and their response was assessed in terms of filtration speed, residual albumin, total protein content and turbidity. The results evidenced that the three variables are involved in the precipitation process. Moreover, the factors displayed significant interactions, indicating that their levels distinctly affect the optimization procedure. The best combination was 3% caprylic acid, 37 C and plasma pH 4.9; under these conditions, all immunoglobulins and only 0.1% albumin remained in the supernatant, in a very fast and simple procedure. After formulation, the antivenom obtained by this procedure presented full lethality neutralizing activity and absence of protein aggregates.
International Journal of Molecular Biology: Open Access, 2020
The treatment of choice for the snake bite is the administration IgG antibodies that are raised in an animal body thorough immunization of the animals. To avoid potential adverse reactions that arises from introduction of animal antiserum in human body, the antibody needs to be purified from the other non-specific proteins like albumin. Optimal conditions for the fractionation of IgG's were studied; ammonium sulfate was added to plasma up to 40% concentration to separate non-specific proteins, iso electric separation was carried out at pH 5.4, and the final IgG's fractionation by the addition of ammonium sulfate up to 50% concentration. A highly purified and concentrated mixture of IgG's was obtained after removal of ammonium sulfate through dialysis. The resulting anti venom was of good quality in terms of purity and was found to be highly potent against venoms of all four types of snakes. Owing to the high purity and greater potency of the anti venom the method has potential to be used for lab and commercial scale production of anti venom.
Refinement strategy for antivenom preparation of high yield and quality
PLOS Neglected Tropical Diseases
Antivenoms from hyperimmune animal plasma are the only specific pharmaceuticals against snakebites. The improvement of downstream processing strategies is of great interest, not only in terms of purity profile, but also from yield-to-cost perspective and rational use of plasma of animal origin. We report on development of an efficient refinement strategy for F(ab') 2-based antivenom preparation. Process design was driven by the imperative to keep the active principle constantly in solution as a precautionary measure to preserve stability of its conformation (precipitation of active principle or its adsorption to chromatographic stationary phase has been completely avoided). IgG was extracted from hyperimmune horse plasma by 2% (V/V) caprylic acid, depleted from traces of precipitating agent and digested by pepsin. Balance between incomplete IgG fraction breakdown, F(ab') 2 over-digestion and loss of the active principle's protective efficacy was achieved by adjusting pepsin to substrate ratio at the value of 4:300 (w/w), setting pH to 3.2 and incubation period to 1.5 h. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. Developed manufacturing strategy gave 100% pure and aggregate-free F(ab') 2 preparation, as shown by size-exclusion HPLC and confirmed by MS/MS. The overall yield of 75% or higher compares favorably to others so far reported. This optimised procedure looks also 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 in each purification step enabling accurate estimation of the process cost-effectiveness.