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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.