Research On The Method Of Rapid Diagnosis Of Brucellosis Based On AC Electrokinetics-Enhanced Capacitive Immunosensor

Brucellosis is one of the most prevalent and hannful amphixenosis in the world caused by Brucella,which threatens the lives and health of people and more than 60 kinds of animals.The epidemic situation of brucellosis in China has risen sharply in recent years,and brucellosis has become a serious public health problem.So the prevention and control of brucellosis is particularly important.At present,the main laboratory diagnostic techniques of brucellosis include RBPT,SAT,ELISA and CFT,etc.RBPT and SAT are easy to operate and observe,but artificial judgment of test results will lead to subjective problems,and the specificity and sensitivity is low.ELISA is suitable for large-scale serological investigation,but it is inconvenient to operate on site due to the high requirement of testing conditions.The sensitivity(23.0-97.1%)and specificity(30.6-100.0%)of complement binding test are unstable,and the reagent is expensive,so its application is greatly limited.Therefore,it is very important to explore a low-cost,rapid and specific detection method for the prevention and control of brucellosis.The existing serological detection methods are mainly based on the reaction of antigens and antibodies.At present,the quality of Brucella antibodies in China are uneven in quality,there are mature commercial antibodies abroad.So the antibody reagents mainly depend on imports,but the price is very expensive.The preparation of monoclonal antibodies against Brucella with high titer,specificity and low-cost can provide effective reagents for the prevention and control of brucellosis.The molecules movement control chips based on AC electrokinetic effect has the advantages of small size,low cost,wide coverage,high sensitivity and easy operation.The traditional ELISA test time can be shortened from several hours to one minute by using the molecules movement control technology based on AC electrokinetic effect,and the detection limit is very low.The Molecules Movement Control electrode chips based on AC electrokinetic effect has the advantages of small size,low cost,wide coverage,high sensitivity and easy operation.The traditional ELISA test time can be shortened from several hours to one minute by using the the molecules movement control technology based on AC electrokinetic effect,and the detection limit is very low.Overseas researchers have used this technique to detect BPA,Zhaika virus RNA,human D-Dimer biomarkers and Gram-negative bacteria in serum,and the ideal experimental results were obtained.This technology is expected to be used in the real-time detection for clinical diseases.We has successfully applied this technology to detect Newcastle disease and porcine circovirus.In this study,Brucella BP26 and Ompl6 protein antigens were prepared by E.coli expression system,and Omp16 monoclonal antibodies were prepared by PEG 1500 cell fusion technology,which provided effective reagents for the prevention and control of brucellosis.In this experiment,11-mercaptoundecanoic acid(MUA)was self-assembled on the surface of the chip to form a molecule film.Its terminal thiol group could form a gold-sulfur bond with the metal on the surface of the chip.Carboxyl groups were activated by 1-(3-dimethylaminopropyl)-3-ethylcarbamide(EDC)and N-hydroxy succinimide(NHS),then the recombinant proteins BP26 and Omp16 were covalently fixed on the surface of the chip electrode,and the non-binding sites were blocked to reduce non-specific adsorption.A diagnostic method of brucellosis antibody based on molecules movement control technology is preliminarily established,which provides a new detection technology for the diagnosis of brucellosis.The main contents of this paper are as follows:(1)The codons of Omp16 and bp26 genes were optimized to synthesize the whole genes of Omp16 and bp26.The restriction sites Nde I and Xho I were designed at 5'and 3'segments of the BP26 gene,respectively.The synthesized BP26 gene was digested by Nde I and Xho I,and the digested products were recovered.The recombinant plasmid pET30a(+)/bp26 was constructed by inserting the BP26 gene into the pET30a(+)vector which was also digested by Nde I and Xho I.At the same time,the recombinant plasmid pET28a(+)/Omp16 was constructed.The recombinant plasmid was transformed into BL21(DE3)to construct recombinant genetic engineering bacteria pET30a(+)-BP26/BL21(DE3)and pET28a(+)-Omp16/BL21(DE3).By screening the induction conditions of IPTG(temperature,concentration of inducer,induction time),the optimum induction conditions of recombinant genetic engineering bacteria were obtained.The recombinant proteins were induced to express in large quantities according to the optimum induction conditions.The recombinant protein were purified by His-tag Nickel-affinity chromatography column.The purity of the purified recombinant protein was analyzed by SDS-PAGE,concentration determination by bicinchoninic acid method(BCA)and specificity identification by Western-blot.(2)BALB/c mice were immunized with purified Omp16 recombinant protein as antigen by long-term immunization.PEG1500 was used as inducer for cell fusion.The positive hybridoma cells were screened by indirect ELISA method about 10 days after fusion.The selected positive wells were subcloned 2-3 times by limited dilution method to obtain monoclonal positive hybridoma cells,then the ascitic fluids were prepared.The purity,titer,subclass,concentration and specificity of the purified monoclonal antibody were determined.(3)The molecules movement control chips based on AC electrokinetic effect were used to detect brucellosis antibody.First,the cleaning conditions and self-assembly modification conditions of the chips were optimized.Then the purified Omp16 and BP26 antigens were coated on the surface of the microelectrode chip,blocking the chips surface for the detection of brucellosis antibody.The sensitivity,specificity and repeatability of the method were evaluated.Results:(1)The recombinant genetic engineering bacteria pET30a(+)-BP26/BL21(DE3)and pET28a(+)-Omp16/BL21(DE3)were successfully constructed.The Omp16 and BP26 recombinant proteins were expressed in the form of inclusion bodies.The optimum induction conditions of pET30a(+)-BP26/BL21(DE3)were 37?,0.5 mmol/L-1 IPTG for 8 hours,and that of pET28a(+)-Omp16/BL21(DE3)was 37? and 0.5 mmol/L-1 IPTG for 6 hours.The recombinant proteins were purified by His-tag nickel column,and they was identified by SDS-PAGE electrophoresis and Westem-blot.The results showed that the purified recombinant proteins reached electrophoretic purity and could be specifically recognized by anti-His-tag monoclonal antibody.lt indicated that the Omp16 and BP26 recombinant proteins were successfully expressed in E.coli BL21(DE3).(2)The purified recombinant protein Ompl 6 was used as the immunogen.Through 2 times of cell fusion,the fusion rates were 65.2%and 89.4%respectively,and the positive rates were 38.5%and 86.5%respectively.A total of 5 hybridoma cell lines secreting anti-Omp16 monoclonal antibodies were screened out,named 5H1,3H1,3A3,5H11and 3A11.The supernatants of hybirdoma cell lines were extracted for isotype determination of mouse monoclonal antibodies.The results showed that 5H1-2H-4D/4G/5F,5H1-4C were IgG2a,3H-1H-4A and 3H-IH-4G were IgG1.The 5H1-4C cell line was inoculated into the abdominal cavity of mice to induce ascites to prepare monoclonal antibodies.The concentration of purified antibody can reach 3.725 mg/mL.SDS-PAGE electrophoresis showed that the purity of the purified antibody was over 90%.Western blot analysis showed that there was an obvious band at 21 Ku,which was consistent with Ompl6 band,indicating that the obtained monoclonal antibodies were specific to Omp16 antigen.The titers of 5H1-4C monoclonal antibodies were determined by gradient ratio dilution.It was found that the titers of 5H1-4C monoclonal antibodies reached 1:1024 000.(3)The optimal chips cleaning method was determined as isopropanol immersion for 20 minutes,absolute ethanol for 10 seconds.ultra-pure water for 10 seconds,ozone cleaner for 20 minutes;the optimal chips modification conditions were incubation at room temperature for 30 minutes at 5 mM MUA and incubation at 23? for 40 minutes at 200 mM EDC-NHS.The recombinant protein Omp16 was coated on the surface of the molecules movement control chips to detect 0.2-500 ng/mL monoclonal antibody diluents.The results showed that the chips could detect 10 ng/mL anti Omp 16 antibody,The recombinant protein BP26 was coated to detect brucellosis negative and positive serum identified by standard competitive ELISA kit.A total of 10 positive sera and 6 negative bovine sera were detected in this experiment,and 5 chips were detected for each sample.Compared with the standard method,the sensitivity(positive rate)and specificity(negative rate)were 96%and 95.7%,respectively,and there was no cross reaction with other common bovine and sheep disease serum.The results showed that the method had high specificity and good repeatability.Conclusion:The recombinant proteins Omp16 and BP26 were successfully expressed in E.coli BL21(DE3).A total of 5 hybridoma cell lines secreting Anti-Omp 16 monoclonal antibodies were screened out.Using the prepared antigens and antibodies,a rapid diagnosis method of brucellosis based on molecules movement control technology was preliminarily established.The method has high sensitivity,high specificity and reproducibility.It can be used for real-time quantitative detection of brucellosis which provides a new detection technology and means for the prevention and control of brucellosis.