Immunoreactivity and neutralization study of Chinese Bungarus multicinctus antivenin and lab-prepared anti-bungarotoxin antisera towards purified bungarotoxins and snake venoms

Abstract

Bungarus multicinctus is the most venomous snake distributed in China and neighboring countries of Myanmar, Laos, north Vietnam and Thailand. The high mortality rate of B. multicinctus envenomation is attributed to the lethal components of α-, β-, γ- and κ- bungarotoxins contained in the venom. Although anti-B. multicinctus sera were produced in Shanghai, Taiwan and Vietnam, the most widely clinic used product was term as B. multicinctus antivenin and manufactured by Shanghai Serum Bio-technology Co. Ltd. In the present investigation, high purity α-, β- and γ-bungarotoxins were separately isolated from B. multicinctus crude venom. Rabbit anti- α-, β- and γ-bungarotoxin antisera were prepared by common methods, respectively. LD50 values of α-, β- and γ-bungarotoxins were systematically determined via three administration pathways (intraperitoneal, intramuscular and intravenous injections) in Kunming mice. LD50 values of β-bungarotoxin were closely related with injection routines but those of both α- and γ-bungarotoxins were not dependent on the injection routines. Commercial B. multicinctus antivenin showed strong immunoreaction with high molecular weight fractions of the B. multicinctus but weakly recognized low molecular weight fractions like α- and γ-bungarotoxins. Although B. multicinctus antivenin showed immunoreaction with high molecular weight fractions of Bungarus fasciatus, Naja atra, Ophiophagus hannah venoms but the antivenin only demonstrated animal protection efficacy against O. hannah venom. These results indicated that the high molecular weight fractions of the O. hannah played an important role in venom lethality but those of B. fasciatus and N. atra did not have such a role.

Fig 1. Molecular weight determination of purified bungarotoxins by MALDI/TOF mass spectrometer.

α-BGT (A). β-BGT (B). γ-BGT (C).

Fig 2. Specificity of three prepared antisera.

SDS-PAGE of B. multicinctus venom under non-reducing conditions, 25 μg/sample (A). Western-blot profile of prepared anti-α-BGT, anti-β-BGT and anti-γ-BGT antisera against crude venom and corresponding bungarotoxins (B). For Western-blot, 2 μg of crude venom or purified neurotoxins were added in each lane. Bm: Crude venom of B. multicinctus.

Fig 3. The immunoreactivity of antisera comparison by indirect ELISA.

Prepared anti-α-BGT antiserum against different antigens (A). Prepared anti-γ-BGT antiserum against different antigens (B). Prepared anti-β-BGT antiserum against different antigens (C). Commercial B. multicinctus antivenin against different antigens (D). The immunoreactivity determination of prepared antisera and the B. multicinctus antivenin (E). Antigens of 1μg/well were coated on a 96-well plate, results were expressed as mean ± SD.

Fig 4. Immunoreactivity of the commercial B. multicinctus antivenin and the prepared antiserum against top Chinese dangerous terrestrial snake venoms.

SDS-PAGE of nine used snake venoms under non-reducing conditions, 25 μg/sample (A). Western blot revealed by B. multicinctus antivenin under non-reducing conditions (B). Western blot revealed by B. multicinctus antivenin under reducing conditions (C). Western blot revealed by normal horse IgG under non-reducing conditions (D). Western blot revealed by prepared anti-α-BGT antiserum under non-reducing conditions (E). Western blot revealed by prepared anti-β-BGT antiserum under non-reducing conditions (F). Western blot revealed by prepared anti-γ-BGT antiserum under non-reducing conditions (G). Western blot revealed by normal rabbit IgG under non-reducing conditions (H). Bm: B. multicinctus; Bf: B. fasciatus; Na: N. atra; Oh: O. hannah; Da: D. acutus; Ts: T. stejnegeri; Dr: Daboia russelii siamensis; Pm: P. mucrosquamatus; Gb: G. brevicaudus. 10 μg/sample was loaded for Western blot experiments.