Vilidation of IgA1 and IgA2 measurements by a solid-phase immunoradiometric assay in serum and secretions

Abstract

We describe specific, sensitive and reproducible immunoradiometric assays to measure total IgA and IgA subclass levels in biological fluids, which take into account the problem that polymeric forms are differently recognized in immunoassays. Sera from subjects totally deficient in one of the IgA subclasses allowed us to ensure the specificity of the subclass assays and to define the proportions of IgA1 (84%) and IgA2 (16%) in the normal pooled serum (from 30 blood donors) used as standard. With purified milk 11-S secretory IgA1 and 11-S secretory IgA2, we determined a correction factor for the corresponding polymeric forms using, respectively, monomeric IgA1 and monomeric IgA2 from pooled serum as standards. With the monoclonal antibodies used, purified 11-S secretory IgA1 was similarly recognized by both the total IgA assay and the IgA1 assay; both total IgA and IgA1 concentrations were underestimated compared with monomeric IgA or monomeric IgA1. In contrast, 11-S secretory IgA2 was better recognized by the IgA2 assay than by the total IgA assay and the values were thus overestimates. Considering this problem of recognition, we fractionated saliva and lung secretions by sucrose density gradient ultracentrifugation before measuring their IgA1 and IgA2 levels.

The Multifaceted Nature of Immunoglobulin A and Its Complex Role in HIV

“…125 With regard to quantitation of molecular forms of IgA, these have been shown to be differentially recognized in immunoassays by antibodies toward the human a-chain, which may present an issue with interpreting IgA levels in mucosal secretions where proportions of molecular forms and subclasses of IgA vary considerably. 126,127 This is compounded by limited availability of pure IgA for each of the molecular forms, which can be used as standards in immunoassays, especially dIgA and SIgA isoforms. Furthermore, determination of IgA subclasses has historically been hindered by the lack of specific reagents as antibodies specific for IgA1 and IgA2 have often been difficult to prepare due to relatively minor structural differences between subclasses, which can show significant cross-reactivity.…”