Production of potent polyvalent antivenom against three elapid venoms using a low dose, low volume, multi-site immunization protocol.

The purpose of this study was to prepare a potent polyvalent antivenom against three elapids namely, the Thai cobra (Naja kaouthia, NK), the King cobra (Ophiophagus hannah, OH) and the banded krait (Bungarus fasciatus, BF). Two groups of horses were immunized. Group 1, comprising five horses, was immunized twice with a mixture of postsynaptic neurotoxins followed by an additional six immunizations with a mixture of crude venoms of the three elapids. Group 2, comprising four horses, was immunized with a mixture of crude venoms throughout the course. For the first immunization, the immunogens were emulsified in Complete Freund's adjuvant and injected using a low dose, low volume multi-site immunization protocol previously developed in this laboratory (Pratanaphon, R., Akesowan, S., Khow, O., Sriprapat, S. and Ratanabanangkoon, K. (1997) Production of highly potent horse antivenom against the Thai cobra (Naja kaouthia). Vaccine 15, 1523-1528). The second immunization was carried out with the immunogens in Incomplete Freund's adjuvant. Blood was drawn to assay the antibody titer by ELISA. Sera at the peak of ELISA titers were pooled and assayed for the median effective dose (ED(50)). The ED(50)'s of antivenom from Group 1 horses against NK, OH and BF venoms were 1.44, 0.22 and 0.23 ml serum/mg venom, respectively, while those from Group 2 horse sera were 0.88, 0.20 and 0.49 ml serum/mg venom, respectively. The potency of sera from Group 2 against BF venom was significantly higher, while the potencies against NK and OH venoms were comparable to those of the corresponding monovalent antivenoms produced under the same protocol. This potent, truly polyvalent antivenom should be useful in saving lives of victims envenomed by these elapids and the immunization protocol should be useful in the production of potent polyvalent antivenoms against other medically important elapids.

Envenomation of mice by Thai cobra (Naja kaouthia) venom: tolerable venom concentration and exposure time.

Naja kaouthia venom appeared in circulation rapidly after intramuscular injection into mice. The venom concentration attained a maximum level with all doses examined after 20 min. The half value of the maximum level was obtained 1 min after injection when a dose of 4LD50 was used. A critical venom concentration endangering mice was assessed from venom concentration in the sera of mice envenomed with sublethal dose (LD50). A fatal condition was produced within 30 min at a venom concentration of 200-300 ng/ml or within 50 min at a venom concentration of 100-150 ng/ml.

Production of highly potent horse antivenom against the Thai cobra (Naja kaouthia).

Naja kaouthia (NK) causes the highest fatality due to snake venom poisoning in Thailand. The specific antivenom produced is of low potency and in short supply. The aim of this study was to improve the antivenom potency. Bentonite and complete Freund's adjuvants (CFA) and various immunogens were compared. Six groups of three to five horses were immunized as follows: Group 1, NK venom adsorbed on bentonite; Group 2, NK venom in CFA; Group 3, NK venom in CFA in multi-emulsion formulation; Group 4, NK venom in 25% CFA; Group 5, NK neurotoxin 3 (NK3) conjugated with tetanus toxoid (NK3-TT) in CFA; Group 6, NK3 conjugated with diphtheria toxoid (NK3-DT) in CFA. Horses in Group 2-6 produced antivenom of very high neutralizing activity, up to four times higher than that of horses of Group 1. CFA (100 or 25%) or as a multi-emulsion formulation, induced comparable neutralizing antibody production with all three immunogens. All horses showed normal weight gain during the course of immunization. Group 1 horses exhibited minimal local reactions while horses in the other five groups had mild and comparable local reactions at the injection sites. No significant differences in the reactions caused by CFA in different formulations or different immunogens were observed. The production of highly potent antivenom against N. kaouthia from these horses should help solve problems associated with the currently available antivenom.

Cross-neutralization of Thai cobra (Naja kaouthia) and spitting cobra (Naja siamensis) venoms by Thai cobra antivenom.

The neutralizing capacity of antivenom prepared against Thai cobra (Naja kaouthia) venom was compared in mice using the homologous venom and that of spitting cobra (Naja siamensis). The amounts of antivenoms neutralizing a dose of 4 LD50 of the test venom were determined. Four antivenom preparations were used: three purified antivenoms and a crude antivenom, which were made using N. kaouthia venom only. Almost the same neutralizing capacity was obtained with the purified antivenoms, whereas a slightly lower capacity was seen with the crude antivenom. However, ratios of the amounts of four antivenoms neutralizing the homologous and heterologous test venoms were almost constant. These results indicated that Thai cobra antivenom possesses neutralizing capacity against spitting cobra venom.