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Updated: Oct 9, 2026
Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting
Published on: December 12, 2011
Different efficacy of various blocking reagents to eliminate interferences by human antimouse antibodies with a
1Zentrum für Frauenheilkunde und Geburtshilfe, Universität Bonn, Germany.
Objective:
The efficacy of three reagents to eliminate interferences with the cancer antigen 125 (CA-125) assay Enzymun CA-125 II by human antimouse antibodies (HAMA) formed by patients after injection of the murine anti-CA-125 antibody OC125 is compared.
Methods:
Apparent CA-125 concentrations of 14 serum samples obtained from 6 patients after multiple injections of 1 mg radiolabeled OC125 F(ab')2 fragments were measured with the Enzymun CA-125 II before and after preincubation, either with nonspecific mouse IgG, with the polymerized mouse IgG MAK-33, or with the commercially available HAMA-blocking reagent IIR.
Results:
In all samples with HAMA concentrations ranging from 341 to 46900 microg/L, false-positive CA-125 values were measured with the Enzymun CA-125 II, which could be reduced by preincubation with the HAMA-blocking reagents. However, although after preincubation with 2 g/L IIR for all samples the CA-125 concentrations measured were reduced to values within the normal range, after preincubation with 0.7 g/L of polyclonal mouse IgG for five samples and after preincubation with 0.7 g/L of MAK-33 for all samples also the reduced values were considerably elevated. Larger amounts of mouse IgG or MAK-33 led only to a slight reduction of the remaining false-positive CA-125 values.
Conclusion:
The present results demonstrate that the polymerized mouse IgG MAK-33 and also the normal murine IgG are not suitable for completely eliminating interferences by HAMAs formed after OC125 treatment. Only the HAMA-blocking reagent IIR seems to be an effective agent to eliminate these interferences. Thus, further studies of this material as a blocking reagent seem to be warranted.
