The Development And Application Of The Competitive ELISA Kit Based On Monoclonal Antibody Against Salmonella O₉ Lipopolysaccharide

Salmonella can cause a series of acute or chronic diseases in infected poultry,which is known as avian salmonellosis.The Salmonella infection can lead to the death of chickens,and the decrease in egg production of adult chickens,in addition,poultry products from chickens infected by Salmonella will become one of the important ways for human to infect Salmonella,which is not only a direct great economic losses to poultry industry,but also a great threat to human health.Because of these harms and hazards,the Chinese National Medium and Long-term Animal Epidemic Control Program(2012-2020)was launched in 2012,it clearly pointed out that Salmonella should be regarded as one of second class of microbes among animal epidemic pathogen,and would be the priority for prevention and control in China,and so the goal which avian salmonellosis should be cleared in large-scale poultry farms in China by 2020 was set up.In order to achieve this goal,the sensitive,accurate and rapid diagnostic methods for Salmonella infection in chicken are indispensable.PAT is a conventionally method for rapid detection of Salmonella antibodies in poultry in China.However its sensitivity and specificity are poor to easily cause false results,which is a main problem in the diagnosis of Salmonella infection.ELISA mothed is considered to be the better than PAT method because of the superiority in the sensitivity and specificity,although ELISA test is more sophisticated and expensive.Based on the monoclonal antibody against O9 antigen of Salmonella(in brief O9 McAb),we developed a competitive ELISA kit for the diagnosis of Salmonella in poultry,which has higher specificity and sensitivity than PAT and is expected to become one of avian Salmonella serological diagnosis methods.1.Development of a competitive ELISA method for monoclonal antibody against Salmonella O9 antigenIn the present study,ascites containing O9 McAb were prepared from 3-47-0 hybridoma cell line previously developed in our lab,and O9 McAb was purified and labeled with HRP.The titer of the HRP-labeled O9 McAb(HRP-O9 McAb)was up to 51,200.Lipopolysaccharide(LPS)was extracted from Salmonella Pullorum by hot phenol water method and used as a coating antigen.The concentration of the LPS was 484,31 ?g/mL by anthracene ketone colorimetry.Reaction conditions for the competitive ELISA were determined by the chessboard method,and an antigen concentration of 320ng/ml for coating,an HRP-O9 McAb concentration of 41.6ng/mL,and the dilution of serum of 1:4 were chosen.After optimization,the optimal conditions for the competitive ELISA included 4? and 24h for antigen coating,37? and 2h for HRP-O9 McAb binding,and 37? and 3mins for TMB substrate reaction.Inhibition rates of up to 94%and 89%were observed using this competitive ELISA method to detect positive serums of Salmonella Pullorum and Salmonella Enteritidis,respectively.Meanwhile,inhibition rates of positive serums of Escherichia coli and Proteus mirabilis by this competitive ELISA method were less than 10%,with significantly negative reactions.2.The development and application of the competitive ELISA kitAfter the optimization of competitive ELISA method,a kit of this competitive ELISA for detection of Salmonella O9 antibody was developed.The critical cut-off value between the positive and the negative of the detection kit was determined by the ROC curve method,the inhibition rate for positive serum was more than 38%,as cut-off value.According the standard of this test,the specificity of the kit reached up to 99.7%and the sensitivity reached up to 96.2%.By using Proclin300 as antiseptic and bacteriostatic agent and HRP protector,the kit can be kept in 4?condition for over 3 months,and maintain good detection effect in the validity period(C.V=1.37),which effectively solved the problem of long storage of ELISA kit.This competitive ELISA kit had good stability.The difference of precision among the kits with in same batch and between batches was small;the coefficient of variation was 6.84%and 9.30%.In double blind clinical trial,the coincidence rate of the detection result by different operators for the same batch of serum samples reached 100%.In addition,we compared the differences between this competitive ELISA kit and other poultry Salmonella detection kit,the results showed that the this kit had no significant difference in detection with the poultry Salmonella detection kit from France ID.vet company,and their coincidence rate was up to 98.1%,nevertheless,there was a little of difference compared to the traditional PAT,their coincidence rate was 88%.This competitive ELISA kit was applied to detect the serum of chicken by the live attenuated vaccine SKLZS06004?spiC.Positive results obtained with the kit could come out at the 21st day at the soonest,and the antibody in the serum was still kept at a high level after 56 days.This competitive ELISA kit was used to detect 776 serum samples from three different breeder farms in Jiangsu,Shandong and Hebei.The results showed average positive rate is 14.04%among this breeder farm,and the breeder farm in Hebei had the lowest positive rate 5.57%.