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

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 (R 2) 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.

An in vitro potency assay using nicotinic acetylcholine receptor binding works well with antivenoms against Bungarus candidus and Naja naja

“…The optimal concentrations of these four reagents used in the in vitro potency assay were described in a previous study 13 . The optimal concentration of NK3 for coating the plates was 15 µg/ml, and 0.707 µg/ml of nAChR for binding to the NK3 coated plate.…”

“…naja (Sri Lanka), a good correlation between the in vitro nAChR-PSNT binding activity and the in vivo murine lethality neutralization activity was obtained. The reason behind this observation could be that the presence of a large amount of cytotoxins in the venom did not affect the results of the in vitro nAChR - PSNT binding assay since the assay has been shown to be specific to PSNT and not to cytotoxins 13 . Furthermore, the results suggested that death is mainly the result of the α-neurotoxins since α-neurotoxins are about 10 times more toxic, with an LD 50 of ca .…”

“…However, because ELISA involves the binding of antivenom antibodies to venom proteins whose toxicity might not be neutralized, results of these in vitro assays usually show poor correlation with results from the murine lethality neutralization assay. We have recently developed a novel in vitro assay 13 based on nAChR binding to determine the potency of antivenom against Naja kaouthia , an elapid that produces, almost exclusively, postsynaptic neurotoxins that are responsible for death in victims. This assay estimates the binding of venom postsynaptic neurotoxins to nAChR, which is the toxicological reaction involved in the lethal effects of cobra envenomation.…”

“…‘Pre-incubation 1′ of the N . naja venom with the F(ab′) 2 antibody was followed by ultrafiltration using a membrane with a MWCO of 50kD, instead of the customary 100 kD membrane 13 . This was done to remove the bound and free F(ab′) 2 antibody, which has a molecular weight of about 100 kDa.…”

“…Recently, a novel in vitro assay using solubilized, purified nicotinic acetylcholine receptor (nAChR) binding has been developed for AV potency assay against the Thai cobra Naja kaouthia 13 . This elapid snake produces mainly postsynaptic neurotoxins (PSNT) which binds specifically and with high affinity to the nAChR at the muscle endplate and thereby inhibits the neuro-muscular transmission causing death in animals.…”