Antigen excess occurs when an antigen is present at such high concentrations that it interferes with antigen-antibody crosslinking, resulting in the formation of smaller immune complexes (Figure 7.4
). This causes immunoassays to underestimate high concentrations of protein.
Serum Hevylite concentrations can range from <0.020 mg/L to >100 g/L. This is a greater range than most other serum proteins. Hevylite IgM assays on the Binding Site SPAPLUS
and Siemens BNII instruments include in built prozone parameters (Chapter 39
). For example, the Binding Site SPAPLUS
instrument monitors the initial reaction kinetics of each sample at three separate time intervals and compares the results with reaction limits set by the manufacturer (through testing an extensive myeloma library Figure 7.5
). Samples detected as being in antigen excess are automatically flagged by the instrument and retested at a higher sample dilution. A very small proportion of samples in antigen excess have normal reaction kinetics so will not prompt the flag. Such cases are considered rare, but cannot be excluded. Hevylite IgA and IgG assays do not include automatic antigen excess detection as the upper limit of the measuring range is higher than that of IgM assays. For these assays, any sample giving unexpected results should be retested at a higher dilution to preclude antigen excess.
The antigen excess capacity is routinely tested during Hevylite assay production. For each HLC assay, a panel of samples containing high concentrations of the corresponding monoclonal intact immunoglobulin type is analysed at the minimum sample dilution over three reagent lots. Samples that give a scatter signal less than the signal produced by the top calibrator are judged to be in antigen excess. Samples are then diluted and the analysis repeated to calculate the antigen excess capacity of each assay. This value is typically between 52 and 100 g/L, depending on the specificity.