Antigen-Down Assay Diluent

1. Coat antibody or antigen onto the ELISA plate using ICT’s Antibody Coating Buffer or Antigen Coating Buffer., 2. Incubate 8–24 hours at room temperature (RT)., 3. Aspirate the coating solution., 4. Wash each Well twice with ICT’s ELISA Wash Buffer., 5. Block the uncoated regions of the ELISA plate by pipetting 300–400 µL of blocking buffer into each Well. Always use an equal or greater volume of blocking buffer than was used for the coating buffer solution., 6. Incubate 8–24 hours at RT. For best blocking, incubate overnight at RT., 7. Aspirate the blocking buffer; do not wash., 8. Run the assay immediately, or dry the plate for long-term storage and seal in a foil storage bag with a desiccant pack. Store dried and packaged plates at 2-8°C.

Minimizes clotting potential and reduces non-specific binding. Antigen-Down Assay Diluent is formulated for testing serum and plasma samples in situations where an undesirable clotting event could occur while performing an antigen-down ELISA. It contains calcium chelating agents and other proprietary additives to eliminate the risk of clotting events in plasma samples and minimize interference from complement and thrombin activity. Antigen-Down Assay Diluent also provides a consistent protein matrix environment that will stabilize the antigen-specific target IgG analyte during high dilution ELISA titration protocols. Assessment of high titer, humoral IgG responses often requires dilutions >100,000-fold. This extensive dilution is often necessary to obtain a chromogen absorbance value that is within the sensitivity detection limits of the ELISA plate reader. This novel assay diluent formulation includes mammalian protein and detergent additives to reduce the non-specific adsorption of generic serum IgG molecules onto the coated and blocked ELISA Well surface. This feature can be especially critical when assessing sera derived from human, porcine, or bovine sources, which show a higher tendency to non-specifically adsorb. By minimizing such non-specific binding events, Antigen-Down Assay Diluent inhibits undesirable background noise and increases the sensitivity of the assay. To use, simply add 50-100 µL to every Well of the ELISA plate, including all Wells designated for standards, controls, and samples. Then add the standards, controls, and samples to the plate. Incorporation of an antimicrobial agent into the formulation allows for room temperature bench-top use and extensive storage stability at 2-8°C.