General 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 IgM-Reducing Assay Diluent into each of the Wells of the ELISA plate using a multi-channel pipettor. The addition of the Assay Diluent is always done prior to adding the standard or test samples to each Well., 2. The standard and test samples should be diluted to the proper Well concentrations using one of ICT’s three different Sample Diluent products to help further equalize matrix complexity parameters between standard/calibrator Wells and the serum/plasma containing sample Wells., 3. Because IgM-Reducing Assay Diluent contains a more diverse composition of additives, it can have some negative impact on the antigen capture step of the ELISA. This leads to reduction in ELISA sensitivity which must be compensated for by using longer antigen capture incubation times or more sensitive HRP substrate components.

Equalizes the sample and standard matrices for a more accurate result. General Assay Diluent is formulated for testing serum, plasma, urine, and cell culture samples in all sandwich ELISA formats. Use of an assay diluent helps minimize matrix complexity differences between the sample (e.g., serum, etc.) and the diluent used to generate the standard curve of the ELISA. Large differences between the sample and the standard diluent matrices will result in under-recovery of the target analyte present in sample Wells. Complex and concentrated protein environments (the matrix) present in serum or plasma samples will greatly reduce the antigen-binding efficiency of the plate-adsorbed antibodies, resulting in a gross underestimation of the amount of target analyte present in the test samples. General Assay Diluent helps equalize the antibody-binding efficiencies between the standard curve and the sample Wells. 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. Mammalian protein additives included in the formulation serve to reduce non-specific interactions between the sample matrix proteins and the plate surface, thereby minimizing background noise. General Assay Diluent also inhibits complement and thrombin activity present in serum and plasma samples. Incorporation of an antimicrobial agent allows for room temperature bench-top use and extensive storage stability at 2-8°C. This assay diluent formulation ensures a more accurate and consistent ELISA performance, regardless of sample type.