Preparation Of Monoclonal Antibodies Against RHDV And Establishment Of An Antigen Capture ELISA

Rabbit haemorrhagic disease (RHD) is an acute fatal disease of rabbits, which was first described in China in 1984. It has been spread all over the world till now. It can cause high economic losses in domestic and commercial rabbits as well as high mortality in wild rabbits.Because RHD is a disease with short course and high mortality, a rapid and simple methods is necessary to clinical diagnosis and ealier period detection.There is no mean to detect antibodies because serological tests couldn't differentiate that the antibody is produced by infection or immunifaction,so the pathogenic detecting are playing important roles.But these pathogenic detecting methods ,including HA/HI, AGP, RT-PCR,are not fit for the field test for their shortages such as low sensitivity or high requirements of technique and equipments.Compared to all methods above,ELISA kit is sensitive,rapid and convenient to operate.So ,developing an ELISA kit detecting RHDV antigen means great significance for the diagnosis and control of RHD.In this test, BALB/c mices were immunized with RHDV local isolated strain named RHDV-TP. Six strains of hybridoma were obtained, which could secret monoclonal antibodies against RHDV.After the identification of these monoclonal antibodies, a McAb named DE₂ with high titre and affinity was selected to be capture antibody to establish a antigen capture ELISA.As the rabbit hemorrhagic disease virus has not been grown in any cell cultures, viruses can be concentrated from the liver.Firstly,big white rabbits were challenged with RHDV-TP strain.Livers from the dead rabbits were collected and homogenated. Then the virus was purified with differential centrifugation and sucrose density gradient centrifugation.After fusions between hybridoma SP2/0 cells and spleen cells from the immunized mice, antibodies in supernatant were detected by indirect ELISA which was established with the only capsid protein–VP60 expressed in insect cells. Six strains hybridoma were prepared and named as AD4,AG10,BC9,BE8,BH3,DE₂.Their antibody titres in ascites ranged from 1:4000 to 1:3×104 in ELISA tests.It was proved in additivity ELISA and Western-blot assays that these six McAbs could specificly identify five different epitopes of RHDV.One McAb named DE₂ with high titre and affinity could cohere with a linear epidope stably.These characters showed that the DE₂ McAb was capable to be a diagnostic reagent.An antigen-capture enzyme-linked immunosorbent assay (Ag-ELISA) for detecting rabbit hemorrhagic disease (RHD)virus was developed with a monoclonal antibody capturing virus ,while the rabbit anti-RHDV serum was used as the second antibody to identify virus. Working conditions of the Ag-ELISA were optimized and its capabilities were evaluated then. In the present study, the optimum working concentration of McAb was 1μg/mL and that of rabbit anti-RHDV serum was 4μg/mL. Using this test , several positive and negtive samples and 67 liver tissue samples from suspectable infected rabbits in local farms were detected.All the known positive samples were detected with positive reslults.62.7% of 67 liver tissue samples had positive results in contrast to 55.2% by HA test. Detection of five positive RHDV samples proved the highest dilution titer by ELISA was 3¹3 times higher than that detected by HA test.The detection limit of this assay is 26ng/mL to purified RHDV.These results showed that the Ag-ELISA based on the McAb-DE₂ could detect out RHDV from liver samples of the infected rabbits correctly. The Ag-ELISA displayed excellent specificity , sensitivity and repeatability.Also it's easy to operate and could save time and labour. The antigen-capture enzyme-linked immunosorbent assay was confirmed an excellent method for rapid diagnosis of RHD. In addition, 6 strains hybridoma cell secreting MAbs of RHDV stably obtained in this test would be used in the related study of RHDV ,such as screening the epitopes and discriminate detection ,in future.