Progress 10/01/20 to 09/30/21
Outputs
Target Audience:Mycoplasma infections continue to be a major concern for swine producers. The ELISA assay being developed is expected to meet the need for a reliable diagnostic assay for the detection of Mycoplasmas in pig production systems. The findings from this development project were presented at the Leman Swine Health conference, the American Association of Swine Veterinarians conference, and the National Veterinary Scholars Symposium in 2021. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided training opportunities for Rachel Bradley (Y2 DVM student University of Minnesota), How have the results been disseminated to communities of interest?Research findings from the project werepresented at the Leman Swine Health Conference (2021),AASV annual meeting (2021) and theNational Veterinary Scholars Symposium (2021) "Development of antigen-based diagnostic ELISA for Mycoplasma hyopneumoniae"Rachel Bradley, Venkatramana D. Krishna, Maria Pieters, Jian-Ping Wang, and Maxim C-J. Cheeran. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We generated several monoclonal antibodies specific to Mycoplasma hyopneumoniae using the hybridoma technique. The primary objective of this present study is to develop a simple, ELISA-based assay for direct detection of M. hyopneumoniae and validate its use with clinical samples obtained from the swine respiratory tract. Two hybridoma clones (MAb 2 and MAb 4) were selected for this study. Initial screening of antibody-producing hybridomas revealed 23 clones secreting antibodies to M. hyopneumoniae with absorbance values in a direct ELISA ranging from 0.5 to 4.00. Based on cross-reactivity studies, three clones that were specific to M. hyopneumoniae were selected. These antibody-producing clones showed no cross-reactivity to M. hyorhinis and M. flocculare antigens. Two of the hybridoma clones (MAb 2 and MAb 4) with the highest absorbance (>1.00 by ELISA), and reactive to different proteins of M. hyopneumoniae, assessed by Western blot, were sub-cloned by limiting dilution and used for further characterization. A sandwich ELISA method to detect M. hyopneumoniae antigens was developed using Protein-A purified monoclonal antibody (Mab 4) as a capture antibody and polyclonal rabbit anti-M. hyopneumoniae as the detection antibody. Monoclonal antibodies from hybridoma culture supernatants were purified using a Protein A IgG purification kit and used as the capture antibody. This sandwich ELISA was specific to M. hyopneumoniae and did not cross-react with M. flocculare, M. hyorhinis, Streptococcus suis, Pasturella multocida, Porcine reproductive and respiratory syndrome virus (PRRSv), or Influenza A virus. The lower limit of detection of this ELISA was found to be 226 ng/mL M. hyopneumoniae antigen in PBS and 453 ng/mL in spiked swine nasal swab samples. Using this newly developed assay, we tested laryngeal swab samples collected from 21 experimentally infected pigs before (0 days post-infection) and 28 days post-infection. The results showed that the sandwich ELISA could detect M. hyopneumoniae in infected pigs. Comparison of ELISA results with PCR showed only 9 out of 21 PCR positive samples were positive by ELISA indicating that the sandwich ELISA may be less sensitive than PCR. More tests are planned with field isolates and comparisons with culture methods for diagnosis. Both MAb 2 and MAb 4 hybridoma clones were sequenced to validate antibody expression and to determine the uniqueness of antibodies produced by these clones. Analysis of sequence obtained from Mab 2 and Mab4 using NCBI IgBLAST (an immunoglobulin variable domain sequence analysis tool) showed each antibody has a single unique clonotype confirming that they are monoclonal and their complementarity determining region 3 (CDR3) is unique. In addition, we also cloned the heavy chain and light chain of MAb 2 and MAb 4 in the eukaryotic expression vector for future recombinant antibody expression in mammalian cells. US Patents will be filed on these clones in the near future.
Publications
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