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Development of Monoclonal Antibody-based Detection of
Mycoplasma Contamination in Cell Cultures
Pitchayapa Kaewchumpon, Naharuthai Inthasin, Nuttawan Kassaket, Jintapa Suesauy, Rawipas Saisuwan,
Wiwit Tantibhedhyangkul, Pattama Ekpo, Patimaporn Wongprompitak*
Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University
Background: *Corresponding Author E-mail: patimaporn.won@mahidol.ac.th
Abstract
Mycoplasma contamination affects cell culture integrity, resulting in unreliable experimental
outcomes and substantial financial losses. These wall-less bacteria evade common antibiotics
and filtration methods due to their small size and flexible membranes. Among six common
contaminating species (M. orale, M. arginini, M. hyorhinis, M. fermentans, M. salivarium,
and A. laidlawii), M. orale is the most prevalent in cell cultures. Current detection methods
face significant limitations: the gold-standard microbiological culture requires 28 days
of incubation, while molecular techniques like PCR demand specialized equipment and
technical expertise. The discovery of elongation factor thermo-unstable (EF-Tu) as a highly
expressed membrane protein in mycoplasmas provides an ideal target for developing rapid
antibody-based detection systems.
Methods: We generated monoclonal antibodies specifically targeting EF-Tu in M. orale. The antibodies’
specificity was validated through binding assays with M. orale lysates, and their detection
capability was assessed using ELISA and immunoblotting across a range of bacterial
concentrations (10⁶-10⁸ CFU/ml). The study focused on developing a simplified protocol
suitable for routine laboratory testing.
Results: The anti-EF-Tu monoclonal antibodies demonstrated specificity for M. orale, showing no
cross-reactivity with other bacterial contaminants. Both ELISA and immunoblotting assays
consistently detected M. orale in cell culture at all tested concentrations (10⁶-10⁸CFU/ml),
with detection limits comparable to existing PCR methods. The assays showed excellent
reproducibility across multiple experimental replicates.
Conclusion: This study establishes EF-Tu as a reliable target for mycoplasma detection and presents
a highly specific monoclonal antibody-based detection system. While the current format
shows good specificity, future development will focus on (1) creating a rapid lateral flow
format for point-of-use testing, (2) expanding the antibody panel to detect additional
mycoplasma species, and (3) validating the method in industrial cell culture applications.
This advancement promises to significantly improve quality control in biomedical research
and biopharmaceutical production by enabling frequent, reliable mycoplasma monitoring.
88 Joint Conference in Medical Sciences 2025