Goat anti rat hrp conjugate

The ability of B. candidus and N. naja venoms to individually inhibit the binding of nAChR to NK3 coated plate were expressed as IC₅₀ (venom concentration inhibiting 50% of the nAChR binding or the median inhibitory concentration). In this experiment, B. candidus or N. naja venom at various concentrations were pre-incubated (25 °C, 1 hr) with a fixed and optimal concentration of nAChR. The mixture was then added to the NK3-coated plate and incubated at 25 °C for 1 h. This was followed by additions of rat anti-nAChR serum at 1:1,600 dilution and incubated at 25 °C for 1 h. Thereafter, 1:4,500 diluted goat-anti-rat-HRP conjugate (Abcam^®) was added and the mixture was incubated for 60 min at 25 ^oC. A parallel experiment, in which purified NK3 was used as the reference standard in place of the venom, was carried out. The concentration of the venom used in the pre-incubation experiment that blocked 50% of the nAChR binding to the immobilized NK3 was the IC₅₀ of that venom.

The ability of an elapid venom (N. kaouthia) which contains PSNTs to inhibit the binding of nAChR to NK3 immobilized plate was studied and was expressed as IC₅₀ (venom concentration inhibiting 50% of the nAChR binding). In this assay, N. kaouthia crude venom at various concentrations was pre-incubated (25°C for 1 hr) with a fixed and optimal concentration of nAChR before the mixture was added to the NK3-coated plate and incubated at 25°C for 1 hour. This was followed by additions of rat anti-nAChR serum at 1:1600 dilution and incubated at 25°C for 1 hr, followed by 1:4500 diluted goat-anti-rat-HRP conjugate (Abcam) and incubated for 60 min at 25 °C. A parallel experiment using purified NK3 as the reference standard in place of the venom was also carried out. The concentration of the tested venom used in the pre-incubation step that inhibited 50% of the nAChR binding to the immobilized NK3 was the median inhibitory concentration (IC₅₀) of that venom.