| Avian leukemia is a general name for a variety of avian tumor diseases caused by avian leukemia virus of avian type C retrovirus family.Avian leukemia virus can spread vertically and horizontally,resulting in chicken growth retardation,egg laying decline,immunosuppression,and so on,which seriously affects the healthy development of poultry industry.In recent years,ALV infection and clinical incidence in chickens occur from time to time.The cost of ELISA and other commonly used methods is high and the extension scope is narrow.Therefore,it is of great practical significance for the prevention and control of AL to establish a new sensitive,specific,rapid and simple detection method of ALV Antigen and antibody.The p27 protein with a molecular weight of about 28KD was expressed in the prokaryotic cells p ET-30a(+)-p27 and purified by Sephadex column affinity chromatography.The immunogenicity of p27 protein was verified by Westernblotting analysis.Mouse ascites of ALV m Ab 2E5 and 3D5 were purified by protein A affinity chromatography column,and paired with another commercial ALV m Ab using colloidal gold immunochromatography method,so as to screen the antibody pairs that can be used for next experiment.In this study,a fluorescent microsphere immunochromatographic method for the detection of ALV Antigen was established.Based on the principle of“sandwich”pattern(antibody-antigen-antibody),to prepare the test strip of ALV,antigen fluorescent microspheres was prepared by coupling m Ab 2E5 with time-resolved fluorescent microspheres,coating the detection line(T)with m Ab 3D5 and coating the and quality control line(C)with Sheep anti-mouse Ig G.The optimal reaction conditions were as follows:The samples were diluted20 times with PBS,then 97?L sample coupled with 3?L fluorescent microspheres,incubated at room temperature for 5 mins,then the samples were added,and the results were read after15 mins.The sensitivity was 30 ng/m L after 10 times serial dilutionof p27 protein;by comparing ALV with NDV,Avian influenza,IBDV and IBV,it was found that ALV was positive and all the others were negative;the within-lot coefficient of variation was not higher than1.00%,and the coefficient of variation between batches was not higher than 2.37%.289clinicalsamples(including meconium and cotton swabs)were detected by the established method.Compared with PCR method,The overall coincidence rate with PCR method is99.31%.In this study,a fluorescent microsphere immunochromatographic method for the detection of ALV antibody was established.P27 protein coupled with fluorescent microsphereswas used as tracer antigen,rabbit anti-chicken Ig G as capture antibody and m Ab3D5 as quality control antibody,the immunochromatographic test strip of ALV antibody fluorescent microspheres was prepared.The optimal reaction conditions were as follows:The sample was diluted 50 times with PBS,97?L sample coupled with 2?L fluorescent microspheres,incubated at room temperature for 5 mins,then the sample was added,and the results were read after 15 mins.Compared with ELISA established in laboratory,the lowest detection level of both was 10-5;by comparing ALV serum with ND serum,Avian Influenza Serum and IBDV Serum,it was found that ALV serum was positive and all others were negative;The T/C value of the Test Strip did not change for 6 months;the repeatability test showed that the within-lot coefficient of variation was less than 1.03%,and the coefficient of variation between batches was less than 2.99%.61 clinical serum samples were collected for the detection of ALV antibody by the fluorescent microsphere immunochromatography method,Compared with commercial ELISA test kit,the coincidence rate was 93.44%.In conclusion,the fluorescent microsphere immunochromatographic Assay for Detection of ALV Antigen and antibody,which is sensitive,specific,rapid,simple and stable,can detect ALV infection quickly and effectively in chickens,it is suitable for rapid detection in chicken field and rapid diagnosis in veterinary clinic.It provides a new technical support for rapid detection of ALV antigen and antibody,and has important significance for prevention,control and purification of ALV in poultry industry in China. |
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