The H5N1 Hemagglutinin (HA) Bovine IgA ELISA Kit employs an indirect ELISA method. In this kit, standard 96-well plates (12 strips with 8 wells/strip) are coated with a pathogen antigen, which combines with the corresponding antibody present in a sample and Positive Control, which is used as a calibration curve for interpretation purposes. When a biotinylated secondary anti-bovine IgA antibody is added, a complex forms between biotinylated secondary antibody and bovine antibody bound to the protein coated plate. A TMB substrate is added, and a blue color is generated. The depth of color is relative to the amount of the human anti-pathogen antigen antibody present. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

The Positive Control consists of an anti-H5N1 Hemagglutinin Bovine Influenza antibody. A biotinylated anti-Positive Control antibody is added to the Positive Control wells and treated identically to the samples. The intensity of the resulting color (measured at 450 nm) is used as a calibration curve for interpretation purposes in the different assays.

• Microplate reader capable of measuring absorbance at 450 nm
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Adjustable 1-25 mL pipettes for reagent preparation
• 100 mL and 1 liter graduated cylinders
• Absorbent paper
• Distilled or deionized water

1. Prepare all reagents, samples and standards as instructed.
2. Add 100 µL positive control, or sample to each well. Incubate 1 hour at room temperature.
3. Add 100 µL prepared Biotinylated Anti-Bovine IgA Antibody into respective wells. Incubate 1 hour at room temperature.
4. Add 100 µL prepared HRP-Streptavidin solution to each well. Incubate 30 minutes at room temperature.
5. Add 100 µL TMB One-Step Substrate Reagent to each well. Incubate 10 to 30 minutes at room temperature.
6. Add 50 µL Stop Solution to each well. Read at 450 nm immediately.