Capture ELISA problems

The issue of which antibody to select for an assay is not a new problem. Certainly anyone involved in the development of an immunoassay has been faced with this choice. Consider attempting to create a multianalyte, microarray-based micro-ELISA of modest density (10 to 100 analytes) and determining which capture antibodies to use based upon their affinities, stabilities, and cross-reactivities. For a sandwich assay, add in the 10 to 100 analyte-specific secondary (reporter) antibodies and determine their levels of cross-reactivity with each other and with the specified antigens and capture antibodies. In other words, achieving high performance for all analytes with a microarray immunoassay is indeed a formidable challenge. 

ELISA of sheep IgM (antigen) was conducted in a PDMS chip. The capture antibody (rabbit anti-sheep IgM-HRP) was added. Then the fluorogenic substrate (HPPA) was added for fluorescent detection. The conventional blocking reagents (0.5% w/v BSA, 0.5% w/v casein, 0.5% v/v Tween-20), which normally worked for the polystyrene ELISA plate, did not work with the hydrophobic PDMS surface. To solve this problem, 4% PEG and 7% normal rabbit serum were included in the above solution for effective blocking [173]. 

The main problem with this form of antigen assay (indirect sandwich I-ELISAs) is that the wells are coated with antibodies that capture antigen. Thus, any subsequent addition of antigen in a test sample will be bound to the wells if it is not fully saturated with the initially added coating antigen. The pretitration of the system then requires that there be no free antibodies coating the wells. Hence, the exact conditions for pretitration may differ from that for the anti-... 

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