ELISA method to assign weight-based antibody concentration to antigen-specific Ig in sera.

The ability to compare results obtained while assessing immune responses to different antigens using different assays is essential. Enzyme-linked Immunosorbent Assay (ELISA) is one of the most widely used assays to detect and quantify antibodies in human and animal samples. However, it carries the limitation of not giving absolute results but relative measures; specifically, ELISA titers are relative to the sample dilutions tested and ELISA Units/mL, assigned to samples, are dependent on the ELISA Units assigned to a standard serum used in the test, posing the question of how to compare results obtained across different ELISA assays. Some comparability limitations between immunoassays can be solved through calibration against International Standards, but they are not always available and they do not enable comparison across antigens. In an attempt to overcome this problem, we have developed a reliable ELISA-based assay that quantifies antigen-specific antibodies in sera with an absolute concentration of μg/mL, thus allowing comparison of results obtained running the assay with different coating antigens. The methodology is based on comparing the signal of the sera of interest against a curve made with quantified IgG. This method has been successfully applied to quantify total IgG in sera from different species: human, rabbit and mouse.