| Background and PurposeNasopharyngeal carcinoma (NPC) is one of the common malignant tumors in south of China and southeast of Asia,which commonly develop in Guangdong,Guangxi,Fujian,Hunan,HongKong,Taiwan and some southeast Asian contries. NPC frequently involves the 35-50 years old of middle-aged and old people, accounting for 60% of the total number of cases. But it is rare in the teens. According to the world health organization (WHO) statistics, about 80% of the NPC happened in China. It harms people’s health and life security seriously, is one of the key prevention and treatment of malignant tumor in China. From 2003 to 2007, the incidence of NPC in China is 4.20/100,000, with a mortality of 2.24/100,000. Incidence in Pearl River basin is more than 10/100,000. Chinese NPC average annual mortality is 2.49/100,000 in men and 1.27/100,000 in women and reaches up to 4.35/100,000 in five southern provinces.The occurrence of NPC associated with environmental, genetic, and virus effect. NPC is the first human cancer that is found to be associated with EBV infection. Study proves that the undifferentiated NPC was associated with EBV infection.Because NPC has no obvious symptoms in early stage and the tumor predilection site is hidden, diagnosis of NPC tends to be delayed usually. Thus it often makes people miss the best time of treatment. Research shows that the misdiagnosis rate is as high as 86.98% in the first diagnosis of NPC. So the early diagnosis of NPC is very necessary.5 years survival rate of early NPC in Ⅰ, Ⅱ stage can be as high as 60%, but in Ⅲ, Ⅳ stage drops to 20%-40%. So, early diagnosis of NPC is the key to improve the effect of treatment and the prognosis. In recent years, research has shown that the markers of infection immunoserology and biology have a unique advantage in NPC early screening, auxiliary diagnosis, clinical stage, the judge of curative effect and prognosis prediction.Epstein-Barr virus belongs to eosinophilic lymphoid virus, γ herpes virus subfamily genera, herpes virus, can cause two different types of infection, namely non productive and productive infection. EBV infection is closely related to many human diseases occurrence and development,including the infectious mononucleosis and B cell lymphoma, T cell lymphoma, NK cell lymphoma and other immune cells diseases and NPC, stomach disorders and other epithelial cells diseases.VCA is viral capsid antigen. The literature reported that serum Epstein-Barr virus VCA-IgA antibodies can appear before 10-61 months in NPC patients. The high level fluctuations of EBV antibody in patients predict the further development of this disease. It may be marker of early diagnosis and differential diagnosis of NPC. In NPC screening program in the future, treating the VCA-IgA antibodies positive as closely monitoring group, and conducting strictly clinical examination and closer follow-up is very necessary. Serum EBV VCA-IgA may as the observation index of evaluating the effect of NPC and monitoring recurrence.EBNA1 can be found in all types of latent infection. It is the only protein which all tumor cells express, is the necessary condition to establish latent infection successfully of EBV. It has to do with viral gene replication and maintain the stability of the virus particles. EBNA1 IgG appears after 3-4 weeks of the infection, peaks after 2-3 months, then drops slightly to a high level and lasts a lifetime. It is the marker of previous infection. Studies have shown that in patients with NPC, the antibodies to EBNA1 can rise to 10 times.In the development of NPC, anti-EBV serum markers mainly predict the short-term risk effect. Through the census detection of VCA-IgA and NA1 IgG antibody levels, can improve the early detection of NPC screening rate, reduce mortality. Detection of the levels of serum anti-EBV antibodies, has been applied to the serological diagnosis of NPC in clinical routine.Virus isolation is the "gold standard"of EBV infection, with a time and people consuming, is less used in clinical application. Serologic test is widely used in clinical. In recent years, with the development of test technology and the improvement of understanding of NPC, detection method of EBV to auxiliary diagnosis of NPC is increasing, such as immunofluorescence and immune enzymatic, enzyme neutral analysis, western blot, immune spots, enzyme-linked immunosorbent assay (ELISA), Polymerase Chain Reaction (PCR) and real-time fluorescent quantitative PCR technology, etc.Among the auxiliary diagnosis of NPC, ELISA and PCR detection of EBV are of the major focus on. The specificity and sensitivity of ELISA is relative high. It is easy to operate and to save the original records. It has an objective judgment results, and can be applied to the blood center of automated test system. But ELISA has some disadvantages, such as, qualitative or half quantitative detection for blood, restrictions of early detection, can’t distinguish patients either in infection stage or in cured stage and has a not enough high sensitivity. PCR method can find the existence of the viral genome and its expression product, directly access to the results, with advantages of high sensitivity, high specificity and quantitative detection. PCR is more directly and accurately to detect pathogenic microorganism infection than immunological methods. But the equipment, time and technology dependence of PCR is higher. So it is difficult to popularize. Developing a more accurate, sensitive, rapid, simple and easy diagnosis and screening method of NPC, will be the development trend of these technologies.Timed-resolved fluoroimmunoassay (TRFIA) using lanthanides of unique fluorescent properties and lanthanides chelates as markers to label antigen, antibody, hormone, polypeptide, nucleic acid probe, etc. After the occurrence of reaction system, TRFIA detector is used to measure fluorescence to judge the concentration of the reaction system, so as to achieve the quantitative analysis. TRFIA has advantages different from other labeling immune technology:high sensitivity (10-18 mol/hole), easy to operate, easy to automate, wide range of standard curve, without sample natural fluorescence interference, preparation of markers is simple, the markers are stable, no radioactive pollution, can be used for multiple labeling, etc.Methods(一) Using the indirect method to develop an EBV viral capsid antigen (VCA) IgA time-resolved immunofluorescence analysis quantitative detection reagent.1. The preparation of the standard and quality control:collecting some high positive samples, after mixing, measure 3 times with IBL ELISA kit to obtain the concentration of the mixing positive sample. Prepare the six series of reference standard (A:0 AU/mL, B:0.5 AU/mL, C:1 AU/mL, D:2.5 AU/mL, E:10 AU/mL, F: 30 AU/mL) and quality-control Ⅰ,Ⅱ,Ⅲ(2.5 AU/mL,,10 AU/mL,20 AU/mL) with sample dilution buffer.2. The preparation of solid phase envelope antigen:Dilute the VCA antigen to 2.5 μg/mL and 100 μL/hole add to the micro-well plate, put in 4℃ over night. Wash the plate, then add 200 μL blocking buffer every hole and put to 4℃ over night. Drain the plate after removing the blocking buffer, then- 20℃ for cryopreservation.3. Preparation and purification of Eu3+ labeled antibody:0.5 mg of IgA antibodies centrifuge for 6 times. According the quality ratio of 5:1, europium labeling reagent is added in and then the mixture is shaking over night. Using column chromatography to separate and purify, then collected with elution buffer, measured the A280 absorbance, collected and mixed the antibodies in peak pip and then added 10% BSA for protection.4. The optimization of the reaction system:4.1 The best concentration of coating:Choose 8 different concentrations to optimize: 0.5 μg/ml,1 μg/ml,1.5 μg/ml,2μg/ml,2.5μg/ml,3μg/ml,3.5μg/ml,4μg/ml.4.2 The best dilution of Eu3+labeling antibodies:Choose 6 different dilutions to optimize:1:400,1:600,1:800,1:1000,1:1500,1:2000.4.3 The best reaction time:Choose 6 different reaction time to optimize:20+20 mim 40+40min、60+60 min、80+80 min、100+100 min、120+120min.5. The determination of reference range:Use the TRFIA reagents to measure 317 normal human serums, make ROC curve after analyzing statistical frequency distribution, then analyze statistical results and combine with the literature to determine the reference range.6. Performance evaluation6.1 Standard curve drawing:Use the double logarithm model Log-Log processing function.6.2 The linear range and sensitivity analysis experiment:Detect the A point (zero dose point) fluorescence value for 20 times, then calculate the average value of A point fluorescent (X) and its standard deviation (SD), put X+2 SD into the above standard curve equation to calculate the analysis sensitivity.6.3 HOOK effect:series dilute of standards, observe the saturation concentration point to dose response curve.6.4 The accuracy of the experiment:The recovery is determined by spiking standards consisting of EBV VCA-IgA at different concentrations into a medium sample containing endogenous EBV VCA-IgA.0.1mL of each of these exogenous EBV VCA-IgA is added into lmL of serum samples while the medium sample is added 0.1mL of the sample dilution. Experiment the 4 samples for 3 repetitions, calculate the mean and recovery.6.5 Precision of the experiment:Repeated measure quality control Ⅰ,Ⅱ,Ⅲ 10 times in 3 different time, then calculate and analyze the intra-and inter-assay variation coefficient (CV).6.6 Interference experiments:Different concentrations of hemoglobin, triglycerides and bilirubin are added into the quality control Ⅰ,Ⅲ, use the VCA-IgA TRFIA detection reagent to test. Each concentration is set of three complex holes. Then calculate the mean value.7. Assessment of clinical trials:Use ZhongShan Biology VCA-IgA ELISA kit as reference reagent, the TRFIA as an analytical reagent, to synchronous detect 171 cases of clinical samples, then use SPSS 13.0 to analyze data.(二) Using the indirect method to develop an EBV nuclear antigen 1 (NA1) IgG time-resolved immunofluorescence analysis quantitative detection reagent.1. The preparation of the standard and quality control:collecting some high positive samples, after mixing, measure 3 times with IBL ELISA kit to obtain the concentration of the mixing positive sample. Prepare the six series of reference standard (A:0 AU/mL, B:2.5 AU/mL, C:10 AU/mL, D:50 AU/mL, E:200 AU/mL, F:500 AU/mL) and quality-control Ⅰ,Ⅱ,Ⅲ(20 AU/mL,,100 AU/mL,400 AU/mL) with sample dilution buffer.2. The preparation of solid phase envelope antigen:Dilute the NA1 antigen to 2.5 μg/mL and 100 uL/hole add to the micro-well plate, put in 4℃ over night. Wash the plate, then add 200μL blocking buffer every hole and put to 4℃ over night. Drain the plate after removing the blocking buffer, then- 20℃ for cryopreservation.3. Preparation and purification of Eu3+ labeled antibody:0.5 mg of IgG antibodies centrifuge for 6 times. According the quality ratio of 5:1, europium labeling reagent is added in and then the mixture is shaking over night. Using column chromatography to separate and purify, then collected with elution buffer, measured the A280 absorbance, collected and mixed the antibodies in peak pip and then added 10% BSA for protection.4. The optimization of the reaction system:4.1 The best concentration of coating:Choose 8 different concentrations to optimize: 0.5 μg/ml,1μg/ml,1.5μg/ml,2μg/ml,2.5μg/ml,3μg/ml,3.5μg/ml,4μg/ml.4.2 The best dilution of Eu3+ labeling antibodies:Choose 6 different dilutions to optimize:1:100,1:150,1:200,1:300,1:400,1:600.4.3 The best reaction time:Choose 6 different reaction time to optimize:20+20min、 40+40min、60+60 min、80+80 min、100+100 mim、120+120min.5. Performance evaluation5.1 Standard curve drawing:Use the double logarithm model Log-Log processing function.5.2 The linear range and sensitivity analysis experiment:Detect the A point (zero dose point) fluorescence value for 20 times, then calculate the average value of A point fluorescent (X) and its standard deviation (SD), put X+2 SD into the above standard curve equation to calculate the analysis sensitivity.5.3 HOOK effect:series dilute of standards, observe the saturation concentration point to dose response curve.5.4 The accuracy of the experiment:The recovery is determined by spiking standards consisting of EBV NA1-IgG at different concentrations into a medium sample containing endogenous EBV NA1-IgG.0.1 mL of each of these exogenous EBV NA1-IgG is added into 1mL of serum samples while the medium sample is added 0.1 mL of the sample dilution. Experiment the 4 samples for 3 repetitions, calculate the mean and recovery.5.5 Precision of the experiment:Repeated measure quality control Ⅰ,Ⅱ,Ⅲ 10 times in 3 different time, then calculate and analyze the intra- and inter-assay variation coefficient (CV).5.6 Interference experiments:Different concentrations of hemoglobin, triglycerides and bilirubin are added into the quality control Ⅰ,Ⅲ, use the NA1-IgG TRFIA detection reagent to test. Each concentration is set of three complex holes. Then calculate the mean value.6. Assessment of clinical trials:Use ZhongShan Biology VCA-IgA ELISA kit as reference reagent, the TRFIA as an analytical reagent, to synchronous detect 148cases of clinical samples, then use SPSS 13.0 to analyze data.Results(一) Using the indirect method to develop an EBV viral capsid antigen (VCA) IgA time-resolved immunofluorescence analysis quantitative detection reagent. The concentration of six standards in this viral capsid antigen (VCA) IgA indirect TRFIA reagent are 0 AU/mL,0.5 AU/mL,1 AU/mL,2.5 AU/mL,10 AU/mL,30 AU/mL, respectively. The best coating concentration, best Eu3+labeled antibody dilution ratio and the optimum reaction time are 2.5μg/mL,1:1000 and 60 +60min. The cut off value of TRFIA is 2.20 AU/mL. The linear rang is 0.029-30 AU/mL, with a sensitivity of 0.029AU/mL. The HOOK effect appears when the concentration is higher than 100AU/mL. Recovery is form 89.44% to 97.81%. The intra-and inter-assay CVs are both less than 10%. There is no cross reaction between VCA-IgA to hemoglobin, triglycerides and bilirubin. And there is no statistical difference between commercial ELISA kit and TRFIA in the coincidence rate of negative and positive.(二) Using the indirect method to develop an EBV nuclear antigen 1(NA1) IgG time-resolved immunofluorescence analysis quantitative detection reagent.The concentration of six standards in this nuclear antigen 1(NA1) IgG indirect TRFIA reagent are 0 AU/mL,2.5 AU/mL,10 AU/mL,50 AU/mL,200 AU/mL,500 AU/mL, respectively. The best coating concentration, best Eu3+ labeled antibody dilution ratio and the optimum reaction time are 2.5μg/mL,1:200 and 60 +60min. The linear rang is 0.162-500 AU/mL, with a sensitivity of 0.162AU/mL. The HOOK effect appears when the concentration is higher than 1765AU/mL. Recovery is from 106.50% to 111.77%. The intra- and inter-assay CVs are both less than 10%. There is no cross reaction between NA1-IgG to hemoglobin, triglycerides and bilirubin. The panel test result is coincidence with panel instruction book which the samples are 7 negative and 14 positive. There is no statistical difference between commercial ELISA kit and TRFIA in the coincidence rate of negative and positive.ConclusionsThe results show that each index (sensitivity, accuracy, precision, specificity, etc.) of quantitative test of the EBV viral capsid antigen (VCA) IgA and nuclear antigen 1 (NA1) IgG TRFIA reagents are met for clinical detection reagent requiement. The 2 TRFIA reagents are similar to the ELISA product performance, are expected to be applied to product after further optimization. |
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