Development Of The Time-resolved Fluoroimmunoassay For Detection Of Rabies Vaccine Glycoprotein And Dual-label For Hepatitis B Virus Surface Antigen And α-fetoprotein

Rabies is an endemic zoonotic disease, fatal but can be prevented. It is caused by rabies virus, a member of the Lyssavirus genus of the Rhabdoviridae family. Rabies occurs in more than 150 countries and territories. Asia, with an estimated 56% of the 55,000 human rabies deaths worldwide, has 31,000 deaths per year. Most of these are in children. This is a very heavy toll. China has the second highest incidence of rabies after India. Prevention and post-exposure therapy require safe and efficacious vaccines. Vaccination is considered one of the most viable and important methods for the prevention of rabies by way of preexposure prophylaxis in high-risk groups, postexposure prophylaxis in contact groups, and preexposure prophylaxis in pet animals that are at risk due to possible contacts with rabid animals. Every year, more than 15 million people worldwide receive a post-exposure vaccination to prevent the disease-this is estimated to prevent hundreds of thousands of rabies deaths annually.Rabies Virus (RV) belongs to Lyssavirus, Rhabdoviridae, bullet-shaped,180nm long,75nm diameter, its head is hemispherical, it gets flat end. The virus particles are composed by two parts of the shell and core. Its genome are consisted of 11928 to 11932 nucleotides, containing five large open reading frame as a single chain, non-segmented negative-strand RNA, to encode five structural proteins. The arrangement sequence from the 3’end to the 5’end of genome are N, P, M, G and L genes, respectively encoding:virus nucleoprotein (NP), phosphoprotein (NS), Matrix Scientific protein (MP), glycoprotein (GP) and polymerase (LP).GP is the only antigen that is able to stimulate the body to produce neutralizing antibodies, and the holding of its antigenicity mainly depends on its spatial conformation. Play an important role in the process of the structural characteristics of the rabies virus GP is closely related to its antigenicity and immunogenicity, which stimulate the body’s immune response. GP is the major protective antigen of the rabies virus. B, and T-cell recognition sites, capable of inducing the body from the cellular and humoral immune response to stimulate the body’s secretion and antibody. Therefore, the carboxy terminus of glycoprotein is missing its main antigenic sites point spatial conformation are broken or missing is another key indicator to measure the the rabies vaccine antigens vitality good or bad.The rabies virus nucleoprotein, glycoprotein content are the main quality indicators of the rabies vaccine, in the process of refining rabies vaccine often need timely detection of the antigen content, has been with the NIH animal Determination all these years. The rabies vaccine is tested using the NIH mouse protection test, which is expensive, of long duration, cumbersome and involves the use of live rabies virus. Assuming that the monoclonal antibody recognizes a correctly folded glycoprotein, the rabies viral glycoprotein content is indicative of the vaccine potency because the rabies viral glycoprotein induces neutralizing antibodies and protection. ELISA based methods have been described which use specific monoclonal antibodies raised against the rabies viral glycoprotein. The determination of glycoprotein content requires a Sensitive, precise and rapid detection assay.Hepatitis B virus (HBV) infection is one of the major causes of both acute and chronic liver hepatitis, liver cirrhosis and primary hepatocellular carcinoma. Hepatitis B surface antigen (HBsAg) is the first virological marker which can be found in filamentous or spherical surface antigen particles in addition to virions in the circulation.The detection amount of HBsAg is approximately 1000 times the amount of complete virus particles. HBsAg can be detected with available assay technology before clinical hepatitis develops which often appears 6-8 weeks after the infection. Moreover, the HBsAg particles found in the serum of patients with HBV infection outnumber mature virus particles, and are strongly immunogenic. The HBsAg test is the most important method to evaluate acute or chronic HBV infection, as in most serological profiles other markers may be absent. Another important use of HBsAg testing is screening of blood donors and blood products to exclude HBV infection.Hepatocellular carcinoma (HCC) has been recognized as the fifth most common cancer and the third most common cause of cancer related death world widely. Because of the global pandemic of hepatitis B and C viral infections, the incidence of HCC is rapidly increasing in Asian and Western countries, and this trend will continue for the next 50 years because of the long latency between infection and the development of HCC. More importantly, some patients are often asymptomatic with early HCC and this often results in late diagnosis and high death rate. The primary marker for HCC is a-fetoprotein (AFP), a single polypeptide chain glycoprotein, which is specific for HCC and is widely used in diagnosis of HCC. Using AFP for the early detection for HCC is highly desirable and important. Now, AFP has been used to screen for HCC in high-risk but asymptomatic groups to identify early curable tumors, decrease disease-related mortality, and to improve survival and cost-effectiveness and much effort has been made to AFP for the clinical significance of patients. The prognosis of advanced HCC remains poor and novel treatment and diagnosis strategies are urgently needed.There are a number of immunoassay methods for human AFP and HBsAg. However, all of them focus on detecting only one analyte and cannot detect more than one biomarkers who possess cross-correlation to each other. As AFP and HBsAg could be detected in the same single assay, the overall procedure becomes simple and the detection time is shortened. The simultaneous detection of AFP and HBsAg could obtain more correlative data and help clinicians to monitor exactly the transformation from chronic hepatitis B to HCC and evaluate the prognosis of HCC. Dual-label has potential applications in various fields. However, conventional fluorescent labeling has a limited success in assay of multiple analytes, which make it difficult to distinguish between the emissions bands of the labellings.Labeling immunoassay is a progressive and comprehensive discipline and is the mainstream core technology of modern immunoassay. Based on the principle of labeling immunology, labeling immunoassay is characterized by high sensitivity and specificity, which has been widely used in medicine research and clinical examinations. In recent years, the development of new theories and new techniques has enormously improved the techniques in labeling immunoassay. The most widely used immunolabeling techniques are fluorescence immunoassay (FIA), enzymatic immunoassay (EIA), radio immunoassay (RIA), colloidal gold immunochromatographic assay (GICA), chemiluminescence immunoassay (CLIA), and time-resolved fluoroimmunoassay (TRFIA). Time-resolved fluorescence immunoassay (TRFIA) is a sensitive quantitative immunoassay technology by using rare earth ion to label antigens or antibodies. Due to the unique fluorescence properties of lanthanide rare earth ions, the technique has advantages of high signal to noise ratio, high sensitivity (10-18mol/well), easy labeling process, long storage time, no radioactive contamination, good repeatability, wide range of detection, short operation process, not sensitive to natural fluorescence interference,multiple labelling and very wide range of applications.TRFIA has higher clinical application value compared with ELISA and RIA, which is widely used in the market for clinical detection of various antigens and antibodies.Here we develop the time-resolved fluoroimmunoassay for detection of rabies virus glycoprotein and dual-label for a-fetoprotein and hepatitis B virus surface antigen, and to explore its feasibility for clinical testing.Method:In this thesis, time-resolved fluoroimmunoassay regants were developed for for detection of rabies vaccine glycoprotein and dual-label for a-fetoprotein and hepatitis B virus surface antigen by using antibody-sandwich method.1. Development and application of human rabies vaccine glycoprotein quantitative detection reagents.1.1 Preparation of rabies virus nucleoprotein antigen:The complete length of nucleoprotein (G) gene of the CTN rabies virus vaccine strain was amplified by PCR using a pair of specific primers designed according to the relevant sequences from GenBank, then it was cloned into plasmid p ET32a (+) to obtain the prokaryotically expressed plasmid p ET32a/G The target gene was then expressed in the Escherichia coli BL21 (DE3) cells by inducted with IPTG and the expression was optimized with a proper induction time of 4 hours. The target protein was identified by SDS-PAGE and Western-blot.1.2 Preparation of monoclonal antibodies:Bal/C mice were immunized with rabies vaccine. When the titer reach 1:64000, prepared monoclonal antibodies by hybridoma fusion method. And then screened antibodies with GP, to sieve out of the 48 uncertain anti-GP monoclonal antibodies.1.3 Purification of monoclonal antibody:Purified with Protein G Sepharose 4 Fast flow affinity chromatography column, and the BCA kit for measuring antibody concentration.1.4 Europium labeled antibody and purification:Purified antibodies were mixed with europium labeling reagent at a mass ratio of 5:1, oscillated at 25℃ overnight, and purified over gel chromatography column of SephadexTM G-50 at the next day. At the same time collect effluent (1 ml/tube). Measured the fluorescence value, and then combined the peak tubes, plused protective agent of 10% BSA,-20℃ preservation.1.5 Monoclonal antibodies screening:First with the rabies virus stock as the standard double-antibody sandwich,48 uncertain anti-GP monoclonal antibody-coated, with another 47 Eu standard, standard curve, sieve out the pairing on singleresistant, then homemade GP and corporate standards as the standard from the standard curve, the final screen pairing on monoclonal antibodies.1.6 Calibrations:The calibrator series were carried out by diluting antigen in the buffer. The desired standard concentrations for G protein were 0,31.25,62.5, 125,250 and 500 mEU/ml. The standards prepared above were lyophilized according to 1 mL aliquot and then stored at 4℃ until used.1.6 Validation experiment1.6.1 Standard curves were obtained by plotting the fluorescence intensity (Y) against the logarithm of the sample concentration(X) and fitted to a four-parameter logistic equation using OriginPro7.5 SR1 (Microcal, USA):LogY=A+B×LogX.1.6.2 Sensitivity:The zero calibration was detected for 20 times repeatedly and the mean signal value and the standard deviation were calculated. The signal value, which was obtain by the mean signal value of zero calibration plus double standard deviation and minus the background value, was substituted into the standard curve equation to acquire the corresponding concentration. This concentration was defined as assay sensitivity.1.6.3 Accuracy test (dilution recoveries):Serial double dilutions of antigen antigen by stander buufer as samples were determinated, and then calculating the measured value, the true value, standard deviation and recovery.1.6.4 High-dose:Increasing concentration of multiple reference standard antigen were determined for signal saturation.1.6.5 Precision testing:Analysis precision:the enterprise standard AF points each doing 10 wells, calculate the mean, standard deviation, and CV values; interassay precision:enterprise standard AF point by three different techniciansto operate, each doing 10 wells, calculate the mean, standard deviation, and CV values.1.6.6 Parallelism:analysis was performed on 3 serum samples diluted serially from 1/2 to 1/32 with standard buffer.1.6.7 Cross reactivity:N protein of rabies vaccine was determined as samples for cross reactivity.1.6.8 Comparison with the measured value of to with Wuhan Institute rabies virus G protein ELISA kit and NIH method:homemade kit with Wuhan as ELISA rabies virus detection kit and NIH method for sample measurement, and calculate the correlation coefficient.2. Development of the dual-label time-resolved fluoroimmunoassay for detection of α-fetoprotein and hepatitis B virus surface antigen.2.1 Prepare standards and controls with AFP and HBsAg antigen, the concentration of AFP (mIU/L)/HBsAg (μg/L) standards were:0/0,1/0.2,10/1,100/5, 500/25 and 1000/150.The standards prepared above were lyophilized according to 1 mL aliquot and then stored at 4℃ until used.2.2 McAbs were diluted with coating buffer and coated into microplate with 96 wells together. The plates were concussed for two hours at 37 ℃,then incubated at 4℃ overnight, closed for three hours at 37℃, vacuum pumped, and stored at 4 ℃.2.3 Preparation of antibodies labelled with Eu3+:Purified antibodies were mixed with europium labeling reagent at a mass ratio of 5:1, oscillated at 25℃ overnight, and purified over gel chromatography column of SephadexTM G-50 at the next day. At the same time collect effluent (1 ml/tube). Measured the fluorescence value, and combined the peak tubes, plused protective agent of 10% BSA,-20℃ preservation.2.4 Preparation of antibodies labelled with Sm3+:Purified antibodies were mixed with samarium labeling reagent at a mass ratio of 2:1, oscillated at 25℃ overnight, and purified over gel chromatography column of SephadexTM G-50 at the next day. At the same time collect effluent (1 ml/tube). Measured fluorescence value, and combined the peak tubes, plused protective agent of 10% BSA,-20℃ preservation.2.5 Evaluation of assay performance2.5.1 Standard curves were obtained by plotting the fluorescence intensity (Y) against the logarithm of the sample concentration(X) and fitted to a four-parameter logistic equation using OriginPro7.5 SRI (Microcal, USA):LogY=A+B×LogX.2.5.2 Sensitivity:The zero calibration was detected for 20 times repeatedly and the mean signal value and the standard deviation were calculated. The signal value, which was obtain by the mean signal value of zero calibration plus double standard deviation and minus the background value, was substituted into the standard curve equation to acquire the corresponding concentration. This concentration was defined as assay sensitivity.2.5.3 Accuracy test (dilution recoveries):Serial double dilutions of antigen antigen by stander buufer as samples were determinated, and then calculating the measured value, the true value, standard deviation and recovery.2.5.4 Precision:The control serum was measured 10 times repeatedly. Each mean concentration and standard deviation was achieved. Thus, the coefficients of variation were calculated.2.5.6 Parallelism analysis was performed on 3 serum samples diluted serially from 1/2 to 1/32 with standard buffer.2.5.7 Interference:The effect of hemolysis, lipemia, and bilirubinemia was assessed by adding hemolysate, bilirubin (unconjugated), and triglyceride and sodium ascorbate to the serum samples of the patient volunteers.2.5.8 Comparison:The samples were determined simultaneously by the CLIA method. The data was analysed by the linear correlation.Result:1. G protein TRFIA:The complete length of GP gene of the CTN strain of rabies virus was amplified by PCR using a pair of specific primers designed according to the relevant sequences from GenBank,then it was cloned into plasmid p ET32a (+) to obtain the prokaryotically expressed plasmid p ET32a/G.The target gene was then expressed in the Escherichia coli BL21 (DE3) cells by inducted with IPTG and the expression was optimized with a proper induction time of 4 hours.The GP was Successfully expressed in the Escherichia coli BL21 (DE3) cells.The antigenicity of this protein was examined by Western blot analysis. A simplified immunization procedure was adopted for the BALB/c mice, we got specific GP antibodies. Then, a novel immunoassay for rapid determination of rabies virus glycoprotein in rabies vaccine was first established by time-resolved fluoroimmunoassay (TRFIA). Based on a sandwich-type immunoassay format, analytes in samples were captured by one monoclonal antibody coating in the wells and "sandwiched" by another monoclonal antibody labeled with europium chelates. The immunocomplex was retained after wash, and then adopted treatment with enhancement solution; fluorescence was then measured according to the number of europiumions dissociated. Levels of the rabies virus glycoprotein were measured in a linear range with a lower limit of quantitation (0.98 mEU/mL) under optimal conditions. The repeatability, recovery, and linearity of the immunoassay were demonstrated to be acceptable. The correlation coefficient of glycoprotein values obtained by our novel TRFIA method and ELISA method was 0.912. The correlation coefficient of rabies vaccine efficacy values obtained by our novel TRFIA method and NIH method was 0.903. The result of the paired T-test for 39 rabies vaccine samples involved was t0.05/2,38=-1.223 (P=0.229>0.05) and indicated that there was no statistical significance between T-NIH and NIH. These results showed good correlation and confirmed that this sensitive, precise and rapid TRFIA was feasible and could be more suitable for the quality control in the process of rabies vaccine production than ELISA and NIH method.2. AFP/HBsAg TRFIA:Based on a two-site sandwich protocol, monoclonal antibodies (McAbs) against AFP and HBsAg were co-coated in 96 microtitration wells, and tracer McAbs against HBsAg and AFP were labeled with europium (Eu) and samarium (Sm) chelates, respectively. After application of diluted serum samples, Eu3+-and Sm3+-McAbs were added and then fluorescence signals of Sm3+and Eu3+ tracers were collected. The detection limitations of AFP and HBsAg were 0.09 mIU/L and 0.01 μg/L, respectively. The measurement ranges of AFP-TRFIA and HBsAg-TRFIA were 1-1000 mIU/L and 0.2-150 μg/L, respectively. The intra- and inter-assay coefficients of variation of the AFP-TRFIA were 3.3-4.1% and 5.7-7.2%, and for HBsAg-TRFIA were 2.9-3.9% and 4.9-6.8%, respectively. The linear correlation of TRFIA and chemiluminescence immunoassay measurements resulted in a correlation coefficient of 0.9949 for AFP and 0.9940 for HBsAg. The results of the paired T-test for samples involved were tAFP 0.05/2,1 n=-0.846, P=0.399>0.05; tHBsAg 0.05/2,82=0.557, P=0.579>0.05 and indicated that there was no statistical significance between the values of TRRIA and CLIA method. For the endurance test, Eu-labeled McAbs were stable for at least 1 year at -20℃, and the results of the TRFIA with the same reagents were also reproducible over 1 year. The availability of a highly sensitive, reliable, and convenient AFP/HBsAg TRFIA will allow the quantification of both AFP and HBsAg that will provide diagnostic value in various clinical conditions, and can be applied for clinical use.Conclusion:The results demonstrate that the TRFIA reagent for detection of rabies vaccine glycoprotein and dual-label for hepatitis B virus surface antigen and a-fetoprotein were developed and met the demand for clinical application and could be the substitute of the import products for basic research and clinical examinations.