Action of bromelain and ficin on horse anti Bothrops sp venom antibodies

The treatment with hyperimmune sera constitute the only specific and effective therapy available against snakebite envenomation, most common in developing countries. Serum quality is an important factor on patient recovery time and in the incidence of death and permanent disability. To date, most sera consist of pepsin digested IgG antibodies harvested from hyperimmune animals. The use of animal derived enzymes, such as pepsin, to digest IgG, constitute a source of adventitious agents and contaminants, such as porcine circovirus. The present study aims to evaluate the use of the plant derived enzymes bromelain and ficin, as an alternative to pepsin. To this purpose, horse serum immunized against Bothrops venoms was purified with caprylic acid and digested with bromelain or ficin. SDS-PAGE results evidence the formation of F(ab)’2 fragments and suggest that a digestion time superior to 8 hours may be required to completely digest the antibodies with bromelain or ficin. F(ab)’2 fragments obtained by digestion with either bromelain or ficin digestion preserved the ability to recognize Bothrops sp. venom in western blotting assays. Therefore, both enzymes are suitable for use in large-scale production, minimizing contamination risks and increasing safety and efficiency of serotherapy treatments.

Action of Bromelain and Ficin on horse anti Bothrops sp venom Antibodies

Abstract The treatment with hyperimmune sera constitute the only specific and effective therapy available against snakebite envenomation, most common in developing countries. Serum quality is an important factor on patient recovery time and in the incidence of death and permanent disability. To date, most sera consist of pepsin digested IgG antibodies harvested from hyperimmune animals. The use of animal derived enzymes, such as pepsin, to digest IgG, constitute a source of adventitious agents and contaminants, such as porcine circovirus. The present study aims to evaluate the use of the plant derived enzymes bromelain and ficin, as an alternative to pepsin. To this purpose, horse serum immunized against Bothrops venoms was purified with caprylic acid and digested with bromelain or ficin. SDS-PAGE results evidence the formation of F(ab)'2 fragments and suggest that a digestion time superior to 8 hours may be required to completely digest the antibodies with bromelain or ficin. F(ab)'2 fragments obtained by digestion with either bromelain or ficin digestion preserved the ability to recognize Bothrops sp. venom in western blotting assays. Therefore, both enzymes are suitable for use in large-scale production, minimizing contamination risks and increasing safety and efficiency of serotherapy treatments.

Vitamins as influenza vaccine adjuvant components.

A number of adjuvant formulations were assayed in mice immunized with 3.75 µg of A/California/7/2009 (H1N1) pdm09 influenza vaccine with vitamins A, D and/or E in emulsions or B2 and/or B9 combined with Bordetella pertussis MPLA and/or alum as adjuvants. Squalene was used as positive control, as well as MPLA with alum. The immune response was evaluated by a panel of tests, including a hemagglutination inhibition (HAI) test, ELISA for IgG, IgG1, and IgG2a and IFN-γ, IL-2, IL-6 and IL-10 quantification in splenocyte culture supernatant after stimulus with influenza antigen. Immunological memory was evaluated using a 1/10 dose booster 60 days after the first immunization followed by assessment of the response by HAI, IgG ELISA, and determination of the antibody affinity index. The highest increases in HAI, IgG1 and IgG2a titers were obtained with the adjuvant combinations containing vitamin E, or the hydrophilic combinations containing MPLA and alum or B2 and alum. The IgG1/IgG2a ratio indicates that the response to the combination of B2 with alum would have more Th2 character than the combination of MPLA with alum. In an assay to investigate the memory response, a significant increase in HAI titer was observed with a booster vaccine dose at 60 days after immunization with vaccines containing MPLA with alum or B2 with alum. Overall, of the 27 adjuvant combinations, MPLA with alum and B2 with alum were the most promising adjuvants to be evaluated in humans.