Absolute quantitation of binding antibodies from clinical samples.

The quantitation of antibody responses is a critical requirement for the successful development of vaccines and therapeutics that often relies on the use of standardized reference materials to determine relative quantities within biological samples. The validity of comparing responses across assays using arbitrarily defined reference values is therefore limited. We developed a generalizable method known as MASCALE (Mass Spectrometry Enabled Conversion to Absolute Levels of ELISA Antibodies) for absolute quantitation of antibodies by calibrating ELISA reference sera using mass spectrometry. Levels of proteotypic peptides served as a proxy for human IgG, allowing the conversion of responses from arbitrary values to absolute amounts. Applications include comparison of binding assays at two separate laboratories and evaluation of cross-clade magnitude-breadth responses induced by an investigational HIV-1 vaccine regimen. MASCALE addresses current challenges in the interpretation of immune responses in clinical trials and expands current options available to make suitable comparisons across different settings.