| Rabies is a zoonotic infection caused by the Rabies virus（RABV）that seriously endangers public health safety and has the highest fatality rate.Rabies has been classified as B class infectious disease and animal blight of the second class.The goal of prevention and control is to purify human rabies.It is the most effective measure to purify human rabies and recognized by the international community to vaccinate dogs against rabies on a large scale,so that the immune density of dogs can reach more than 70%.The monitoring of rabies antibodies is the key to evaluate the efficacy of rabies vaccine use among animals.The search for a rapid,high-specificity,high-sensitivity and high-throughput rabies antibody detection technology is of great significance for the effective prevention and treatment of rabies,providing technical support for the monitoring of the immune effect of rabies vaccine and the detection of rabies antibodies.Before,Rabies Antibody detection methods include Mouse Neutralization Test（MNT）and Fluorescent Antibody Virus Neutralization test Test,FAVNT),Rapid Fluorescent Focus Inhibition Test（RFFIT）,Enzyme Linked Immunosorbent Assay（ELISA）,Gold Immuno Chromatography（GICA）,etc.These methods have different degrees of shortcomings,including:MNT time is long,requires live viruses and animals,sensitivity and repeatability is relatively low;FAVNT and RFFIT have high sensitivity and specificity,but it takes a long time,requires live virus and live cells,and requires high requirements for laboratory and operating personnel.ELISA showed general performance,sensitivity and specificity were inferior to FAVNT,RFFIT,and time was inferior to GICA.GICA is not suitable for high-throughput detection due to its low sensitivity.Chemiluminescence enzyme immunoassay based on AC electrokinetic effect is an electrochemical method in particle manipulation technology,which has the advantages of good specificity,high sensitivity,short time,low cost and high throughput.Using a high-throughput microelectrode chip as a carrier,the AC electrokinetic effect was used to accelerate the specific immune reaction between the object under test and the biological probe on the surface of the microelectrode chip,and the detection signal was received by chemiluminescence enzyme immunoassay to realize the rapid analysis and detection of the sample.In the early stage,our laboratory successfully detected brucella antibodies,porcine circovirus,avian influenza virus,Newcastle disease virus,etc.,but the application of this technology in the detection of rabies antibodies has not been reported.Ⅰ.Research Content:1.Preparation and identification of recombinant nucleoprotein of rabies virus and identification of monoclonal antibodyNucleoprotein（NP）gene sequences of RABV nucleoprotein（NP）provided by Gen Bank are synthesized by chemical methods.p ET28a（+）-NP recombinant plasmid is constructed using p ET28a（+）as prokaryotic expression vector and identified by double digestion.Then the recombinant plasmid was transformed into the receptor cell BL21（DE3）for induced expression,and the recombinant NP was prepared.The purified recombinant NP was obtained after the inclusion body treatment and purification by His-tag nickel column.The purity was identified by SDS-PAGE electrophoresis,the concentration was determined by BCA spectrophotometry,and the specificity was identified by Western blot.Biological characterization of RABV NP monoclonal antibody was performed,including purity identification by SDS-PAGE electrophoresis,concentration determination by BCA,specificity identification by Western blot,antibody titer determination by indirect ELISA and antibody subtype identification by ELISA.2.Rapid detection of rabies antibodies based on microparticle manipulation technologyFirstly,the feasibility of the experimental materials and detection methods were tested and verified.The antigen and chemiluminescence analysis were used as immune detection means,the microelectrode chip was used as the solid phase carrier material for immune reaction,the microelectrode chip mold,the detection of rabies antibody in serum to be tested and the dilution ratio of serum were tested and verified.Then,the experimental parameters were screened and optimized to improve the comprehensive performance of this detection method.In order to improve the encapsulation rate of antigens on microelectrode chips,the plasma activation process,the antigen coating concentration and the antigen coating conditions were screened respectively.In order to improve the specificity and sensitivity of the detection method,the sealing time was screened.In order to improve the acceleration effect of immune response,the acceleration voltage,acceleration time and acceleration frequency were screened respectively.Finally,the detection limit,recovery rate of negative serum,sensitivity,specificity,accuracy,repeatability and stability of this rapid detection method were tested.A rapid detection method for rabies antibody based on AC electrokinetic effect and chemiluminescence enzyme immunoassay was established.Ⅱ.Research results1.Preparation and identification of recombinant nucleoprotein of rabies virus and identification of monoclonal antibody（1）Preparation and identification results of recombinant NP:The recombinant plasmid p ET28a（+）-NP was successfully constructed by double digestion of rabies virus NP plasmid,and the target gene fragment was found at 1359 bp.The purified recombinant NP was purified by Ni column affinity chromatography.SDS-PAGE electrophoresis results showed that the purified recombinant NP was of high purity.The concentration of purified recombinant NP was 0.64 mg/m L by BCA.It has good specificity.（2）Biological identification of recombinant NP monoclonal antibody:The results of SDS-PAGE electrophoresis confirmed that the purity of monoclonal antibody was high,the concentration of monoclonal antibody was 0.8mg/m L by BCA method,and the results of Western blot showed that monoclonal antibody had good specificity.The titer of monoclonal antibody was greater than 1:32000 by indirect ELISA method,and the subtype of monoclonal antibody was lg G1 type by ELISA method.2.Research results of rapid detection of rabies antibodies based on microparticle manipulation technology（1）Preliminary establishment of detection methods Experimental results:NP（before dialysis）has better activity than NP（after dialysis）,and NP（before dialysis）is identified as coated antigen;The sensitivity of chemiluminescence method is about 10times higher than that of Elisa.it only takes 1min,and can be used as the immune sensor of this detection method.It can be successfμLly coated with recombinant NP on high-throμghput microelectrode chips and used as solid carrier material for immune response.Ac electrokinetic effect can accelerate the immune response on microelectrode chip in a short time.The microelectrode chip mold can not only accelerate the immune response of high throμghput,but also reduce the background value of luminescence intensity to about 5000,which can greatly improve the sensitivity of this detection method.The reasonable dilution ratio of serum samples was determined to be 1:10.（2）Test method parameter optimization experimental results:The optimized plasma activation process was N2 activation once,the coating concentration of screened antigen was 20μg/m L,and the coating condition was 37℃for 2 h,which improved the coating rate of antigen to a certain extent.The screening and sealing time was 0.5 h,and the luminescence intensity was reduced to about 2000,which further improved the sensitivity.The acceleration voltage was 6 V,the acceleration time was 2 min,the acceleration frequency was 5 KHz,and the accelerated immune response tended to be saturated.（3）Performance test results:The recovery rate of negative serum was low,and the serum samples showed strong immunosuppression in the reaction system of this detection method.The linear range of detection was 1 ng/m L-200 ng/m L,the detection limit of LOD was about 1ng/m L,and the serum samples had no significant effect on the detection limit.The sensitivity was 85.00%,the specificity was 100.00%,the accuracy was 91.43%,and the reproducibility of qualitative determination was91.43%.0.5 IU/m L)had false negative test results.Ⅲ.ConclusionIn this study,the rabies virus nuclear protein with high purity and strong specificity was successfully prepared,and the monoclonal antibody of the rabies virus nuclear protein with high purity,strong specificity and high titer was identified and obtained.A rapid detection method for rabies antibody was developed by microparticle manipulation technique based on AC electrokinetic effect and chemiluminescence enzyme immunoassay.This method has high specificity,high sensitivity,good repeatability and good stability.It can achieve high throughput qualitative detection of rabies antibodies within 15min,which makes up for some defects of ELISA and GICA methods.It can be used for mass preliminary screening and protective evaluation of rabies antibodies,and improve detection efficiency.This study also provides a new method and an effective reagent for the rapid detection of rabies antibodies in batches. |
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