Improving an anti-beta₂ GPI ELISA by reducing the influence of a blocking agent

There are still considerable interlaboratory differences in positivity rate in anti-β₂GPI ELISA. We have already shown that BSA as ablocking agent could introduce a substantial interference effect in an anti-β₂GPI ELISA.

The aim of this study was to validate and possibly reduce an interference effect of different blocking agents on the detection of IgG anti-β₂GPI antibodies by ELISA.

We used Costar high binding plates coated with affinity purified human β₂GPI and blocked with 1% BSA or 3% gelatin in PBS.Selected sera (20 NHS, 20 APS sera and 10 sera from children with atopic diseases) were diluted in PBS containing 0.05% Tween (PBS-T) or in 0.1% BSA/PBS-T or in 1% gelatin/PBS-T.

When plates were blocked with BSA and samples diluted in PBS-T, 11/50 sera expressed values above the cut-off level in the wells coated with β₂GPI and also substantial binding in sample blanks wells (SB) mostly exceeding the binding to the antigen, therefore these samples were considered negative (average SB for all sera = x ± SD=63 ± 127 mOD). The specificity of IgG antibodies yielding high background bindings was confirmed by direct binding to BSA on solid phase (correlation with SB: P < 0.001, R²=0.88) and efficient inhibition by fluid phase BSA. Further, the sera were diluted in 0.1% BSA/PBS-T, which resulted in negligible binding to BSA either directly coated on the plates or used as the blocking agent and hence lowered SB to insignificant levels (SB=13 ± 9 mOD). Following this modification, 3/11 sera previously found negative due to high SB values, clearly expressed low positive IgG anti-β₂GPI values. The inhibition of anti-BSA with 0.1% BSA in fluid phase was almost complete in 3 minutes, suggesting that longer preincubation time may be unnecessary.

1% gelatin/PBS-T as the sample diluent buffer did not prevent the substantial binding to BSA used as the blocking agent either (SB for 20 sera with the highest binding to BSA =203 ± 262 mOD). The same was true even when the plates were blocked with 3% gelatin and samples diluted either in PBS-T (SB=117 ± 120 mOD) or in 0.1% BSA/PBS-T (SB=127 ± 122 mOD) generating substantial SB values in 18/38 tested sera. Similarly to BSA, significantly lower background bindings were reached only when gelatin was used as the blocking agent and 1% gelatin added to the sample diluent buffer (SB=30 ± 21 mOD).

To reduce the interference effects of a blocking agent it was essential to dilute sera in a buffer containing the same agent. Since the binding to BSA or gelatin was detected in both normal human and patients' sera we suggest to follow this general guideline in anti-β₂GPI ELISA to better define the cut-off points and to more accurately verify not only high, but also most of low positive results.