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Preparation And Application Of Monoclonal Antibody Against Human Procalcitonin

Posted on:2013-04-04Degree:MasterType:Thesis
Country:ChinaCandidate:H WangFull Text:PDF
GTID:2230330395975333Subject:Bio-engineering
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Procalcitonin (PCT) is a116-amino-acidpolypeptide with a molecular weight of13kDa.It is the precursor of calcitonin(CT). In healthy individuals, PCT is secreted by the C cells ofthe thyroid, then it is degraded to calcitonin by protease, and its concentration in serum isbelow0.1ng/ml. But on the severity of infection, the concentration of PCT in the circulationincreases up to several hundred-fold compared to physiological concentrations. Compared tosome other inflammatory factors including white blood cells, interleukin-6(IL-6), tumornecrosis factor-α(TNF-α) and C-reactive protein(CRP), PCT is more sensitivity and higherspecificity. Therefore, serum PCT levels can be used as biomarker for microbial infectionsand monitor the effectiveness of the associated antibiotic therapy.Herein, human PCT gene was alloyed and was successfully cloned into E.coli BL21(DE3) for recombinant expression. SDS-PAGE analysis suggested PCT recombinantexpression mainly in a soluble form. The recombinant protein was purified by one-stepNi-NTA chelating affinity chromatography, and it has been proved a high activity throughindirect Elisa by monoclonal antibody for PCT.Balb/c mouse were immunized with the hrPCT-his, after three immunizations, the titer ofmouse serum achieved integration requires. Mouse spleen cells were harvested and fused withSP2/0myeloma cells. Hybridomas that produce antibodies against PCT were subcloned bylimiting dilution until stable clones were established. Five cell strains F6, G2, C2, D2, E5were screened and ascites fluid was obtained from the intraperitoneal cavity of inoculatedmouse. The subclass of five monoclonal antibodies was IgG1subclass and the molecularweight was all about170kDa. The titer of five mAbs in ascites was108,107,107,107,108,respectively. After purification by caprylic acid ammonium sulfate precipitation method, Thetiter of five mAbs were107,106,106,106,107. The affinity constant of five mAbs were2.25×109、1.02×108、2.31×107、1.75×108、1.28×108, respectively. The concentration of fivemAbs were6.26mg/ml,4.347mg/ml,3.95mg/ml,3.864mg/ml,4.439mg/ml, respectively. Thepurity of five mAbs were89.71%,94.71%,87.96%,97.13%,91.47%.We are interested in the development of laboratory diagnostic reagents and techniquesthat are applicable in the clinic. In this study, the five antibodies were used for ELISA(horseradish peroxidase-marked) platform and immunochromatography (fluorescence latex-marked) platform pairing, to establish clinical rapid diagnostic methods. F6-G2and E5-G2were proved as a better sensitivity antibody pairing in the ELISA platform, F6-C2was pair can be uesd in the immunchromatography platform.The high sensitivity of antibody pairing were used for clinical verification, F6-G2andE5-G2clinical relative coefficient (Clinical samples n=32) were0.975and0.985, generalspecificity were96.5%and96.43%respectively. Three times clinical were detected in theimmunochromatography platform, the relative coefficients (n1=28,n2=46,n3=35) of F6-C2toclinical truth value were0.964,0.976,0.955, specificity were97.8%,96%,95.5%,respectively.
Keywords/Search Tags:procalcitonin, monoclonal antibody, affinity, point-of-care-testing, Enzyme-linked immunosorbent assay, antibody pairs, immunochromatographic
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