IgG detection by enzyme-linked mass spectrometric assay versus color, fluorescent, ECL in buffer and serum

The detection of human IgG (hIgG) in tris buffer versus fetal calf serum (FCS) served as a model system to explore the factors that contribute to sensitivity of enzyme-linked immunosorbent assay (ELISA) with and without capture reagents. Enzyme assays in the liquid phase showed that enhanced chemiluminescence (ECL), fluorescence or liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS) detection methods reached the femtogram range (fg/well) and were more sensitive than colorimetric detection (pg/well). The four detection methods were compared in the solid phase as a full-sandwich ELISA with affinity purified and cross adsorbed capture antibodies against the fragment crystallizable (Fc) domain of hIgG and biotinylated anti fragment-antigen-binding (Fab) antibodies served as detection reagents. After coating 96 well plates with 100 ng of capture antibody for full-sandwich ELISAs colorimetric, ECL, fluorescence and LC-ESI-MS/MS detection all reached low picogram range of hIgG whether in buffer or serum. In direct half-sandwich ELISA with hIgG in tris buffer the detection methods reached the low picogram levels. The direct assays in serum showed a strong matrix effect and the different detection methods showed robust agreement across the nanogram range. Direct ELISA assays in buffer in 1536 well plates using only 10 μL reaction volumes, with 0.1 μL injected on column showed good linearity, accuracy and low variation within and between days with significant signals by 100 pg/well. Background binding and the matrix effect, not the detection method, governed the sensitivity of ELISA. However, LC-ESI-MS/MS provides similar results to other methods at much smaller sample and reaction volumes.