Study On Rapid Detection Method Of Liver Cancer Serum Markers Based On Microparticle Manipulation Technology

Hepatocellular carcinoma(HCC)is the most common and highly fatal malignant tumor,ranking the third in the global malignant tumor mortality rate,seriously threatening human’s health and life.HCC has strong invasion,The five-year survival rate is only 70% after surgical resection and liver transplantation,coupled with the poor prognosis of liver cancer and the high risk of recurrence,so the early diagnosis of HCC is of great significance.Alpha-fetoprotein(AFP)as marker of liver cancer has been widely used in clinical medical diagnosis,the diagnosis sensitivity of AFP is only 60%～70%,which is affected by the degree of differentiation of liver cancer cells,so the clinical diagnostic value is very limited.Many studies have shown that there are significant differences in Soluble programmed death factor ligand 1(s PD-L1)of different clinical stages of liver cancer serum,with significant positive correlations with the level of AFP.Liver cancer experts say that s PD-L1 may be an auxiliary serum marker for HCC clinical diagnosis.Combining detection of AFP and s PD-L1 can improve the sensitivity of cancer diagnosis.Nowadays,the methods of diagnosis of serum markers mainly include colloidal gold(GICA),enzyme linked immunosorbent assay(ELISA),chemiluminescence immunoassay(CLIA),radioimmunoassay(RIA)and so on.Among them,the repeatability of ELISA is poor,it needs repeated antibody incubation and cleaning,results are easily affected by temperature and time.GICA has high detection cost,difficult measurement.CLIA requires professional equipment,it cost highly and not suitable for field testing.RIA have the problems of cross reaction and false positive reaction,different ways of processing samples,degrading enzymes,salts and PH will lead to inaccurate detection results.Therefore,it has great significance to explore a rapid,accurate and low-cost immunoassay for the diagnosis of liver cancer.The detection of serum liver cancer markers is mainly based on the immune binding reaction of antigen and antibody.At present,the quality of serum liver cancer markers produced in China is different,and the price of import is so expensive.Therefore,the preparation of serum liver cancer marker protein with high titer,specificity and low cost can provide an effective reagent for early rapid detection of liver cancer.Microspheres manipulations technology based on AC electrodynamic and impedance immunosensing has the advantages of high sensitivity,good specificity,no marking,low cost,simple operation and portability.The technology can successfully detect the f M level of BPA and the quantitative detection of zika virus RNA.Our laboratory successfully detected the Newcastle disease virus(NDV),porcine circovirus(PCV),avian influenza virus(AIV),the antibodies of brucellosis and so on.Appliing this technique to detect the liver cancer markers in serum has not been reported.The technology using the AFP,s PD-L1 antigen and antibody as detection reagent,Optimizing the cleaning method of the microelectrode chip,the optimal packaging condition,sealing time and AC electrical testing conditions to build a rapid detection method of liver cancer based on AC electric effect and impedance immune.In the later stage,the chip wrapped in the antibody can be treated for vacuum sealing.When use,only connecting a micro-impedance like a mobile phone can complete the site 1min rapid detection.The contents of this paper are as follows:(1)Expression and purification of AFP in prokaryotic systemAccording to the AFP whole gene sequence provided in the Gen Bank(Genbank Login Number:NM＿001134.2),AFP whole gene was synthesized by codon preference analysis and optimization of Escherichia coli.The whole AFP gene was connected to the plasmid vector p ET28a(+),and then the recombinant plasmid AFP-p ET28a(+)was transformed into Escherichia coli receptive cells,and the p ET28a(+)-AFP/BL21(DE3)was successfully constructed.The optimal induction conditions for AFP recombinant proteins were screened from induction time,induction temperature,IPTG inducer concentration,respectively.The Pure AFP recombinant protein was purified by His-tag nickel column after obtaining a large amount of AFP recombinant protein with the best induction conditions.The Pure AFP recombinant protein was identified by SDS-PAGE electrophoresis for analysis purity,BCA determination of recombinant protein concentration,and Western-blot for identification of protein specificity.(2)Preparation and biological identification of AFP monoclonal antibodiesThe AFP recombinant protein was used as immunogen to immunize BALB/c mice.After 4 times of immunization,the orbital blood of immune mice was taken for the determination of serum titer before cell fusion.Double the antigen dose three days before cell fusion to strengthen the immune mice.After strengthening the immunity,the spleen of the mice was made into splenic cell suspension and mixed with myeloma cells to promote cell fusion by PEG1500.After that,the fusion cells were subcloned by finite dilution method,and the supernatant of subclonal cells was screened by ELISA method,So we can got a hybridoma positive cell line that could stably secrete antibodies.At least two times subclones for the positive cell line were performed to ensure the screening of AFP monoclonal hybridoma cell line and expand the training.Coperitoneal injection of monoclonal hybrioma cells into mice to induce afp ascites antibody,and then purified with the protein a-pillar.The biological characteristics of AFP ascites monoclonal antibody were identified,including SDS-PAGE purity identification,BCA concentration determination,monoclonal subtype identification,western-blot specific identification,measurement of monoclonal antibodies by enzyme-linked immunosorption test(ELISA),etc.(3)Establishment and optimization of microparticle manipulation technology apply to specific detect AFP、s PD-L1 antigensFirst of all,the microelectrode chip is pretreated,including chip frequency sweep and microscope to observe whether the resistance wire has short circuit or open circuit,optimizing the cleaning mode of the chip surface,and then ozone activated the chip to facilitate the binding of hydrophilic active groups to AFP、s PD-L1 antibodies.The coating concentration,coating time and optimal sealing time of AFP、s PD-L1 antibody were optimized,and then the AFP、s PD-L1 antigen was detected by chip-connected impedance meter.Optimizing the electrical parameters of impedance meter include AC voltage and current frequency,and establishing microspheres manipulations technology for specific detection of AFP、s PD-L1 antigen.microparticle manipulation technology were evaluated from sensitivity,specificity and repeatability.Result:(1)The AFP recombinant plasmid was identified by enzyme digestion and the result showed that the AFP recombinant plasmid was successfully constructed.When the recombinant plasmid is transformed into BL21 receptive cells,the recombinant protein can be expressed in large amounts at 30℃,0.1 mmol/L IPTG concentration and 8 hour induction.Purified by Ni column affinity chromatography,SDS-PAGE results showed that the purity of purified AFP recombinant protein was high,and the concentration by BCA concentration determination was 0.9239 mg/m L.Western blot results confirm that the purified AFP recombinant protein can strongly bind to the commercially AFP antibodies.(2)Two cell fusions were carried out in this AFP antibody preparation experiment.According to statistics,the fusion rate of the first cell fusion was 78.6% and the positive rate was 73.2%.After three subclones,three AFP monoclonal hybridoma cell lines were obtained.The second fusion rate was 100% and the positive rate was 99.7%,Four cell lines with stable secretion of monoclonal antibodies were selected by four subclones and named 3-6C、4-5C、3-9F、6-10 F respectively.After repeated cryopreservation and resuscitation,the above-mentioned cell lines can still secrete high titer antibodies.The1-10D、2-8D、6-3C of the first cell fusion were identified by subtype,the results showed that all three cells were Ig G2 a type.Taking 1-10 D and 2-8D to enlarge the culture and intraperitoneal injection of mouse induced ascites monoclonal antibody.protein A column purification and SDS-PAGE electrophoresis analysis and identification.The results showed that there were bands at 55KD、25KD,which was consistent with the size of heavy chain and light chain of igg antibody,and the purification effect was better.The antibody concentration can reach 2.0180 mg/m L by BCA.Western blot results showed that purified ascites monoclonal antibody and AFP recombinant protein can bind specifically,the ELISA results showed that AFP monoclonal antibodies can react strongly with AFP proteins purchased and AFP recombinant proteins,the titer can reach1:2048000.(3)The microparticle manipulation technology was established and optimized to detect AFP、s PD-L1 antigens.Finally determine the best cleaning way of the chip: at first,useing isopropanol ultrasonic cleaning 10 minutes,and then washed with anhydrous ethanol ultrasonic for 10 minutes,Finally,ultrasonic cleaning of 10 minutes.The optimal modification condition of the antibody was 10μg/m L AFP Mc Ab coated 1h at 37℃ and sealed 30 minutes with 1%superblock at 25℃.10μg/m L s PD-L1 Mc Ab coated 3h at 37℃and sealed 30 minutes with 1%superblock at 25℃.The best detection parameters of impedance meter are 100 m V AC voltage and 100 k HZ AC frequency.AFP、s PD-L1 antigen diluents at 0.1,1.0,10,100,1000ng/m L respectively were detected under the optimal alternating current parameters.The results showed that the detection limit of AFP、s PD-L1 antigen can stablely reach 1ng/m L.In the specific experiment,the chip was coated with AFP、s PD-L1 monoclonal antibody to detect normal human serum and clinical samples,All the results showed that the specificity(negative detection rate)of AFP、s PD-L1 antigen could reach 96.7% by the microparticle manipulations technology.In repetitive experiments,A total of 20 liver cancer serum samples and 4 normal human serum samples were detected(three parallel groups per sample),sensitivity of AFP Mc Ab load chip up to 95%(specific positive detection rate),s PD-L1 Mc Ab chip sensitivity up to 96.6%.Clinical experimental correlation results showed a positive correlation with afp and spd-l1 expression in liver cancer serum.Conclusion:This study successfully constructed p ET28a(+)-AFP/BL21(DE3)genetically engineered bacteria.After a large amount of induced expression and purification,high purity AFP recombinant protein was obtained.Using this as immunogen,Through long-range immunization and PEG1500 promote cell fusion,limited dilution and subclonal screening,7 cell lines that can stably secrete AFP monoclonal antibodies were obtained.A monoclonal cell line was injected intraperitoneally to induce ascites monoclonal antibody,and a high titer and specific AFP antibody was obtained by purification.Using the AFP prepared and the s PD-L1 preserved in the laboratory,establishing the rapid detection method of AFP、s PD-L1 antigen,and the technique was evaluated from sensitivity,specificity,repeatability and clinical sample detection.The results showed that the detection of serum liver cancer markers by this technique has high sensitivity,specificity and good repeatability.This study provides effective reagents and low cost detection techniques for early field rapid diagnosis of liver cancer.