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Published on: September 7, 2018
Development of an enzyme-linked immunosorbent assay for serotyping ureaplasma urealyticum strains using monoclonal
F Echahidi1, G Muyldermans, S Lauwers
1Department of Microbiology, Academisch Ziekenhuis, Vrije Universiteit Brussel, Brussels, Belgium.
Abstract:
Ureaplasma urealyticum comprises 14 serotypes. The existing serotyping methods all use polyclonal antibodies. These methods are time-consuming and labor-intensive, and they cannot always be performed on primary isolates; in addition, the results are difficult to interpret. We developed a new enzyme-linked immunosorbent assay (ELISA) method using serotype-specific monoclonal antibodies (MAbs) to enable the serotyping of U. urealyticum isolates from primary broth cultures. Each of the 14 serotype reference strains was tested against 14 selected MAbs. Homologous reactions were very strong, while heterologous reactions were negligible. Three cross-reactions were observed: MAb 5 cross-reacted with serotype 2, MAb 14 cross-reacted with serotype 3, and MAb 8 cross-reacted with serotype 13. Despite the cross-reactions observed, all the serotype reference strains of U. urealyticum could be identified and differentiated using a combination of MAbs. Reproducibility was analyzed with a fractionated antigenic preparation and with several freshly prepared antigens of the same strain. No significant interrun variation was found with the fractionated antigen, but significant variations in optical density (OD) values were found when freshly prepared antigens were tested. However, the variation in OD values did not influence the overall interpretation of the ELISA: reactions with homologous MAbs were always prominent compared to those of the negative controls. This newly developed ELISA using MAbs seems promising for serotyping of U. urealyticum clinical isolates.
Insights
A new enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (MAbs) offers a faster and more accurate method for serotyping Ureaplasma urealyticum. This improved diagnostic tool can identify all 14 serotypes from primary cultures.
Area of Science:
- Microbiology
- Immunology
Background:
- Ureaplasma urealyticum presents 14 serotypes, traditionally identified using polyclonal antibodies.
- Current serotyping methods are laborious, time-consuming, difficult to interpret, and not always suitable for primary isolates.
Purpose of the Study:
- To develop a novel, efficient enzyme-linked immunosorbent assay (ELISA) for Ureaplasma urealyticum serotyping.
- To utilize serotype-specific monoclonal antibodies (MAbs) for improved accuracy and ease of use.
Main Methods:
- Developed an ELISA utilizing 14 serotype-specific MAbs against 14 Ureaplasma urealyticum reference strains.
- Tested homologous and heterologous reactions, noting three specific cross-reactions.
- Assessed reproducibility using fractionated and freshly prepared antigens.
Main Results:
- Strong homologous reactions and negligible heterologous reactions were observed.
- Identified cross-reactions between MAb 5/serotype 2, MAb 14/serotype 3, and MAb 8/serotype 13.
- Despite cross-reactions, a combination of MAbs enabled differentiation of all serotypes.
- ELISA demonstrated good reproducibility, with variations in fresh antigens not affecting overall interpretation.
Conclusions:
- The developed ELISA method using MAbs is a promising tool for serotyping Ureaplasma urealyticum clinical isolates.
- This method offers a more efficient and reliable alternative to existing polyclonal antibody-based techniques.
