Antigen-Down Assay Diluent

1. Once the capture antigen has been coated onto the plate and the plate has been properly blocked with one of ICT’s stabilizing blocker products, pipette 50 – 100 µL of Antigen-Down Assay Diluent into each of the Wells of the ELISA plate using a multi-channel pipettor. The Assay Diluent is always added prior to adding the test and control serum/plasma samples to plate Wells., 2. The control and test serum/plasma samples should be diluted to the proper Well concentrations using one of ICT’s three different Sample Diluent products. Using an ICT Sample Diluent to dilute the serum and plasma samples will also serve to help minimize the degree of nonspecific IgG binding to the antigen coated and blocked ELISA plate Wells. The test (sera and plasma) samples, diluted typically to 1:40 or greater in a sample diluent, should be incubated in the antigen coated ELISA plates for 30 – 60 minutes at room temperature or 37°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.