LEAP Testing Service

INHIBITION ELISA (ASTM D6499-03)

I. Scope
This ELISA is an ASTM standard test method for the immunological measurement of antigenic protein in natural rubber and its products.

II. Procedure

1. Two different plates are used in the inhibition ELISA. They are referred to as the competition or dilution plate and the assay plate.
2. In the competition plate, serial dilutions of a latex protein standard and product extracts are mixed with latex specific anti-sera and incubated for one hour. If latex proteins are present, they will bind with the antibody making it unavailable for binding to the latex antigen coating the wells of the assay plate.
3. Each well of the assay plate has the same amount of latex antigen bound to it. Once blocked, the reaction mix from the dilution plate can be transferred to the assay plate.
4. Those samples that have high levels of latex protein will bind all available antibody. Consequently, no free antibody will be available to bind to the antigen on the assay plate. When the plate is processed for color, no color will develop. Conversely, in those samples with little or no latex protein, little or no antibody molecules will be bound to soluble antigen. Therefore the antibody is free to bind to the antigen coating the assay plate. When these wells are processed, high color levels will be observed.
5. Antigen levels are determined from a standard curve using known amounts of latex protein.

Note: In the inhibition ELISA, the lack of color is proportional to the amount present in the extract. This is the opposite of the LEAP assay.