Development Of Colloidal Gold Test Strip For Detection Of Subgroup A Of Avian Leukemia Virus(ALV-A)

Avian leukemia subgroup A is an infectious neoplastic disease caused by avian leukosis virus subgroup A?ALV-A?,which mainly causes long-term viremia,growth retardation,immunosuppression,decreased production performance,tumors and death in chickens.The infection rate of the disease is very high in Chinese chickens,which is second only to the subgroup J avian leukemia,it causes huge economic losses to the chicken industry in China every year.There are no efcient vaccines or drugs to treat ALV to date culling positive chickens from breeder chicken flocks is the most effective measure in clinical practice.It is very important and necessary to develop sensitive and rapid detection methods for screening ALV-A positive chickens from breeding chickens.There are some traditional methods to detect ALV-A is cumbersome,requires special equipment,and takes long time,these detection methods cannot meet the requirements of clinical large-scale detection.This study aims to develop a new colloidal gold test strip mediated by monoclonal antibody?mAb?and polyclonal antibody?pAb?,and compares performance of the strips.To evaluate the applicability of test strips in clinical samples and provide scientific basis for clinical application.In this study,ALV-A gp85 protein was prepared by E.coli expression system.The protein producted after purifcation was inoculated into rabbits three times to product polyclonal antibody.ELISA and IFA were used to detect antibody titer and specificity respectively.The ALV-A monoclonal hybridoma cell line A14GZ prepared in laboratory was inoculated into Balb/c mice to prepare ascites,then the titer and specificity of the antibody were detected.The reaction conditions were optimized and the colloidal gold-labeled antibody solution was prepared by using a certain size of colloidal gold particles and monoclonal antibodies,polyclonal antibody coated nitrocellulose membrane?NC membrane?,assemble ALV-A colloidal gold test strip according to assembly steps of test strip.The specifcity of the colloidal gold test strip was evaluated using different strains of ALV subgroups,including ALVA,ALV-B,ALV-J,and ALV-K.The sensitivity of the test strips was assessed using purifed ALV-A gp85 protein and serially diluted cell supernatant samples containing ALV-A virus.The detection stability of the test strips at 4°C,25°C,and 37°C after180 d was evaluated.The developed test strips were used to test clinical cloacal swab samples,serum samples and egg album samples from layer chickens artifcially infected with ALV-A.These samples were also detected using IDEXX ALV P27 Antigen ELISA Kits to evaluate the accuracy of the test strip.The results showed that:?1?the size of the prepared ALV-A gp85 protein was 46 KDa,and the titers of the obtained polyclonal antibody and monoclonal antibody were 1:10⁶ and1:2¹³,respectively.The results of IFA and Western-blot showed two antibodies that both specifically bind to ALV-A and gp85 protein.?2?The size of colloidal gold particles in colloidal gold solution prepared by reduction of chloroauric acid and trisodium citrate was 20nm.Monoclonal antibody and polyclonal antibody were used as labeled antibodies and detection antibodies respectively.Optimum pH value of colloidal gold-antibody complex reaction system was 8.3.The optimum working concentration of antibody was 16.8?g/mL.?3?The test strip could specifically recognize ALV-A,but could not recognize ALV-B/J/K;the detection limit of ALV-A was 64 TCID₅₀/mL,and the detection limit of ALV-A gp85 protein was 80 ng/mL;it had good reactivity when stored at 4?for 180 days,at 25?for 60 days and at 37?for 15 days.?4?The results of comparison between the colloidal gold strip and the international commercial ELISA kit for ALV antigen showed that the positive and negative coincidence rates of cloacal swab samples were 94.06%?190/202?,blood samples were95.2%?119/125?and egg white samples were 95.31%?61/64?.;and the results of PCR showed that the false positive rate of test strip was much lower than that of ELISA.At the same time,the test strip has the advantages of rapid detection,low cost and no special equipment.These results indicate:the colloidal gold test strip of ALV-A was developed.The strip shows high specifcity,sensitivity and rapid detection of ALV-A,These features are very helpful for detection of ALV-A from many chicken tissue samples in avian clinical production.