A human procalcitonin immunoassay kit and its preparation method and application
A human calcitonin and immune detection technology, applied in the biological field, can solve the problems of insufficient detection sensitivity, long reaction time, expensive experimental equipment, etc.
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Embodiment 1
[0113] The preparation of embodiment 1PCT antigen
[0114] (1) Take 1 mg PCT full-length antigen;
[0115] (2) Prepare 2ml of enzymatic solution: Prohormone Convertase (PC), Carboxypeptidase (CP), Aminopeptidase (AP), Pepeitdyl Glycine Amidating Monooxygenase (Pepeitdyl Glycine Amidating Monooxygenase) -oxygenase, PAM) each 30IU / g dissolved in 20mM Tris, 500mMNaCl, pH 7.4 buffer.
[0116] (3) Add enzymatic hydrolysis solution, enzymatic hydrolysis in water bath at 37°C for 2 hours;
[0117] (4) Separately couple three monoclonal antibodies against N-terminal (N-proCT), calcitonin (CT) and calciferin (KAT) antigens to the chromatographic column, and enzymatically digest The solution was sequentially filtered for affinity purification to obtain three antigens: N-terminal (N-proCT), calcitonin (CT) and anti-procalcitonin (Katacalcin, KAT). The specific steps are as follows:
[0118] a The above antibodies were bound to protein A microbeads respectively. 2 mg of monoclonal ant...
Embodiment 2
[0130] The detection of embodiment 2PCT epitope peptide
[0131] Specific identification of immune response to PCT antigen polypeptide by Western blot: take the final purified procalcitonin polypeptide for 15% SDS-PAGE identification, and use the anti-PCT monoclonal antibody produced by Abcam to perform western blot analysis on it, using Millipore Immobilon The Western Chemiluminescent HRP Subscrate system developed the color, and the results showed that a clear band can be seen where the anti-PCT monoclonal antibody binds to the PCT protein.
Embodiment 3
[0132] The preparation of embodiment 3PCT epitope peptide antigen
[0133] The related antigen obtained in Example 1 is coupled with related macromolecular substances to improve its immunogenicity: the PCT N-terminal (N-proCT), calcitonin (Calcitonin, CT) and calcitonin (Katacalcin, KAT) is coupled with latex microspheres, KLH, BSA, GST, etc., and part of the coupling needs to add a cross-linking agent to promote the improvement of its cross-linking rate. The obtained antigenic polypeptide fragments can be used to immunize animals.
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