The study aimed to establish a rapid and simple assay to detect Shiga-like toxin IIe(SLT-IIe).Monoclonal antibodies(McAbs) against SLT-IIe A subunit were produced by using Escherichia coli(E.coli) expressed recombinant SLT-IIe A subunit protein as antigen to immunize BALB/c mice.A competitive ELISA for detecting antibodies against SLT-IIe was developed on the basis of recombinant protein expressed by E.coli and HRP-labelled McAbs against SLT-IIe A subunit.The optimum conditions of the ELISA were developed as following: the concentration of recombinant for coating ELISA plates was 0.32 μg·mL-1;the best dilution of serum to be tested was 1∶2;the working titer for HRP-labelled McAbs was 1∶3 200;and the inhibition rate above 40% was selected as the positive judging standard.A total of 60 serum samples were detected in parallel by both competitive ELISA and vitro neutralization assay.33.8% of them were detected as positive by competitive ELISA,while 30.9% of them were positive by vitro neutralization assay.The coincidence rate of the two assays was 88.2%.The results showed that the competitive ELISA had the advantages of higher sensitivity,specificity,reproducibility,stability and easy operation.It would be very useful in diagnosis,surveillance of SLT-IIe antibodies and epidemiological survey.

According to the Bordetella bronchiseptica fimD sequence(X75811) of GenBank,we designed a pair of primer,and 1 098 bp DNA fragment of fimD was amplified from swine Bordetella bronchiseptica identificated in our laboratory.The acquired PCR products were inserted into pET-28a and expressed in BL21(DE3).Results of Sodium docecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot assay showed that the fimD gene was expressed in the form of inclusion body,and the recombinant protein was about 42 kD,and can react with the swine antisera to Bordetella bronchiseptica.The fusion protein was purified and used as coating antigen to develop the indirect Enzyme-Linked Immunosorbent Assay(ELISA).When using this ELISA to detect 668 clinical sera,30.7% positive sera were detected.Parallel examination of the same 102 serum samples was conducted by using the ELISA and micro-agglutination test(MAT),the result showed that the ELISA is more sensitivity than MAT.

OBJECTIVE:We developed an indirect ELISA method for detecting Bordetella bronchiseptica (Bb) pertactin antibodies based on the recombinant pertactin protein expressed in Escherichia coli (DE3) strain.METHODS AND RESULTS:The prn gene encoding Bb pertactin was fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pGEX-prn. SDS-PAGE showed that the GST-PRN fusion protein was expressed in high level in BL21 carrying pGEX-prn. The strong reactivity of the GST-PRN fusion protein, specifically with antiserum against porcine Bordetellosis caused by Bb HH0809, was identified by Western blot. The recombinant protein fragment of rPRN was purified from the GST-PRN fusion protein digested by protease thrombin with the purity of 93.1%. The rPRN-based indirect ELISA was developed for detecting antibodies against PRN. The ELISA could detect positive samples in experimentally infected pigs fourteen days post inoculation and the degree of sensitivity was over 4 times higher than the latex agglutination test with the coating antigen of killed Bb. Thirty-two point seven percent of positive samples were detected in 1,229 clinical samples while no false positive results were found in detecting 7 antisera against porcine bacterial diseases. Sera samples from two bordetellosis-positive pig fields were tested by the indirect ELISA method and the results indicated that pigs were infected by Bb during the nursery periods.CONCLUSION:The assay showed excellent specificity, sensitivity and reduplication, and can be useful for epidemiological survey and clinical diagnosis of swine bordetellosis.

Monoclonal antibodies against Pasteurella multocida toxin(PMT)were produced using Escherichia coli expressed recombinant PMT proteins as immunogens.On the basis of the recombinant protein and HRP-lablled monoclonal antibody AH12 against Pasteurella multocida toxin,a competitive ELISA was developed to detect antibodies against Pasteurella multocida toxin. Optimium conditions of the ELISA were as following:the concentration of recombinant protein for coating ELISA plates was 223 ng/mL;the best dilution of serum was 1:2;The working titer for HRP-labelled monoclonal antibody AH12 was 1:3 200;More than 50% of inhibition rate was positive judging standard.Stability test showed that both inter-assay and intra-assay coefficient variation were below 10%.Both competitive ELISA and in vitro neutralization assay were used to detect 50 known and 82 unknown sera.The results indicated that the positive coincidence and the negative coincidence of 50 known sera were 90% and 100%,respectively; 40.2% of 82 unknown sera were positive detected by competitive ELISA,while 36.6% of them were positive by in vitro neutralization assay,and the coincidence rate was 91.5%.These results showed that the competitive ELISA possessed the advantages of high sensitivity,specificity,stability and briefness.It will be practical for pig diagnosis,immunity effect evaluation and epidemiological survey.

Monoclonal antibodies against FMDV vp2 protein were prepared and a competitive ELISA based on the monoclonal antibodies and vp2 protein was established. Balb/c mice were immunized with Escherichia coli expressed fusion protein. The splenocytes from immunized mice were fused with myeloma cells SP2/0. The hybridism cells were screened by indirect ELISA and limited dilution method. Two hybndoma cell Iines secreting mAbs against Asia I type foot-and-mouth disease were obtained. The titer and relative affinity of mAbs were determined by ELISA. Specificity of mAbs was analyzed by Western blotting. The ELISA titers of the ascites induced by the two hybridism cells were above 100 x 2(9).A competitive ELISA for the use of FMDV antibody detection was established using E. coli expressed fusion protein as coating antigen and HRP-labled mAb as detecting antibody. Clinical tests showed the method had 89.0 percent agreement with UBI Kit to detection of FMDV antibodies and 86.5 percent agreement with LPB- ELISA kit (Ceditest kit) for detection of antibodies against Foot-and-Mouth Disease Virus respectively.

On the basis of structural analysis of Escherichia coli Shiga-like toxin Ⅱe (SLT-Ⅱe) A subunit by TMpred software, the subunit A of SLT-Ⅱe was expressed in the soluble form which makes it easy to be purified. The strong reactivity of the recombinant SLT-Ⅱe subunit A (re-SLT-ⅡeA) was identified by Western blot. The expressed protein did not demonstrate the lethal effect to mice in mice infection experiment and cytotoxicity to Vero cells. Rabbit anti-serum against recombinant protein could neutralize, up to 90.4 % of efficacy, the cytotoxicity of natural SLT-Ⅱe to Vero cells in vitro. The mice immunized with recombinant SLT-Ⅱe A did not produce protective immunity after challenge with virulent Escherichia coli ED1 strain in mice model. The re-SLT-ⅡeA-based indirect ELISA was developed for detection of antibodies against the subunit A of SLT-Ⅱe. The ELISA could pick up the positive samples in infected pigs eight days post inoculation and 20.9% of positive samples were detected in 1 083 clinical samples while no false positive results were found in detection of nine antisera against porcine bacterial diseases.