| Background and PurposeRubella virus and cytomegalovirus (CMV) both are the important viruses of TORCH screening.Rubella is an acute respiratory infectious disease caused by rubella virus, which clinical signs are mild. But a maternal rubella virus infection can result in serious consequences,especially infection occurs during the first three months of pregnancy. Rubella virus can be transmitted through the placenta and may result in fetal death or may cause serious malformation fetus, which called congenital rubella syndrome (CRS). CRS is the main reason of blind, deaf, congenital heart disease and low intelligence.CMV infection is usually accompanied with mild or no symptoms. However, CMV infection during pregnancy can cause intrauterine transmission, which is harmful, can lead to severe fetal injury, including growth disorder, low spirit, jaundice and central nervous system abnormalities. Children born with severe infection can appear sleepiness, convulsions, respiratory distress syndrome, etc., might die in the days or weeks. Survivors can appear sequelae, such as mental retardation, learning disabilities, movement disorders and deafness, etc. CMV in patients with low immune function,such as blood diseases patients, cancer patients, transplant recipients, and people infected with HIV, can cause serious infection and even life-threatening.Serology detection of IgG and IgM antibodies can initially determine the maternal infection status. IgG antibody can exist for several years or even a lifetime after produce. Only IgG is positive suggests previous infection. From IgG negative to positive, which called seroconversion, prompts recent primary infection. The antibody titer of recovery phase is 4 times or more than 4 times higher than acute phase, can also make a diagnosis of recent acute infection. Determining of IgG antibody titter, can also understand the people recessive infection level of rubella virus and CMV and examine the immune effect of vaccine. IgM antibody appears within several days after virus infection, usually lasts for several weeks to months. IgM antibody is positive, is an important index of acute viral infections.Time-resolved fluorescence immunoassay (TRFIA) takes advantage of 3 valence rare earth ions and their chelates which possess unique fluorescence properties as tracers to label antigen or antibody, instead of fluorescent substances, enzymes, isotopes, chemical luminescence materials, etc. After the antigen-antibody reaction, fluorescence intensity of reaction products is determined by TRFIA detector. According to the fluorescence intensity of the product and the ratio of relative fluorescence intensity, the detector determines the concentration of the analytes in reaction system, so as to achieve quantitative analysis. The dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) is currently the most widely used TRFIA which possesses a biggest bright spot of multi-labeled technique.This study applies the characteristics of TRFIA dissociation enhanced technique and the advantages of multi-labeled, to develop three quantitative detection reagents for rubella virus IgG antibody, CMV IgG antibody and rubella virus/CMV IgM antibody. We evaluate various performance indicators, and compare them with foreign reagents to evaluate their clinical applicability.Methods1. Using the indirect TRFIA method to quantitatively detect the rubella virus or CMV IgG antibodies of human serum, respectively.1.1 Coating the specific antigen:Dilute the rubella virus antigen or CMV antigen to 3μg/mL using coating buffer, 100μL/hole add to the 96 micro-well plate, then block, swab-off, vacuum packaging,2～8℃ for storage.1.2 EU3+-labeled goat anti-human IgG polyclonal antibody:According to the label proportion of 5:1,1 mg of goat anti-human IgG polyclonal antibody is pretreated, such as removed impurity, then concentrated to 4～5 mg/mL and mixed with 0.2 mg of DTTA-Eu.4℃ reacted over the night, after purification treatment, add the final concentration of 0.1%BSA as protectant,-20℃ for storage.1.3 Preparation of standard:Mix several rubella virus or CMV IgG antibody positive samples which possess a high value, dilute to 10,20,50,100,250 IU/mL and 5,10,20,40,100 PEIU/mL using sample dilution buffer. After calibrated to be qualified,2～8℃ for storage.2. Using capture dual-labeled TRFIA to quantitatively detect rubella virus IgM and CMV IgM simultaneously in human serum.2.1 Coating anti-μ chain monoclonal antibody:Dilute the anti-μ chain monoclonal antibody to 3 μg/mL using coating buffer, 100uLl/hole add into the 96 micro-well plate, then block, swab-off, vacuum packaging,2-8℃ for storage.2.2 EU3+-labeled rubella virus monoclonal antibody:According to the quality labeled proportion of 5:1,1 mg of rubella virus monoclonal antibody is pretreated, such as removed impurity, then concentrated to 4-5 mg/mL and mixed with 0.2 mg of DTTA-Eu.4℃ reacted over night. After purification treatment, add the final concentration of 0.1% BSA as protectant,-20℃ for storage.2.3 Sm3+ labeled streptavidin:According to the labeled quality proportion of 2:1,0.4 mg of streptavidin is pretreated, such as removed impurity, then concentrated to 4-5 mg/mL and mixed with 0.2 mg of DTTA-Sin,4℃ reacted over night. After purification treatment, add the final concentration of 0.1% BSA as protectant,-20℃ for storage.2.4 Biotinylation of CMV antigen:According to the molar ratio of 1:25,1 mg of CMV antigen is pretreated, such as removed impurity, then concentrated to 2 mg/mL and mixed with 281～μg NHS-LC-Biotin. React at room temperature for 1h. After purification treatment, add the final concentration of 1 mg/mL BSA and 0.05% NaN3 as protectant,-20 癈 for storage.2.5 Preparation of standard:Mix several rubella virus IgM antibody positive samples or cytomegalovirus IgM antibody positive samples which possess a high value, dilute to 2,5,15,40,80 AU/mL and 5,20,80,200,400 AU/mL using sample dilution buffer. After calibrated to be qualified,2～8℃ for storage. 3. Performance evaluation3.1 Coincidence rate of the negative/positive reference product:Detect the commercial serum panel (PTR201 or PTC203) and the national serum panel to calculate the coincidence rate.3.2 Dose-response curve:Use the logarithmic concentration of the reference standard as the abscissa and the logarithmic value which calculated by the signal value minus the background signal as the ordinate. Use the double logarithm model Log-Log processing function.3.3 Analytical sensitivity:Detect the zero dose point fluorescence value for several times, then calculate the average value of zero point fluorescent (X) and its standard deviation (SD), put X+2 SD into the above standard curve equation to calculate the analysis sensitivity.3.4 Linearity:Test the samples which are obtained by seriesly dilute the serums of high or low concentration. The mean results value and dilution ratio are conducted straight line fitting using least squares. Then calculate the correlation coefficient r.3.5 Recovery experiment:In routine inspection samples, add the standard buffer contained same volume but different concentrations of specific antibody to prepare recovery samples to calculate the recovery.3.6 Precision experiment:In three experiments at different times, test positive serum samples of different concentrations (high, medium and low), calculated the variation coefficient in one experiment and coefficient variation among three experiments.3.7 Cross reaction:Choose the samples which might cross-react with the purpose antibody to test.3.8 Interference experiment:Test the positive samples which are added of hemoglobin, triglycerides or bilirubin, then compare concentration changes before and after adding distractors.3.9 Comparison with foreign reagent:parallel test samples by homemade TRFIA reagent and foreign kit, use the statistical software SPSS 13.0 for data analysis.Results1. Rubella virus IgG TRFIA:Analytical sensitivity is 0.12 IU/mL, with a detection range of 0.12～250 IU/mL. The accuracy is good that the recovery rates are all among 90% to 110%. Inter- and intra-CV both are less than 10%. Compared to the foreign kit, the coincidence rate of specificity is 97.10%(67/69), of sensitivity is 99.42%(340/342). After matching chi-square test, P=1.000, there is no significant difference between the two detection methods. Combined with kappa coefficient test, κ=0.965, P=0.000, result shows inosculation of two test method has statistical significance and is strong.2. CMV IgG TRFIA:Analytical sensitivity is 0.18 PEIU/mL, with a linear range of 0.18～100 PEIU/mL. The accuracy is good that the recovery rates are all among 90% to 110%. Inter- and intra-CV are both less than 10%. Compared to the foreign kit, the coincidence rate of specificity is 95.35%(41/43), of sensitivity is 100.00%(368/368). After matching chi-square test, P=0.500, there is no significant difference between the two detection methods. Combined with kappa coefficient test, κ=0.973, P=0.000, result shows inosculation of two test methods has statistical significance and is strong.3. Dual-labeled TRFIA of rubella virus IgM and CMV IgM:Analytical sensitivity of rubella virus IgM and CMV IgM is 0.15AU/mL and 1.12AU/mL, with a linear range of 0.15-80.00AU/mL and 1.12-400.00AU/mL, respectively. The accuracy is good that the recovery rates are all among 90% to 110%. Inter-and intra-CV are both less than 10%. Compared to the foreign kit, the coincidence rates of specificity of rubella virus IgM and CMV IgM is 99.15%(352/355) and 98.27% (340/346), the coincidence rates of sensitivity is 96.43%(54/56) and 93.85% (61/65), respectively. After matching chi-square test, P rubella virus=1.000、 PCMV=0.754, there is no significant difference between the two detection methods. Combined with kappa coefficient test, κ rubella virus=0.949、P rubella virus=0.000;κ CMV=0.910? Pcmv=0.0000, shows inosculation of two test methods has statistical significance and is strong.ConclusionsThis study using time-resolved fluorescence immunoassay technology, successfully develope three quantitative detection reagents, which can be applied to detect rubella virus IgG/IgM antibody and CMV IgG/IgM antibody in the serum. After evaluation, sensitivity, accuracy, detection range, precision, specificity and other various performance indexes all met the requirements. Compared with the commercial foreign reagent, there is no significant difference in clinical specificity and sensitivity, which reaches the requirement of clinical detection reagent. Especially the dual-labeled TRFIA of rubella virus IgM/CMV IgM, which can test two different serum markers in one experiment at the same time, greatly reduces the workload of clinical detection, has a wide clinical application potential. |
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