Proteases Present in Some Pancreatic Cyst Fluids May Affect Mucin Immunoassay by Degrading Antibodies and Antigens

Abstract

Biomarker detection in pancreatic cyst fluids is of importance to improve the diagnosis of mucinous cystadenoma, a precancerous lesion. However, assay protocols are generally established for serum testing. Immunoradiometric assay of gastric M1/MUC5AC mucin was performed on pancreatic cyst fluids with well-characterized monoclonal antibodies. Among 1466 pancreatic cyst fluids tested, about 10% to 15% of samples presented abnormal behaviors: (i) radioactivity measured after immunoradiometric assay much lower than the blank of the assay and (ii) increasing dilution of the fluids leading to apparent increase of M1/MUC5AC concentration. In contrast, none of the 109 hepatic cyst fluids tested presented interference.We demonstrate that some (n = 54) interfering fluids cause mucin degradation as well as antibody degradation. Western blot analysis showed that the C-terminal part of the M1/MUC5AC apomucin is most sensitive to degradation. The presence of proteases that degrade antibodies as well as mucin may explain the pitfalls observed in 3.6% of the samples. To detect this interference, each fluid has to be systematically tested at 1:100 dilution in the presence of a saturating concentration of M1/MUC5AC mucin standard and in the absence of antiprotease reagents. Detection of interference could prevent false results caused by mucin degradation in situ.