Differential assay reactivity of immunglobulin A anti-ß2 glycoprotein I antibodies: implications for the clinical interpretation of antiphospholipid antibody testing.

Abstract

The routine measurement of IgA anticardiolipin (aCL) and IgA anti-β2 glycoprotein I (anti-β2 GPI) antibodies remain controversial despite several studies demonstrating an association with thromboembolic disease in patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). This controversy may be a contributing factor for the current under use of IgA antiphospholipid antibodies. We aimed to investigate the nature of discrepant IgA anti-β2 GPI reactivity to help define the diagnostic value of IgA antiphospholipid antibodies. Four sera selected from SLE/APS patients and positive for antiphospholipid antibodies but having discrepant IgA anti-β2 GPI reactivity on two commercial assays were studied. IgA antibodies were affinity purified to investigate anti-β2 GPI reactivity. Column wash through and eluent fractions were tested on both IgA anti-β2 GPI assays. Results were normalized to total protein. Assay conjugates and standards from the discrepant assays were interchanged. The diseased samples were strongly positive in one assay [144-388 IgA antiphospholipid (APL) units] and negative or weakly positive in another assay (9.9-53 APL units). IgA eluents from IgA anti-β2 GPI positive samples reacted 10 times stronger on the reactive assay. When normalized to protein content, the eluents showed no cross-reactivity for IgG or IgM anti-β2 GPI antibodies, confirming IgA isotype specificity. Conjugate interchange confirmed that both assays bound IgA anti-β2 GPI antibodies, but the anti-IgA conjugate from the reactive assay was 4 times stronger, suggesting that its ability to detect IgA anti-β2 GPI antibodies was partially dependent on the anti-IgA conjugate and calibration. These results confirm not only the presence of IgA anti-β2 GPI antibodies in the selected patient samples but also highlight an IgA conjugate issue for the unreactive assay, causing an underestimation of IgA anti-β2 GPI. This finding may assist in the ongoing standardization efforts of APS antibody testing. In addition, conclusions from published clinical studies may need to be revised as some assays may understate IgA significance.