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Updated: May 2, 2026
Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae
Published on: February 24, 2023
Identification of Mycoplasma suis antigens and development of a multiplex microbead immunoassay
Ana M S Guimaraes1, Andrea P Santos, Jorge Timenetsky
11Joanne B. Messick, Department of Comparative Pathobiology, Purdue University, 625 Harrison Street, VPRB, West Lafayette, IN 47909. jmessic@purdue.edu.
Abstract:
The aims of the current study were to identify Mycoplasma suis antigens and develop a multiplex microbead immunoassay (MIA). A M. suis-expression library was screened for immunogens using sera from infected pigs. Based on bioinformatics, putative antigens were identified within positive inserts; gene fragments were expressed and purified as polyhistidine fusion proteins, and immunoreactivity was confirmed by Western blot. Selected antigens were used to develop a MIA. Sera from noninfected and infected pigs were used to set the median fluorescent intensity (MFI) cutoffs and as positive controls, respectively. Assay specificity was tested using sera from pigs seropositive for other pathogens (2 different pigs seropositive for each pathogen). Samples from 51 field pigs and 2 pigs during the course of acute (pig 1) and chronic (pig 2) infections were tested using MIA, indirect hemagglutination assay (IHA), and quantitative polymerase chain reaction (qPCR). Sixteen reactive plaques (52 genes) were detected. A heat-shock protein (GrpE), a nicotinamide adenine dinucleotide-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPN), and 4 proteins from paralogous gene families (PGFs) were identified as antigens by Western blot. While GrpE, GAPN, and 1 PGF protein were strong antigens, the others were not suitable as MIA targets. A MIA using GrpE, GAPN, and the strongly reactive PGF protein was developed. Cross-reactivity with sera from pigs infected with Mycoplasma hyopneumoniae, Porcine circovirus-2, Porcine parvovirus, Porcine reproductive and respiratory syndrome virus, and Porcine respiratory coronavirus with this MIA was not observed. Pig 2 was consistently positive by MIA and qPCR, whereas pig 1, initially negative, seroconverted before becoming qPCR positive. Only 2 samples (from pig 1) were IHA positive. Five (9.8%) field samples were qPCR positive and 40 (78.43%) were positive for all 3 MIA antigens; however, all were IHA negative. In summary, the MIA is specific and more sensitive than qPCR and IHA, providing simultaneous evaluation of antibody response to M. suis antigens.
Insights
This study identified key Mycoplasma suis antigens and developed a sensitive multiplex microbead immunoassay (MIA) for detecting pig antibodies. The new MIA is more accurate than existing methods for Mycoplasma suis diagnosis.
Area of Science:
- Veterinary Immunology
- Bacteriology
- Diagnostic Assay Development
Background:
- Mycoplasma suis causes significant economic losses in the swine industry.
- Accurate diagnostic tools are crucial for controlling M. suis infections.
- Existing diagnostic methods like IHA and qPCR have limitations in sensitivity and specificity.
Purpose of the Study:
- To identify novel Mycoplasma suis antigens for diagnostic purposes.
- To develop and validate a multiplex microbead immunoassay (MIA) for detecting M. suis antibodies.
- To compare the diagnostic performance of the developed MIA with existing assays (IHA, qPCR).
Main Methods:
- Screening of an M. suis expression library to identify immunogenic proteins.
- Expression and purification of candidate antigens (GrpE, GAPN, PGF proteins).
- Development of a MIA using selected antigens and validation with pig sera and field samples.
Main Results:
- Identified heat-shock protein (GrpE), glyceraldehyde 3-phosphate dehydrogenase (GAPN), and a paralogous gene family (PGF) protein as key M. suis antigens.
- Developed a specific MIA with no cross-reactivity against other common swine pathogens.
- The MIA demonstrated higher sensitivity and specificity compared to indirect hemagglutination assay (IHA) and quantitative polymerase chain reaction (qPCR).
Conclusions:
- The developed MIA is a highly specific and sensitive tool for simultaneous detection of antibody responses to multiple M. suis antigens.
- This novel MIA offers improved diagnostic capabilities for Mycoplasma suis infections in pigs.
- The MIA can aid in better disease management and control strategies for M. suis.
