Preparation Of A Monoclonal Antibody Against Des-γ-carboxy Prothrombin And Preliminary Development Of An Electrochemical Immunosensor For Determing DCP

Hepatocellular carcinoma（HCC）is a common malignant tumor with a significant mortality rate,which threatens human life.China is a country with a high incidence of hepatocellular carcinoma,and the death rate of hepatocellular carcinoma is the second highest among malignant tumors in China.The prognosis is unsatisfactory and has a high risk of recurrence,so it is very important for early diagnosis of HCC.methemoglobin（AFP）,a marker for early diagnosis of HCC,is widely used,but due to the influence of the degree of development of liver cancer cells and other reasons,it results in poor sensitivity of AFP,which is only 60%-70%,and its clinical diagnostic value is limited.Des-gamma-carboxy prothrombin（DCP）is a newly discovered marker for hepatocellular carcinoma.When used alone in clinical practice,the sensitivity and specificity of DCP are better than those of AFP.At present,domestic and international liver disease guidelines have listed DCP as an extremely important indicator for hepatocellular carcinoma detection.At present,the detection of DCP has been continuously developed from the initial enzyme immunoassay（EIA）to the current fully automated chemiluminescent enzyme immunoassay（CLEIA）.Although CLEIA has improved the response sensitivity based on EIA,there are still some problems in practice,such as the preservation of enzyme use and reaction time problems.These drawbacks limit the application of DCP in the diagnosis of liver carcinoma.Therefore,the development of a highly specific and sensitive method for detecting DCP is of great significance for the premature diagnosis of hepatocellular carcinoma.DCP serological detection methods are mainly based on antigen-antibody binding,and at present,the quality of DCP antigen produced in China varies and the import price is expensive.The preparation of highly efficient,strongly specific,and low-cost antibodies to DCP antigens can provide the basis for early and rapid detection of hepatocellular carcinoma.Electrochemical detection methods are widely used in different fields such as safety control of food,analysis of drugs,and detection of the environment due to their specificity,simplicity of operation,and short response time.Among them,disposable screen-printed electrodes（SPE）are widely used in biosensor development.SPE electrodes are of an integrated type and have been able to be commercialized.Nanomaterials can be easily functionalized in their working area,which makes SPE electrodes have great superiority as substrates.So far,methods based on SPE electrodes for DCP detection have been reported.In this research,DCP protein was prepared by prokaryotic expression system,and DCP monoclonal antibody was prepared by using the PEG cell fusion method to provide effective reagents for DCP electrochemical sensor detection method;Au@TiO2 composite material was chemically synthesized,and the composite material was modified to the working area of SPE electrode,and the working area was modified by amination,and DCP monoclonal antibody was immobilized by covalent bonding on The surface of the material was closed with 5%skimmed milk powder,and a biosensor platform based on SPE electrode as a substrate was built by the above steps,and then the sensor was evaluated,mainly including the optimal reaction time of DCP antigen-antibody,sensitivity,specificity,reproducibility,and serum samples for analysis and evaluation.The contents of the paper as follows.（1）Prokaryotic expression and identification of de-γ-carboxy prothrombin（DCP）The paper referred to the CDS sequence of the DCP gene published in Gen Bank（LX095963）,and on the basis of ensuring the correct amino acid sequence of DCP,the codon of this gene was optimized and the CDS sequence was synthesized.It was also integrated into the plasmid pET28a（+）vector to establish the pET28a（+）/DCP recombinant plasmid,and then the recombinant plasmid was introduced into the receptor state BL21（DE3）for induced expression.The DCP protein was purified using Ni columns,and the DCP reconstituted protein purity assay,concentration assay,and protein blotting analysis were performed.（2）Preparation of DCP monoclonal antibody and its biological characterizationThe prepared DCP reconstituted protein was taken by dialysis to clear the denaturant and then injected into the peritoneal cavity of BALB/c mouse after sufficient emulsification with the same volume of adjuvant.Spleen cells from mice that had completed 4 immunizations and after 1 booster immunization were scraped off and induced to fuse with myeloma cells（SP2/0）using PEG.An indirect ELISA was established and the supernatant was screened repeatedly.The screened cell lines capable of secreting the target monoclonal antibody were expanded and cultured,injected into the peritoneal cavity of mice to prepare ascites,and collected,and the antibody isotypes were identified using an isotype identification kit.The ascites monoclonal antibody was purified by Protein G columns and the biological properties of the DCP antibody were identified.（3）Study of DCP detection method based on Cys/Au@TiO₂ composite electrochemical immunosensorThe Cys/Au@TiO₂ composite material was used as a signal amplifier,and Au nanoparticles were compounded on the surface of TiO₂ nanoparticles by reduction method to obtain the target material Au@TiO2;then it was added to the working electrode region by dropwise addition,and self-assembled Cysteine on the composite material after drying,and the DCP antibody was bound to the material by covalent binding to obtain the antibody modified Cys/Au@TiO₂ electrode,connect the functionalized modified electrode sheet to the electrochemical workstation to detect DCP antigen,optimize the optimal reaction time of DCP antigen-antibody,and evaluate the repeatability,sensitivity,stability,specificity,and serum spiked samples of the sensorResults.（1）The results showed that after digestion of plasmid pET28a（+）/DCP using NdeⅠand XhoⅠenzymes,a gene fragment at 1875 bp appeared with the expected results,indicating the successful construction of DCP recombinant plasmid.The optimum induction conditions for the DCP protein of the engineered bacteria were 25°C,0.6 mmol/L IPTG concentration,and 8 h.The purified DCP protein reached electrophoretic grade purity and the concentration of 1.1923 mg/m L was determined by the BCA method,and the obvious band appeared at 72 k D by Western blot analysis.The results showed that when the DCP antigen was encapsulated at a concentration of 4μg/m L and the antibody was diluted to 1:512000at 2 mg/m L,the reaction results were still relatively obvious,indicating that the DCP recombinant protein has good immunoreactivity.（2）During the preparation of the DCP monoclonal antibody,two cell fusion assays were performed,and the fusion rates of the two assays were 98.1%and 100%,correspondingly.The initial positive tests were 95.4%and 46.7%,respectively.7 DCP monoclonal hybridoma cell lines,named 1C7,1D2,1D11,1F6,2D4,5D6,and 3H6,were sieved after repeated cloning and repeated screening of the fused cells.these cell lines could still produce highly effective antibodies after repeated freezing and recovery operations.The subtypes of 5D6 and 3H6 were detected by the subtype identification kit,and the results of IgG1 type were obtained by expanded culture and inserted into the ascitic cavity of BALB/c mice to make ascites,which was purified by protein G column and detected by electrophoretic analysis.The results were found to be consistent with the size of the standard results of heavy and light chains of IgG-type antibodies,indicating that the purified monoclonal antibodies were of high purity.The concentration was detected by the BCA method up to 1.1996 mg/m L,and the Western blot results showed that the monoclonal antibody could bind specifically to DCP recombinant protein,and the ELISA showed that the validity of DCP monoclonal antibody could reach 1:1024000.（3）An electrochemical biosensor platform was established and applied to the detection of DCP antigens.In this study,the optimal reaction time for antigen-antibody was determined to be 40 min,the optimal scanning voltage for cyclic voltammetry（CV）was from-0.7 v to 1 v with a scan rate of 50m V_·S^-1,and the differential pulse voltammetry（DPV）scan voltage was from-0.3 v to+0.5 v.The detection range of the sensor was also determined and the results were between 1 pg/m L and 10 ng/m L;by 5replicate tests,the current variation of the same concentration was small and reproducible.The specificity of the sensor was measured by testing fetal bovine serum,anti-Müllerian test tube hormone,methemoglobin,DCP antigen,and mixed samples,and the results showed that only samples with DCP antigen or samples containing DCP target substances showed large current changes,and the rest of the samples without target substances showed no significant response.This indicates that the biosensor has excellent specificity.By testing the DCP serum spiked samples,the results showed that the sensor has potential for clinical application.Conclusion.In this study,DCP recombinant protein was successfully prepared by the E.coli expression system.After compounding as an immune antigen,seven cell lines capable of secreting DCP monoclonal antibodies were obtained by long-range immunoassay,PEG method to promote fusion,and after several subcloning and screening.Using the homemade DCP antigen and antibody,an electrochemical immunosensor based on Cys/Au@TiO₂ composite was developed for the detection of DCP antigen,and the method was evaluated in terms of sensitivity,reproducibility,and serum sample detection of the sensor.The results showed that this method has high sensitivity,good reproducibility,and strong specificity,and can be used for rapid diagnostics of liver carcinoma at an early stage.This study provides a reagent basis and a new assay for DCP detection.