Establishment Of Human Procalcitonin Double Antibody Sandwich Elisa Detection System

Human procalcitonin(PCT)is a specific marker for bacterial and fungal infection that was found in the nineties.Commonly it can be detected in severe systemic bacterial,fungal,parasitic,acute malarial infections,systemic response syndrome(SIRS)and multiple organ dysfunction syndrome(MODS).PCT is the new index with high sensitivity and specificity during diagnosis.Procalcitonin(PCT)is a 116-amino-acidpolypeptide with a molecular weight of 13kDa.It is the precursor of calcitonin(CT).In healthy individuals,PCT is secreted by the C cells of the thyroid,then it is degraded to calcitonin by protease.In this paper,human PCT sandwich ELISA detection system was established through experimental study.First,human PCT N-terminal polypeptide antigen was used to immunize New Zealand white rabbits to obtain antibody serum.Purified polyclonal antibody was obtained using immunoaffinity chromatography.Polyclonal antibody titer was 128000,and there was no cross reaction between the antibody and similar protein mentioned in this paper.Then,this study chose human PCT N-terminal polypeptide as the antigen.High titer and highly specific ascites were prepared using mice immunization,cell fusion and cell cloning to obtain the anti-human PCT monoclonal antibody.Three-types of monoclonal antibodies,3H5,2D7,3C9 were successfully prepared.3C9 which has the highest titer was selected to prepare mouse ascites.Then the purified PCT monoclonal antibody with high specificity was successfully prepared.A sodium periodate method was employed to conjugate polyclonal antibody with horseradish peroxidase(HRP)to prepare enzyme labeled antibodies,which were optimized through a variety of ELISA conditions.The coating amount was 350 ng/well,with 0.1 mol/L PBS as a coating solution.The coating condition of 4℃ over night.The antibody was diluted at 1:4000 to establish a human PCT double antibody sandwich ELISA detection system.Double antibody sandwich ELISA method’s detection limit was 0.05 ng/mL,with intraplate and interpolate variation coefficient being less than 10%.The established ELISA test system was preliminarily verified.The results showed that the sensitivity was 93.2%and specificity was 97.9%.A comparative study was conducted between the sandwich ELISA and bioMerieux VIDAS PCT kit.The results showed that the two methods had a good correlation.This paper further studied the stability of coated ELISA plate,standard and enzyme labeled antibody.Results showed that antibody and enzyme labeled antibody could be stored at 4℃ for more than 6 months.This has laid a foundation for the further development of early and rapid human PCT detection kits.