| Background and PurposeEpstein-Bar virus, a y herpesvirus addicted a human B lymphocytes with a genome of 184 kb, was first discovered in African Burkitt lymphoma cell cultures by Epstein and Barr in 1964. It infects more than 90% of the adult, and is associated with a spectrum of chronic or malignant diseases, including infectious mononucleosis syndrome, EBV-associated hemophagocytic lymphoproliferative syndrome, chronic active EBV infection, EBV-related immunodeficiency disease and EBV infectin-associated tumors (such as nasopharyngeal cancer(NPC), stomach cancer, primary kitt lymphoma, T/NK cell lymphoma,etc.). Among them, NPC was the first human cancer to be found associated with EBV, studies confirmed that more than 95% undifferentiated nasopharyngeal carcinoma tissue could found EBV. Since Old LJ et confirmed that EBV was related to NPC serology in a immune diffusion experiment, in home and abroad, there were a lot of researches were done and found that the immunoglobulin against to EBV antigen were specificity for the diagnosis of NPC.After infecting human, EBV exists in two states:① Incubation period:the virus does not replicate this period, cells were mainly synthetic core antigen (Epstein-Barr virus associated nuclear antigen, EBNA) and latent membrane protein (LMP);② Cleavage stage:viral gene is completely expression this stage, the synthetised viral proteins are immediate-early protein (Zta), early intracellular antigen (EA), Epstein-Barr virus capsid antigen (VCA) and late protein. These antibodies against the antigens all can be discovered in NPC patient.EBNA1 antigen expresses quite early in EBV infected cells, it mainly regulates EBV genes replication, enhances virus gene transcription, and maintains the presence of virus additional sub; in addition, EBNA1 can combined with the repetitive sequence family of the replication origin of EBV latent infection, to activate LMP1 promoter and promote its transcription. It will continue to exist in the final stages of the viral replication cycle, studies has been reported that the sensitivity and specificity in the detection of NPC were 91.9% and 91.4%, respectively, and confirmed that EBNA1 was an ideal serological marker for the detection of NPC. Currently, ELISA specific for EBNA1 IgA are commonly used in the clinical diagnosis of EBV infection, compared with smear methods, ELISA method is more simple and fast, and more easy to judge the test results. However, there are also some disadvantages, such as the low sensitivity and poor repeatability, in addition, the enzyme-labeled antibodies are unstable because the purity of the enzyme and reaction process is susceptible to environmental factors. In this study, we developed a time-resolved fluorescence immunoassay technology (TRFIA) for the detection of EBNA1 IgA. TRFIA is characteristic of high sensitivity, wide detection range, long fluorescence lifetime, substantial Stokes shift, easy automation, less susceptibility to matrix interference and no radioisotope pollution. It could alternative ELISA to use in clinical diagnosis. In addition, due to the advantage of TRFIA, in the next study of EBNA1 IgA time-resolved fluorescence immunoassay for quantitative detection kit, it has obvious advantages than other methods.Zta, is the most important immediate-early protein which was considered as an switching elements in the switch from EBV latency to replication. As an important transcription factor of EBV, it leads EBV switch from the latency to the lytic by starting the waterfall transcriptional expression of a series of early and late proteins. In the 1990s, studies had found that EBV Zta antibody can be used in the diagnosis of NPC, and there were also some researches evaluated the accuracy of EBV Zta antibody used for the detection of NPC, they confirmed that the detection of EBV Zta antibody used in the diagnosis of NPC had high accuracy, especially with high specificity in NPC screening, so the EBV Zta antibody has a high diagnostic value and application prospects. To research and develop the vitro diagnostic kit for Zta antibody is the focus of clinicians and diagnostic reagents manufacturers concern, but there is only one commercial product used in clinical, and TRFIA used in the detection of EBV Zta antibody has not been reported.In this study, we developed a new immunoassay for the detection of Zta IgA based on TRFIA. According to evaluate the performance indicators and compare with the other detection reagent, we evaluated its feasibility in clinical testing applications.Method:Ⅰ. Development of Time-resolved fluorescence immunoassay kit for detection of EBNA1 IgA in human serum.1.1 Optimizing the production method of EBNA1 IgA TRFIA diagnostic kit1.1.1 Using indirect method developed EBNA1 IgA time-resolved fluorescence immunoassay reagent.1.1.2 Preparation of NA1 IgA negative control and positive controls:the negative EBNA1 IgA antibody samples detected by ELISA were diluted with dilution buffer, they were as negative control; the positive EBNA1 IgA antibody samples were diluted with dilution, they were as positive control.1.1.3 Europium labeled antibody and purification:0.5mg anti-human IgA antibody was added to ultrafiltration tube (50KD), and centrifuged for 6min at 9000rpm. The antibody was washed six times using labeling buffer repeatedly. Then, we mixed the antibody and DTTA-Eu in 200μL of labeling buffer. The mixture was incubated at 25℃overnight with shaking. Labeled mouse anti-human IgA antibody was separated from excess free chelates on a Sephadex G-50 column using elution buffer. Fractions were combined,1 ml/tube, then to measure the fluorescence value of each tube and combined the high value tube. The concentration of the europium labeled antibody were detected by BCA method. At last, 1%o of BSA was added to the labeled antibody as protein agent. They were stored at-20 ℃ until required.1.1.4 Determination of coating concentration:in order to choose the optimum coating concentration of NA1 antigen, we set a total of seven coating concentrations, they were 0.5μg/ml, 1μg/ml,1.5μg/ml,2μg/ml,2.5μg/ml,3μg/ml,4μg/ml, respectively. At different coating concentration, we analyzed the fluorescence value of samples and controls. At the optimum coating concentration, the fluorescence of the samples and controls tended to be stable.1.1.5 Determination of Europium-labeled antibody dilution ratio:we diluted the labeled antibody with assay buffer to 1:400,1:600,1:800,1:1000,1:2000,1:4000, 1:6000. At different Eu-labeled antibody dilution ratio, there were differences in fluorescence between positive and negative samples. Under the condition of the low background luminescence value, we determined the optimum dilution ratio depend on the ratio of the fluorescence value of the positive sample and the negative sample.1.1.6 Determination of reaction time:under the determination of the other reaction conditions, we set 15min,30min,45min,60min,90min,120min, respectively. We observed the fluorescence value of the negative and positive samples changing with the reaction time increase. At last, we chose the reaction time as the optimum one when fluorescence of the positive and negative samples tended to be stable.1.2. Evaluate the performance of EBNA1 IgA time-resolved fluorescence immunoassay detection reagent.1.2.1 Precision test:We tested three samples (S1, S2, S3), we calculated the mean, standard deviation, intra-and inter-assay coefficient of variation of each sample.1.2.2 Specificity:We selected ten samples of CMV-positive samples, herpes simplex virus type I positive samples, herpes simplex virus type Ⅱ positive samples and hepatitis B, hepatitis C positive samples, repectively. Then, we detected them using the TRFIA kit developed by us to evaluate the cross-reactivity.1.2.3 Interference test:Triglycerides, bilirubin, hemoglobin were added into negative and positive controls. Then, we tested these controls and calculated the means.1.3. Evaluate the clinical application performance of the kit1.3.1 Dtermination of the reference range:A total of 420 healthy human samples were tested by EBNA1 IgA time-resolved fluorescence immunoassay diagnosis kit. We analyzed the fluorescence value distribution of these samples and calculate the ratio between the samples and the negative control. Boundary value was taken to cover over 99% of normal samples, initially identified as a normal reference value of the kit, and then combined with the ROC curve to determine the reference value range at last.1.3.2 Comparison of TRFIA and control kit ELISA:A total of 509 samples were measured by EBNA1 IgA TRFIA kit and control kit simultaneously. We compared the results obtained from two analysis method and calculated the negative and positive coincidence rates.II. Development of Time-resolved fluorescence immunoassay kit for detection of Zta IgA in human serum.2.1 Optimizing the production methods of Zta IgA TRFIA diagnostic kit2.1.1 Using indirect method developed Zta IgA time-resolved fluorescence immunoassay reagent.2.1.2 Preparation of Zta IgA negative control and positive control:the negative Zta IgA antibody samples detected by ELISA were diluted with dilution buffer, they were as negative control; the positive Zta IgA antibody samples were diluted with dilution, they were as positive control.2.1.3 Europium labeled antibody and purification:0.5mg anti-human IgA antibody was added to ultrafiltration tube (50KD), and centrifuged for 6min at 9000rpm. The antibody was washed six times using labeling buffer repeatedly. Then, we mixed the antibody and DTTA-Eu in 200μL of labeling buffer. The mixture was incubated at 25 ℃overnight with shaking. Labeled mouse anti-human IgA was separated from excess free chelates on a Sephadex G-50 column using elution buffer. Fractions were combined,1 ml/tube, then to measure the fluorescence value of each tube and combined the high value tube. The concentration of the europium labeled antibody were detected by BCA method. At last, 1‰ of BSA was added to the labeled antibody as protein agent. They were stored at-20 ℃ until required.2.1.4 Determination of coating concentration:in order to choose the optimum coating concentration of Zta antigen, we set a total of seven coating concentration, they were 0.5μg/ml, 1μg/ml,1.5μg/ml,2μg/ml,2.5μg/ml,3μg/ml,4μg/ml, respectively. At different coating concentration, we analyzed the fluorescence value of samples and controls. At the optimum coating concentration, the fluorescence of the samples and controls tended to be stable.2.1.5 Determination of Europium-labeled antibody dilution ratio:we diluted the labeled antibody with assay buffer to 1:400,1:600,1:800,1:1000,1:2000,1:4000, 1:6000. At different Eu3+-labeled antibody dilution ratio, there were differences in fluorescence between positive and negative samples. Under the condition of the low background luminescence value, we determined the optimum dilution ratio depend on the ratio of the fluorescence value of the positive sample and the negative sample.2.1.6 Determination of reaction time:under the determination of the other reaction conditions, we set 30min、45min、60min、90min、120min, respectively. We observe the fluorescence value of the negative and positive samples changing with the reaction time increase. At last, we chose the reaction time as the optimum one when fluorescence of the positive and negative samples tended to be stable.2.2 Evaluate the performance of Zta IgA time-resolved fluorescence immunoassay detection reagent.2.2.1 Precision test:We tested three samples (S1, S2, S3), we calculated the mean, standard deviation, intra-and inter-assay coefficient of variation of each sample.2.2.2 Specificity:We selected ten samples of CMV positive samples, herpes simplex virus type I positive samples, herpes simplex virus type Ⅱ positive samples and hepatitis B, hepatitis C positive samples, respectively. Then, we detected them using the TRFIA kit developed by us to evaluate the cross-reactivity.2.2.3 Interference test:Triglycerides, bilirubin, hemoglobin were added into negative and positive controls. Then, we tested these controls and calculated the means.2.3. Evaluate the clinical application performance of the kit2.3.1 Dtermination of the reference range:A total of 445 healthy human samples were tested by Zta IgA time-resolved fluorescence immunoassay kit. We analyzed the fluorescence value distribution of these samples and calculate the ratio between the samples and the negative control. Boundary value was taken to cover over 99% of normal samples, initially identified as a normal reference value of the kit, and then combined with the ROC curve to determine the reference value range at last.2.3.2 Comparison of TRFIA and control kit ELISA:A total of 501 samples were measured by Zta IgA TRFIA kit and control kit simultaneously. We compared the results obtained from two analysis method and calculated the negative and positive coincidence rates.Results:I. Development of Time-resolved fluorescence immunoassay kit for detection of EBNA1 IgA in human serum.The optimum coating concentration of NA1 antigen was 2.5μg/ml, the ratio of Eu3+-labeled antibody was determined as 1:2000, the reaction time was determined as 60min. Through analyzing the results of the 420 healthy human samples, we found that there cover over 99% of samples when the ratio between the sample fluorescence value of sample and negative control was 2.7. ROC curves showed that the sensitivity and specificity were 97.7% and 95.5%, respectively. Depended on the above results, we decided the cutoff value=the fluorescence value of negative control ×2.7. Inter-and intra CVs were 1.56%-4.99% and 3.92%-6.95%, respectively. No cross-reaction with CMV, HSV Ⅰ type, HSV type Ⅱ, HBV, HCV positive serum. Interference test showed that no interferences were detected from triglycerides, bilirubin and hemoglobin. Compared the results obtained from the TRFIA kit and ELISA kit, the results of two analytical method were highly consistent, which indicated that the EBNA1 IgA TRFIA reagent met the requirements of clinical testing.Ⅱ. Development of Time-resolved fluorescence immunoassay kit for detection of Zta IgA in human serum.The optimum coating concentration of Zta antigen was 2.0μg/ml, the ratio of Eu3+-labeled antibody was determined as 1:1000, the reaction time was determined as 60min. Through analyzing the results of the 445 healthy human samples, we found that there cover over 99% of samples when the ratio between the sample fluorescence value of sample and negative control was 3.1. ROC curves showed that the sensitivity and specificity were 99.2% and 93.6%, respectively. Depended on the above results, we decided the cutoff value=the fluorescence value of negative control ×3.1. Inter- and intra CVs were 2.45%-3.69% and 3.80%-4.80%, respectively. No cross-reaction with CMV, HSV Ⅰ type, HSV type Ⅱ, HBV, HCV positive serum. Interference test showed that no interferences were detected from triglycerides, bilirubin and hemoglobin. Compared the results obtained from the TRFIA kit and ELISA kit, the results of two analytical method were highly consistent, which indicated that the Zta IgA TRFIA reagent met the clinical requirements.Conclution:These results indicated that the EBNA1 IgA TRFIA reagent and the Zta IgA TRFIA reagent developed in this study had good performance, including precision, specificity and interference etc. they were all met the requirements of clinical application. It is expected to be used in clinical detection widely. |
PDF Full Text Request