A novel in vitro potency assay of antisera against Thai Naja kaouthia based on nicotinic acetylcholine receptor binding

Kavi Ratanabanangkoon,Kae Yi Tan,Sirida Youngchim,Pavinee Simsiriwong,Kritsada Pruksaphon,Nget Hong Tan,Choo Hock Tan,Bunkuea Chantrathonkul,Sukanya Eursakun,Wongsakorn Wongwadhunyoo

doi:10.1038/s41598-017-08962-3

Abstract

Snake envenomation is an important medical problem. One of the hurdles in antivenom development is the in vivo assay of antivenom potency which is expensive, gives variable results and kills many animals. We report a novel in vitro assay involving the specific binding of the postsynaptic neurotoxins (PSNTs) of elapid snakes with purified Torpedo californica nicotinic acetylcholine receptor (nAChR). The potency of an antivenom is determined by its antibody ability to bind and neutralize the PSNT, thus preventing it from binding to nAChR. The PSNT of Naja kaouthia (NK3) was immobilized on microtiter wells and nAChR was added to bind with it. The in vitro IC50 of N. kaouthia venom that inhibited 50% of nAChR binding to the immobilized NK3 was determined. Varying concentrations of antisera against N. kaouthia were separately pre-incubated with 5xIC50 of N. kaouthia venom. The remaining free NK3 were incubated with nAChR before adding to the NK3 coated plates. The in vitro and in vivo median effective ratio, ER50s of 12 batches of antisera showed correlation (R2) of 0.9809 (p < 0.0001). This in vitro assay should be applicable to antisera against other elapid venoms and should reduce the use of live animals and accelerate development of life-saving antivenoms.

Highlights

Snake envenomation is an important medical problem especially in the developing world
By using a different reaction scheme and conditions from that studied above[12], we report here the development of a novel in vitro potency assay of monospecific antisera against the venom of the Thai monocled cobra Naja kaouthia based on nicotinic acetylcholine receptor (nAChR) binding
It is reported here the first successful development of an in vitro potency assay for antiserum against an elapid snake based on nicotinic acetylcholine receptor binding

Summary

Introduction

Snake envenomation is an important medical problem especially in the developing world. Numerous investigators have studied and reported the use of ELISA for AV potency determinations together with the correlation between the results of the in vitro and in vivo neutralization assays[14,15,16,17,18,19,20]. Ibrahim and Farid[21] studied the lethality-neutralizing potency, ELISA antibody level and the avidity indexes of a polyvalent antivenom against seven snake venoms. They showed poor correlation between the in vivo and in vitro assays with the in vitro assays always giving high values. ELISA has at times been criticized on the grounds that the antigen-antibody ‘binding’ reaction measured cannot be assumed to be the same as the ‘neutralization’ reaction of the antigen

Methods

Results

Conclusion