[Preparation and application of monoclonal antibodies against trypsinogen activation peptide].
OBJECTIVE
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).


CONCLUSIONS
The mAbs against TAP were prepared successfully and a competitive ELISA method for detecting TAP concentration was established.
Xu
Xia
X
Department of Clinical Immunology, Faculty of Laboratory Sciences, Guangzhou Medical College, Guangzhou 510182, China. yy-xuxia@163.com
Wang
Hui
H
D000208:Acute Disease; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D007075:Immunoglobulin M / Q000096:biosynthesis; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D009842:Oligopeptides / Q000276:immunology* / Q000652:urine; D010195:Pancreatitis / Q000175:diagnosis*
D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M; D009842:Oligopeptides; C080396:trypsinogen activation peptide