intracellulariscultures. techniques to concentrate bacteria that are extracted from infected cell monolayers (3,6). During the routine cultivation ofL. intracellularisin the laboratory, the authors found that (i) inMycoplasma-contaminated cell cultures,Mycoplasmawas inevitably concentrated withL. intracellularis, (ii) the growth ofL. intracellulariswas inhibited byMycoplasmacontamination, and (iii)L. intracellulariswas eventually lost from the culture. Mycoplasmawas also concentrated during the passaging ofL. intracellularis, inducing cytopathic changes, including the death of cells required for the sustained growth ofL. intracellularis. This report describesMycoplasmacontamination ofL. intracellulariscultures that led to the unintended acquisition of a monoclonal antibody againstMycoplasmaspp. during the attempted generation of a monoclonal VX-222 antibody againstL. intracellularis. A strain ofL. intracellularis, PIA/MyCoyL1 (Animal, Plant, and Fisheries Quarantine and Inspection Agency [QIA], Republic of Korea), was used to generate a monoclonal antibody. Briefly, McCoy cells were infected with anL. intracellularissuspension. The passage of infected cells was performed using a 0.1% KCl treatment followed by removal of infected cells from the flask using a cell scraper (6). The scraped cells were ruptured by passing the infected cells six times through a 20-gauge needle. The resulting bacterial suspension was used to infect fresh McCoy cells in 25-cm2flasks. Infected McCoy cells were harvested on a weekly basis using the aforementioned technique to maintain theL. intracellularisculture. The bacterial suspensions were stored at 4C until they were used to challenge BALB/c mice later in the same day. The mice were primed and boosted twice every 3 weeks with an extractedL. intracellularissuspension combined with Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO). To PRKM10 generate hybridomas, spleen cells were harvested 3 days after the intravenous boost and fused with SP2/O myeloma cells using polyethylene glycol. The immunoperoxidase monolayer assay (IPMA) was used to screen the supernatant from hybridoma subclones. For the IPMA, theL. intracellularisculture plate (or slide) was fixed, incubated with the supernatant from the hybridoma subclones for 30 min at 37C, and washed 5 times with phosphate-buffered saline (PBS) (pH 7.2). Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG was diluted 1:600 in 1% bovine serum VX-222 albumin (BSA) in PBS and added at a concentration of 30 l/well. The plate was incubated for 45 min at 37C and then washed, and a chromogen (3-amino-9-ethyl-carbazole) solution was added to each well. The plate was then incubated at room temperature for 20 min. The plate was washed with distilled water 3 times, allowed to dry, and examined using an inverted light microscope.L. intracellularis-positive and -negative antisera were included on each plate as controls. The positive hybridoma subclones were preliminarily chosen based on IPMA staining data that matched those typically observed (6) withL. intracellularis-positive antisera (Fig. 1A). Several dozen monoclonal antibodies that targetedL. intracellulariswere developed. VX-222 However, two typically different patternsone pattern characteristic forL. intracellularisand one pattern characteristic forMycoplasmaspp.were observed using supernatants from some hybridoma subclones. Many of the granules were aggregates composed of several smaller particles in IPMA-stainedL. intracellularisculture plates (Fig. 1B). The particles were irregularly shaped, small, and round. In contrast, theL. intracellularisparticles were regular, large, and rodlike. We found that there was an accidental contamination of the inoculum used to immunize mice during the monoclonal antibody production process. We presumed that one hybridoma (F9) culture produced a monoclonal antibody of the IgG2b isotype againstMycoplasmaspp. A species-specific PCR assay that targeted a presumed mollicute-specific sequence of the 16S rRNA gene ofMycoplasmaspp. was used as previously described (10), and the amplified PCR product was sequenced. Importantly, the immunohistochemistry test using the F9 monoclonal antibody and the species-specific PCR assay enabled the labeled granules observed by light microscopy to be identified as morphologically distinct organisms, a result which allowed us to more confidently conclude that the granules detected under bright-field conditions were fromMycoplasmaspp. This F9 antibody was finally identified as a monoclonal antibody againstMycoplasma hyorhinisby using an immunohistochemistry test forMycoplasmaspp. in a cell culture and sequencing analysis. == Fig 1. == Immunoperoxidase and immunofluorescence assays forL. intracellularis(A and C) andM. hyorhinis(B and D). Red-labeledL. intracellularis(A) andM. hyorhinis(B). Fluorescently labeledL. intracellularis(C) andM. hyorhinis(D). Bars = 50 m. The detection of other contaminatingMycoplasmaspecies, such asMycoplasma oraleandMycoplasma fermentans, inL. intracellulariscultures stored in our laboratory was also possible with the IPMA method and the monoclonal antibody produced in this study. The monoclonal antibody reacted with 12 species ofMycoplasma(ATCC) which exhibited the staining pattern characteristic ofMycoplasmaspp. The detection of VX-222 12 otherMycoplasmaspecies, such asM. oraleandM. fermentans, was also possible using the immunofluorescence staining method. However, this antibody did not react withMycoplasma-free McCoy cells. Nonspecific staining was also not observed when this antibody was tested on other cell lines, such.