Preparation Of Monoclonal Antibody Against Procalcitonin And Preliminary Development Of An Immunosensor For Determing PCT

Procalcitonin（PCT）has been used as a differential diagnostic biomarker with high sensitivity and specificity in clinical for bacterial infections and viral infections.There are a variety of PCT detection methods,such as radioimmunoassay,projection immune turbidity method and double antibody sandwich chemiluminescent immunoassay,enzyme-linked immunosorbent assay and colloidal gold colorimetric method,etc.Although these methods have the advantages of high accuracy,high sensitivity and good specificity,most of the reagents required are dependent on imports,and the required instruments are expensive,and the operation procedure is tedious.Therefore,to develop a rapid,accurate and low-cost PCT detection method is a problem that needs to be solved urgently in clinical laboratory medicine.The Immunosensor based on excessively tilted fiber grating（Ex-TFG）has the advantages of high sensitivity,good specificity,free labeling,portable equipment,and low operation costs,and has the potential for clinical detection.In the previous period,our laboratory has successfully applied the immunosensors based on Ex-TFG to detect heart Failure biomarker N terminal terminal natriuretic peptide（NT-proBNP）,porcine circovirus type 2（PCV2）and Newcastle disease virus（NDV）.In this paper the PCT protein was first prepared by prokaryotic expression,and the BALB/c mice were immunized four times with the purified PCT protein,then the PEG1500 reagent was used to induce cell fusion to prepare monoclonal antibody against PCT protein.Based on the principle of specific binding of SPA and IgG,the SPA was immobilized on the immunosensor,then the SPA and the antibody were combined to detect the PCT,which provided a new detection method for PCT.The content of this paper is as follows:1.In order to realize the procalcitonin（PCT）high expression in E.coli,in this paper the gene sequences of PCT were optimized and synthesized,then connected into the pET28a（+）vector to construct recombinant plasmid pET28a（+）/PCT,which was transformed into E.coli BL21.The IPTG was used to induce the expression of the recombinant protein.Three factors include induction temperature,induced agent concentration and induction time were set up,and the best induction conditions were screened out to induce the expression of PCT recombinant protein in large quantities.The PCT recombinant protein was purified by Ni affinity chromatography,then he concentration,purity,and specificity of the recombinant PCT protein were analyzed respectively by BCA method,SDS-PAGE electrophoresis,and Western blot.2.The BALB/c mice were immunized four times with the purified PCT protein,then the spleen cells of the immunized mouse and SP2/0 cells were fused with PEG-1500.An indirect enzyme-linked immunosorbent assay（ELISA）was developed to detect monoclonal antibodies（Mabs）secreted by the hybridoma cell lines.The selected positive wells were subcloned at least twice or more to obtain monoclonal positive hybridoma cells,then the ascitic fluids were prepared.The mouse ascitic fluids were first purified by by caprylic acid ammonium sulfate precipitation method and then purified by Protein A column.The biological characteristics of the purified monoclonal antibody were identified.3.The excessively tilted fiber grating was modified on surface,followed by surface hydroxylation modification using concentrated H₂SO_4,silanization modification of APTES,and immobilization of SPA on the surface of the grating.Then SPA was combined with PCT monoclonal antibody for the detection of PCT,and the stability,repeatability,clinically,and specificity of the sensor were evaluated.Results:1.The results showed that the recombinant E.coli was induced by 0.5mM IPTG at25℃for 12 hours,the PCT protein was the highly expressed in E.coli.The SDS-PAGE showed that the purity of PCT protein is over 90%.The Western blot showed that there was an obvious band at 17KD,in line with the expected.The results showed that the PCT protein was highly expressed in E.coli,which laid the foundation for the preparation of PCT monoclonal antibodies and the research of the detection methods.2.To prepare monoclonal antibody（Mab）against PCT protein,cell fusion was made twice.The cell fusion rates were 79.69%and 60.41%,and the positive rates were 4.17%and 16.88%,respectively.Two hybridoma cell lines secreting monoclonal antibodies,named 8D3 and 10F9,were developed by twice subclones.After repeated frozen storage、thawed revival and generation,Two hybridoma cell lines were steadily producing high titer of Mab to PCT.The results of ELISA test showed that the antibodies secreted by 8D3 and10F9 cells belongs to IgG2b,IgG1 isotype,respectively.The specificity of antibody against PCT was identified by Western-blot.Ascites antibody was prepared and purified by caprylic acid ammonium sulfate precipitation method and then followed by Protein A column chromatography.The purity of the purified antibody reached over 90%,select the highest peak collected solution in the same batchand,and its concentration was 1.178mg/m L,1.617 mg/mL,and 1.094 mg/mL by BCA.3.Optical fiber gratings were subjected to hydroxylation,silylation,and immobilization with SPA and PCT-conjugated antibodies.The spectrum of each surface modification was examined.Corresponding resonance wavelength shifts of the first surface modification and subsequent ones such as hydroxylation,silylation,and immobilization with SPA and PCT-conjugated antibodies were 0.015 nm,0.08 nm,0.094 nm,and 1.008nm,respectively,which indicated that each step was successfully performed.The minimum limit of detection of PCT by this immunosensor was 0.5 ng/mL,and the highest limit of detection was 200 ng/mL.The results of the three repeated tests showed that the repeatability of the sensor is good and it can be reused.The specific experimental results showed that the detection could only specifically identify PCT antigen,and the specificity of the sensor is good,The clinicalexperimental results show that PCT in patient serum can be detected and the sensor is clinically good.Conclusion:In this paper,the PCT recombinant protein with high specificity and high purity was prepared by prokaryotic expression.Two hybridoma cell lines steadily producing high titer of Mab to PCT were prepared by cell fusion and subsequent ones.A PCT immune sensor based on Ex-TFG was established by the prepared PCT antigen and antibody.The PCT immune sensor has the advantages of high detection sensitivity,good experimental repeatability,high detection specificity,and it can realize real time quantitative detection of samples,which lays a foundation for the clinical detection of procalcitonin.