Aim: To prepare monoclonal antibody against trypsinogen activation peptide(TAP), and develop an immunoassay of TAP for the early diagnosis of acute pancreatitis.
Methods: Chemically synthesized TAP was conjugated to KLH as immunogen to immunize BALB/c mice. The spleen cells were fused with Sp2/0 cells. Clones secreting specific monoclonal antibody were screened by indirect ELISA. After cloning, several hybridoma cell clones stably producing anti-TAP monoclonal antibody were obtained. The mAbs were identified and the detection of TAP concentration by competitive ELISA was established.
Results: Ten hybridoma cell clones which could produce monoclonal antibodies to TAP were obtained. The isotypes of the ten mAbs were all IgM, except one (IgG1). Ascites titers were between 1:10(5) to 1:10(6). Specificity analysis proved that all these mAbs reacted only with TAP and had no cross-reaction to bovine serum albumin(BSA), human albumin, trypsinase, amylase. Neutralisation test showed that these mAbs could be evidently neutralized by TAP. A competitive ELISA for the measurement of TAP was developed, with a sensitivity of 0.69 microg/L. The average intra- and inter-assay coefficient of variation (CV) were 9.10% and 10.33% respectively. The median urinary TAP concentration was 57.35 microg/L for acute pancreatitis patients and 9.30 microg/L for controls (P<0.01).
Conclusion: The mAbs against TAP were prepared successfully and a competitive ELISA method for detecting TAP concentration was established.