Rabbit anti horse igg conjugated with horseradish peroxidase hrp

The protein (50 ng) was diluted with 50 µL PBS and then coated onto 96-well polystyrene microplates (Corning Inc., Corning, NY, USA), followed by incubation overnight at 4 °C. The plates were washed six times with 100 µL of PBS-T (0.1% Tween-20) and blocked with 100 µL of 1% ovalbumin in PBS for 2 h at room temperature. After six rounds of additional washing, equine plasma samples (antivenom) were diluted (1:20,000) in PBS and added to each pre-coated well for another 2 h incubation at room temperature, and excess antibodies were removed by washing 6 times with PBS-T. Afterwards, rabbit anti-horse IgG conjugated with horseradish peroxidase (HRP) (Bethyl Laboratories, Montgomery, TX, USA) was added to each well and incubated at room temperature for 1 h. After final washing six times, 50 µL of tetramethylbenzidine (TMB) substrate (Clinical Science Products Inc., Mansfield, MA, USA) was added to each well and reacted for 10 min. The reaction was terminated by adding 25 µL of 2N H₂SO₄ (J.T Baker, Radnor, PA, USA), and absorbance was measured using a SpectraMax M5 microplate reader (Molecular Devices, San Jose, CA, USA) with excitation and emission wavelengths of 450 and 540 nm, respectively. Each assay and sample extraction was performed in triplicate.

Cobra venom proteins (10 ng) or NTX-derived peptides (100 ng) were diluted in 50 μL PBS and coated onto 96-well polystyrene clear microplates (Corning Inc., Corning, NY, USA) with incubation at 4 ℃ overnight. Plates were washed six times with 100 μL phosphate-buffered saline containing 0.05% Tween-20 (PBST) and blocked with 200 μL of 1% ovalbumin in PBS at room temperature for 1 h. After washing wells with PBST six times, horse plasma was diluted (1:20,000) and added to each well, followed by incubation of the plate at room temperature for 1 h. After six washes with PBST, rabbit anti-horse IgG conjugated with horseradish peroxidase (HRP) (Bethyl Laboratories, Montgomery, TX, USA) was added to each well and incubated at room temperature for 1 h. Plates were further washed six times with PBST and 50 μL of tetramethylbenzidine (TMB) substrate (Clinical Science Products Inc., Mansfield, MA, USA) was added to each well for 10 min. The reaction was terminated with 25 μL of 2N H₂SO₄ (J.T Baker, Radnor, PA, USA) and absorbance of each well measured with a SpectraMax M5 microplate reader (Molecular Devices, San Jose, CA, USA) at excitation and emission wavelengths of 450 and 540 nm, respectively. Each assay was performed in duplicate, and the mean of absorbance value was used for further statistical analysis.