Quantifying Specific Antibody Concentrations by Enzyme-Linked Immunosorbent Assay Using Slope Correction

Abstract

Assessing the magnitude of an antibody response is important to many research and clinical endeavors; however, there are considerable differences in the experimental approaches used to achieve this end. Although the time-honored approach of end point titration has merit, the titer can often be misleading due to differences in how it is calculated or when samples contain high concentrations of low-avidity antibodies. One frequently employed alternative is to adapt commercially available enzyme-linked immunosorbent assay kits, designed to measure total antibody concentrations, to estimate antigen-specific antibody concentrations. This is accomplished by coating the specific antigen of interest in place of the capture antibody provided with the kit and then using the kit's standard curve to quantify the specific antibody concentration. This approach introduces considerable imprecision, due primarily to its reliance on a single sample dilution. This “single-point” approach fails to address differences in the slope of the sample titration curve compared to that of the standard curve. Here, we describe a general approach for estimating the effective concentration of specific antibodies, using antisera against foot-and-mouth disease virus VP1 peptide. This was accomplished by initially calculating the slope of the sample titration curve and then mathematically correcting the slope to that of a corresponding standard curve. A significantly higher degree of precision was attained using this approach rather than the single-point method.