Journal of Immunological Methods, 131 (1990) 203-212 Elsevier
203
JIM05641
Fast and simple procedure for the detection of cell culture mycoplasmas using a single monoclonal antibody * R o m a n Blazek, K a r l Schmitt, U t e K r a f f t a n d U l r i c h H a d d i n g Institutefor MedicalMicrobiologyand Virology,Heinrich-Heine-University,4000Diisseldorf F..R.G. (Received2 October 1989, revisedreceived16 February 1990, accepted 12 April 1990)
The detection of mycoplasmas in cell cultures is still a problem, especially in those laboratories in which the detection and identification of microorganisms is not established as a routine procedure. In our laboratory, monoclonal antibodies (mAbs) have been prepared to Acholeplasma laidlawii, Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma arginini and Mycoplasma salivarium. 30 mAbs were obtained and one of these, designated CCM-2, was shown to bind to all five mycoplasma species. It also bound to Mycoplasma fermentans, Mycoplasma hominis and to all wild types (n = 54), isolated from cell cultures submitted to our laboratory. The mAb was used in a immunofluorescent assay (IF) and the method correlated with the microbiological assay. Using this mAb immunofluorescent staining of cells is a fast and simple procedure for mycoplasma detection in cell cultures.
Key words: Cell culture mycoplasma; Mycoplasmadetection; Monoclonal antibody to mycoplasmas
Introduction Since a wide range of biological, immunological and medical research has become dependent on cells growing in vitro, good cell culture conditions are of great importance if valid and reproducible results are to be obtained. Mycoplasma infections of continuous cell lines remain a serious problem; these organisms may not be obvious with ordinary light microscopy and they may not overgrow and kill the cells as do bacteria and fungi. For that
Correspondence to: K. Schmitt, Institut fiir Medizinische Mikrobiologie und Virologie, Heinrich-Heine-Universit~it, Moorenstrasse 5, 4000 Diisseldorf, F.R.G. * This work contains essential parts of the MD thesis of Roman Blazek
reason mycoplasmas can persist undetected in cell cultures for months. Since they are able to alter a great variety of biologic, immunologic and biochemical properties of cells, mycoplasmas are a source of artifacts and false interpretation of results (McGarrity et al., 1984). Although there are several methods to detect mycoplasmas in cell cultures, most of them give unreliable results when not used by experts e.g., the detection of mycoplasmas by microbiological culture media (Chen, 1977). To be sensitive enough, some methods are dependent on an indicator cell line, such as D N A staining with diamidinophenolindol (DAPI) or Hoechst stain 33258 (Russel et al., 1975; Chen, 1977) and mycoplasma mediated cytotoxicity using 6-methylpurine desoxyriboside (6MDPR) (McGarrity et al., 1982, 1986). Thus, it may take several days to obtain results. Except for the detection of cell culture mycoplasmas by genetic probes (McGarr-
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (Biomedical Division)
204 ity et al. 1986), most of the methods take too long and are too complicated to be used in laboratories which are inexperienced in mycoplasma research. The purpose of this study was to generate mAbs to cell culture mycoplasmas and to investigate whether these could be used for the detection of such contaminants. In studies reporting the identification of mycoplasmas which were obtained from cell cultures the species Mycoplasma orale, Mycoplasma argininL Mycoplasma hyorhinis, Acholeplasma laidlawii and Mycoplasma salivarium accounted for 96% of the isolates (McGarrity et al. 1985). With this in mind we prepared mAbs to these five mycoplasma species and tested them for their potential use in the diagnosis of these mycoplasmas. The most important outcome of the present study was that one of the mAbs generated was able to bind to all of the common mycoplasma contaminants of cell cultures. This mAb was designated CCM-2 (CCM: cell culture mycoplasmas) and was used in IF tests for the organism.
Materials and methods
Mycoplasmas The following five mycoplasma species were used for immunization: A. laidlawii (NCTC 10116: from the National Collection of Type Cultures, London, U.K.), M. hyorhinis (ATCC 17981: from the American Type Culture Collection, Rockville, MD), M. orale (ATCC 23714), M. arginini (NCT C 10129) and M. salivarium (NCTC 10113). Additional species were used for subsequent assays, namely M. pneumoniae strain FH, M. fermentans (ATCC 19989), M. gallisepticum (ATCC 15302) and M. hominis (NCTC 10111). The source of these mycoplasma species and the components of the media for their propagation have been described previously (Schmitt et al., 1988). 200 ml broth cultures in logarithmic phase were centrifuged three times at 15,000 x g for 30 min, the pellets were washed in PBS and the final suspension of antigen was adjusted to a protein concentration of 1 mg/ml. Depending on the mycoplasma species the preparation, which was usually stored at - 70 o C, contained 108-109 cfu/ml.
Immunization Female BALB/c mice 3-4 months of age were immunized intraperitoneally. The initial immunization was carried out with 200 /xg of the mycoplasma protein, emulsified in complete Freund's adjuvant (CFA, Difco Laboratories, Detroit, MI, U.S.A.). The four subsequent booster injections of 100 ~tg of antigen without CFA were given every 2 weeks. Fusion and selection of hybridomas 3 days after the last immunization the spleen cells were fused with the myeloma cell line Xg3 Ag8 653 using a procedure initially described by K~Shler and Milstein (1975) and modified by Fazekas De St. Groth and Scheidegger (1980). After fusion the cells were distributed into 96-well microtiter plates (Greiner, Niirtingen, F.R.G.). The supernatant fluids of the wells with proliferating hybridomas were tested for antimycoplasma antibodies by ELISA. The hybridomas from positive wells were cloned by limiting dilution and after retesting for mAb they were expanded into bulk cultures and frozen in liquid nitrogen. The supernatants of these cultures were collected and used for further characterization of the mAbs. Ascites Ascitic fluid was produced in BALB/c mice following an intraperitoneal injection of 0.5 ml pristane (Aldrich-Chemie, Steinheim, F.R.G.). After 2 weeks the mice were injected with bulk cultures of cloned hybridomas (app. 5 x 106 cells). Ascitic fluid was withdrawn after 10-14 days. Conjugation of mAbs with biotin succinimide ester The mAbs for conjugation were purified by ammonium sulphate fractionation and FPLC ionexchange chromatography. The mAbs were conjugated as described by Goding (1983). ELISA Freeze-thaw disrupted mycoplasmas were diluted to give 20/~g/ml of protein in coating buffer (borate-buffered saline (BBS), 50 mM NanB407 + 150 mM NaC1, pH 9.0). 30/~1 volumes were then applied to each well of a microtiter plate (Falcon, Dickinson Labware, Oxnard, U.S.A.) and incubated for 60 min at room temperature (RT).
205
bodies (Dianova), (1 h for each incubation). The NC was rinsed between the steps three times with PBS. The developing solution consisted of 30 mg 4-chloro-l-naphthol in 10 ml methanol, 50 ml TBS (TBS: 50 mM Tris, 200 mM NaC1, pH 7.5) and 25 /~1 30% H202.
Free binding capacity was blocked with 1% bovine serum albumin (BSA) in PBS for 2 h. 30 /~1 of antibody solution were added per well and left to incubate for 1 h. The bound antibody was detected by goat anti-mouse peroxidase-labelled antibodies (Dianova, Hamburg, F.R.G., 1/2000, 20 /zl/well) and the substrate ABTS (Boehringer, Mannheim, F.R.G.) + H202. In order to determine the antibody class of the generated mAbs, class-specific peroxidase-labelled antibodies to IgG1, IgG2a, IgG2b and IgG3 were used (Dianova). In order to detect cross-reactivity of the mAbs with the PPLO-broth medium the ELISA was also performed with the medium as antigen at a dilution of 1/5 in BBS.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting (Western blot) The mycoplasma preparation (1 mg/ml of protein) diluted 1/2 in sample buffer (2% SDS, 5% mercaptoethanol, 12.5% glycerol, 0.5 mM Tris, pH 6.8) was heated at 100 °C for 5 min. Electrophoresis was then performed by the method of Laemmli (1970) with 9% or 12% acrylamide gels using appropriate molecular weight markers (Bio-Rad, Munich, F.R.G.). Nitrocellulose filter blots were prepared by the procedure of Towbin et al. (1979), BS was applied and the assay completed as previously described for IB.
Immunobinding assay (IB) This IB assay was modified from the procedure described by Kotani and McGarrity (1985). The assay was performed at room temperature with agitation. 5 btl of antigen suspension (app. 1 mg/ml) were applied as a spot to a nitrocellulose sheet (NC, Schleicher & Schiill, Dassel, F.R.G.) and left to dry. The NC was incubated successively in a blocking solution (BS: 5% dry nonfat milk in PBS), then with undiluted supernatants from hybridoma producing mAbs or alternatively with ascitic fluid diluted 1/1000 in BS, and finally with rabbit anti-mouse peroxidase-labelled anti-
Trypsin treatment of mycoplasma antigen A preparation of M. hominis (1 mg/ml, clinical isolate no. 25) was treated with trypsin (250/~g/ml, Sigma, Daisenhoven, F.R.G.) for time periods ranging from 1 s to 15 h. The reaction was terminated with trypsin inhibitor (750 /tg/ml, Roth, Karlsruhe, F.R.G.) and the samples prepared for subsequent immunoblotting.
TABLE I SPECIFICITY
O F 30 G E N E R A T E D
M y c o p l a s m a species as a n t i g e n
mAbs, TESTED IN ELISA
R e a c t i v i t y o f the m o n o c l o n a l a n t i b o d i e s 2219h 2220h 2222 h
22241 2226 j 2228 I
2207° 2206° 2202 o 2208 o
2209a 2210a 2211a 2214 a
2231s 2232s
22251
22271
2199° 2201 o
2198 °
22231
2205 ° 2203 o
22291
2200° 2221 h
+
+
+
-
+
+
2215 a 2216 a 2217 a M. A.
hyorhmis laidlawii
+
.
-
+
.
. -
. -
.
g.
orale
-
-
+
-
-
g.
arginini
-
-
-
+
.
M.
salivarium
.
.
.
.
mAbs were obtained from mice immunized with
.
-
+ h M.
hyorhinis,
.
+ .
. + I A.
.
.
+
-
+
+
. laidlawii,
+ o M.
. -
orale,
+
+
+
+
+
+
-
+
-
+
-
+
+
+
+
a
M.
arginini,
s M.
salivarium.
206
Triton X-114 phase partitioning of mycoplasma antigen Using a slight modification of the method of Bordier (1981) which was applied to M. hyorhinis by Riethman et al. (1987), a preparation of M. orale (1 m g / m l of protein) was separated into hydrophobic, hydrophilic and nonsoluble fractions. Three repetitions of the phase fractionation resulted in fractions of sufficient purity for subsequent SDS-PAGE and immunoblotting. IF performed with fixed cells Cell suspensions to be assayed for mycoplasmas were applied without any kind of pretreatment as a spot (50 /~1) to coverslides. The slides were dried at 50-60 ° C for 45 min. The cells were then fixed and permeabilized with ethanol (1 min in 70% ice-cold ethanol/water). In order to avoid subsequent nonspecific staining, the slides were not allowed to dry out after fixation (Goding, A M
1
2
3
4
5
6
7
M
1983). The cells were counterstained with Evans Blue (0.005% in H20, 30 min at 37°C) and then exposed to antimycoplasma antibodies for 60 min (50/~l/cover slide of either undiluted supernatant fluid or ascites 1/1000 in BS + 10% FCS or purified mAb conjugated with biotin in BS + 10% FCS). The slides were rinsed three times in PBS. Depending on whether unconjugated or biotinconjugated mAbs were used, the bound antimycoplasma antibodies were detected by either FITC-conjugated goat anti-mouse Abs or FITCconjugated streptavidin (Dianova). After three washes in PBS the coverslides were airdried and mounted on slides for microscopy with Fluoroprep (BioMrrieux, Charbanirres-lesbains, France). For the examination of the slides a Zeiss Photomicroscope-III, equipped for light fluorescence microscopy, was used. A total of 500 × (40 × 12.5) magnification was generally sufficient for the detection of mycoplasmas. In cell cultures
1
2
3
B 4
5
6
7
MW x 10 - 3
-- 200 --116
--97
--66
43
t --
31
Fig. 1. Reactivity of mAb CCM-2 with common cell culture mycoplasmas in an immunoblot. A: patterns of mycoplasma proteins separated by SDS-polyacrylamide (9%) gel electrophoresis and stained with Coomassie Blue. B: immunoblots of the separated mycoplasma components prepared from an identical gel to that in A and probed with the mAb CCM-2 (ascitic fluid 1/1000 in BS). The bound mAb was detected by goat anti-mouse peroxidase-labelled antibodies. Lanes: M, molecular weight markers; 1, M. fermentans; 2, A. laidlawii; 3, M. hyorhinis; 4, M. orale; 5, M. arginini; 6, M. salivarium; 7, M. hominis.
207
contaminated with only a few mycoplasmas, a total of 800 × (64 × 12.5) magnification was used.
IF performed with unfixed cells For this procedure mycoplasma infected and mycoplasma free AtT-20 cells (ATCC-CCL89) adherently grown on coverslides were stained with mAbs and with FITC-conjugated goat anti-mouse Abs (1 h for each incubation time). They were then fixed with 2% formaldehyde in PBS for 10 min, mounted and examined as above.
Results
A total of 30 stable hybridoma producing IgGclass antibodies to mycoplasmas were obtained after five fusions. The reactions of the mAbs generated are detailed in Table I. Out of the 30 mAbs, 19 were monospecific. The remaining 11 mAbs were able to bind to two or
more species. In contrast to a similar study by Buck et al. (1982), we decided to investigate both groups of mAbs. Ten out of the 19 mAbs, which appeared species-specific in ELISA, were selected for further studies, particularly the identification of mycoplasmas isolated from cell cultures. One mAb of IgG1 isotype described in Table I as mAb no. 2200 and later designated as CCM-2 became the main subject of the present study. As Table I shows, this mAb reacted with all five mycoplasma species. Ascitic fluid was produced and the antibody further tested by immunoblotting using two additional mycoplasma species: M. hominis and M. fermentans. The epitope recognized by the mAb CCM-2 was present on a molecule with an apparent molecular weight of 42,000-47,000, depending on the mycoplasma species. Except for M. hyorhinis and A. laidlawii, all of the mycoplasma species also showed additional components of lower molecular weight (Fig. 1). B
A M
1
2
3
4
5
6
1
2
3
4
5
6
MWxl0
-3
116
J--N 97
--66
--
43
:/!iiiill i!ii ....i:!;ii:i:i~ ii i;i!
m31 !ii!il:!!!! :~i
.... i !iiiii~ii
--22
Fig. 2. Electrophoretic patterns and immunoblot of trypsin treated and untreated M. hominis antigen. A: components of M. hominis antigen separated by SDS-PAGE (12% gel stained with Coomassie Blue). B: immunoblot from the separated components using the mAb CCM-2. Lanes: M, molecular markers; 1, untreated M. hominis antigen; 2-6, M. hominis treated with trypsin (250 # g / m l ) for 1 s (lane 2), 20 s (lane 3), 1 min (lane 4), 2.5 h (lane 5), 15 h (lane 6).
208 For further epitope analysis trypsin treatment and Triton X-114 phase fractionation of the mycoplasma antigen preparation were performed. The 44 kDa component of M. hominis antigen showed decreased staining with mAb CCM-2 after brief treatment with trypsin. This was accompanied by the appearance of a new immunoreactive band with an estimated molecular weight of 36,000. Incubation times up to 15 h resulted in significantly diminished staining of the 44 kDa component and the complete removal of the new 36 kDa band. The epitope bearing component was shown to be trypsin sensitive (Fig. 2) and in addition, to partition into the aqueous phase (Fig. 3). In order to use the mAb CCM-2 in IF, the assay was first performed on unfixed cells: AtT-20 cells were permitted to adhere to cover slides and then infected with M. orale. After 48 h the unfixed cells were subjected to the immunofluorescent
M
1
A 2
staining procedure using anti-M, orale mAb 2208 as a positive control. Since the mAb CCM-2 failed to stain unfixed mycoplasmas the epitope was assumed to be expressed inside these bacteria. Therefore subsequent IF testing was performed on ceils fixed and permeabili.zed with ethanol. For this test the mycoplasma free cell line Xg3 Ag8 653 was separately infected with six mycoplasma species: M. fermentans, M. hyorhinis, M. orale, M. arginini, M. salivarium and M. horninis. After 2-3 cell passages, immunofluorescent staining of the contaminated cells was performed. In all cases except the negative control (mycoplasma free cell line), a bright fluorescence was seen, localized either on the cells, between the cells or on the glass, depending on the contaminating mycoplasma species (Fig. 4). Previous experiments demonstrated the reactivity of mAb CCM-2 with several mycoplasma
B
3
4
1
C
2
3
1
2
3
MWxlO
-3
--116 -- 97
--
l
g
v
66
-43
--31
Fig. 3. Electrophoretic patterns and immunoblot of Triton X-114 phase partitioned M. orale antigen. A: componentsseparated with SDS-PAGE (12% gel stained with Coomassie Blue). B: immunoblot of the separated components incubated with mAb CCM-2. C: immunoblot incubated with the anti-M, orale mAb 2208 which bound to a detergent phase antigen and served as control for the phase partition experiment. Lanes: 1, untreated M. orale antigen; 2, detergent phase; 3, aqueous phase; 4, insoluble proteins.
0
0
210
species originating from the American Type Culture Collection, or from the National Collection of Type Cultures. In the following assays m A b CCM-2 was tested for binding to mycoplasmas isolated from cell cultures and clinical specimens. In view of the large number of isolates to be tested, IB was chosen as the most advantageous method. Over a period of 3 years cell cultures submitted to our laboratory were assayed for mycoplasmas by the cultivation method in PPLO broth and under anaerobic conditions on agar. 54 wild type mycoplasmas were isolated and collected to be used as antigen for lB. MAb CCM-2 produced a positive reaction with all of them.
TABLE
II
DETECTION STUDY
OF MYCOPLASMAS
ON 20 DIFFERENT
Cell lines tested
IN CELL
CELL
CULTURES,
LINES
Method used
for mycoplasmas
IF
Culture A
D
Agar
Human Cell lines B cell l i n e s : R a j i
+
+
-
+
Griese B
+
+
-
+
FH-B
.
-
+
.
.
.
.
Winkler B
+
Darius BLCL
.
MP-B
+
+
-
+
B JAB
+
+
-
+
T cell l i n e s : M o l t 4
Macrophage
. + .
+
+
-
+
CEM
+
+
-
+ +
L 428
+
+
-
Jurkat
+
+
-
+
H u t 78
+
-
+
+
B9
+
+
-
+
K 562
+
+
-
+
+
+
-
+
C o l o n i c c a r c i n o m a cell l i n e : DETA
+
+
-
+
-
+
-
+
Mouse cell lines B cell l i n e :
L 1210
+
T cell l i n e s :
3 D O 54.8
.
.
.
.
BW 5147 cell l i n e :
.
.
.
.
Mastocytoma
P 815-JEK
+
IF:
immunofluorescent
medium
Discussion
cell l i n e : U 937
broth
Another IB test was carried out with 186 mycoplasma isolates from clinical specimens, delivered to the Department of Medical Microbiology, University of Mainz. 146 of these isolates were identified by antisera as M. hominis. MAb CCM-2 reacted with all 186 isolates. When using the m A b CCM-2 for the diagnosis of cell culture m y c o p l a s m a s in the immunofluorescent assay described above, it is important that the m A b does not crossreact with uncontaminated cell cultures. For testing a great variety of cells for crossreactivity with the mAb CCM-2 in a single experiment, the IF assay was performed with h u m a n peripheral blood leucocytes (PBL) and mouse spleen cells (SC). PBL and SC were infected with M. hominis and incubated for 24 h in order to provide a positive control. Except for the positive control, no fluorescent staining of the blood cells occurred. At the next stage the m A b CCM-2 employed in the IF assay was used to diagnose mycoplasma contamination of cell cultures. Anaerobic incubation of agar and broth media, routinely used in our laboratory for the detection of mycoplasmas, was chosen as the reference method. As shown in Table II, 20 different cell lines submitted to our laboratory were assayed. All 20 cell lines originated from one laboratory, in which the cells were screened for mycoplasmas for the first time. 16 cell cultures were positive for mycoplasmas by the IF assay and this result correlated with the microbiological assay. The cells were retested 2 months later. They were still positive for mycoplasmas in both tests.
medium
+
assay with mAb
supplemented
supplemented
+
CCM-2.
with arginine.
with dextrose.
D:
A: PPLOPPLO-broth
The frequency of mycoplasma contaminated cell cultures - about 15% of cell lines are believed to be infected (McGarrity et al., 1985) - and the quick spread of the infection within the laboratory imposes a requirement for regular screening of continuous cell lines. The immunofluorescence assay described could be carried out in most cell culture laboratories. The m A b CCM-2 was shown to bind to the following mycoplasma contaminants of cell cultures: M. fermentans, A. laidlawiL M. hyorhinis, M. orale, M. argininL M. salivariurn
211
and M. hominis. Together, these account for more than 96% of the reported isolates (McGarrity et al., 1985). Furthermore, the mAb reacted with all 54 wild types isolated from contaminated cell cultures. In addition the mAb CCM-2 reacted with all tested (n = 186) clinical mycoplasma isolates, of which 146 were identified as M. hominis, a very heterogenous mycoplasma species (Andersen et al., 1987). When an immunobinding assay was performed with M. pneumoniae strain F H and M. gallisepticum (ATCC 15302) as antigen (data not shown), it became clear that the mAb CCM-2 did not bind to these two mycoplasma species. However, since M. pneumoniae and M. gallisepticum are almost never isolated from cell cultures, this result does not seriously detract from the value of this mAb for the detection of most cell culture mycoplasmas. In a 9 year study (Pollak-Vogelzang et al., 1987) only two isolates of M. pneumoniae accounted for 0.1% of the isolates and they were detected in hybridoma cultures originating from one laboratory. The antigen recognized by the mAb CCM-2 was shown to be trypsin-sensitive and to partition efficiently into the aqueous phase following Triton X-114 fractionation. Moreover, the mAb was not able to bind to native mycoplasmas in IF tests. These results suggested that the epitope is comprised of, or linked to a trypsin-sensitive polypeptide, neither exposed on the surface of the mycoplasmas, nor associated with integral membrane proteins, i.e., a cytoplasmic component. The reaction of antibodies with antigens localized within cells requires fixation of the cells in order to render them permeable to proteins (Goding, 1983). In preliminary studies ethanols fixation was found to be optimal for fluorescence staining of mycoplasmas. A troublesome technical aspect was nonspecific staining of fixed cells by the secondary antibody but this artifact was virtually eliminated if the cells were not allowed to dry after fixation and counterstained with Evans Blue. Even better results were obtained using purified and biotin-conjugated mAb CCM-2. The advantage of this was that the biotin-conjugated mAb could be used to screen hybridomas producing mouse immunoglobulin.
The IF correlated with microbiological assay, but more cell cultures should be tested. The high sensitivity of the test was due to the fact that the IF revealed even single mycoplasmas on the cell surface. N o indicator cell line was necessary and therefore the complete IF could be performed in about 5 h. Thus the major advantages of the assay are sensitivity, simplicity and rapidity.
References Andersen, H., Birkelund, S., Christiansen, G. and Freundt, E.A. (1987) Electrophoretic analysis of proteins from Mycoplasma hominis strains detected by SDS-PAGE, two dimensional gel electrophoresis and immunobinding. J. Gen. Microbiol. 133, 181. Bordier, C. (1981) Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem. 256, 1604. Buck, D.W., Kennett, H.R. and McGarrity, G. (1982) Monoclonal antibodies specific for cell culture mycoplasmas. In vitro 18, 377. Chen, T.R., (1977) In situ detection of mycoplasma contamination of cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res. 104, 255. Fazekas De St. Groth, S. and Scheidegger, D. (1980) Production of monoclonal antibodies: Strategy and tactics. J. Immunol. Methods 35, 1. Goding, J.W. (1983) Monoclonal Antibodies: Principles and Practice. Academic Press, London, p. 223. KiShler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibodies of predefined specifity. Nature 256, 495. Kotani, H. and McGarrity, G.J. (1985) Rapid and simple identification of mycoplasmas by immunobinding. J. Immunol. Methods 85, 257. Laemmfi, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. McGarrity, G.J. and Carson, D.A. (1982) Adenosine phosphorylase-mediated nucleoside toxicity. Exp. Cell Res. 139, 199. McGarrity, G.J. and Kotani, H (1985) Cell culture mycoplasmas. In: S. Razin and M.F. Baffle, (Eds.), The Mycoplasmas, IV, Mycoplasma Pathogenicity. Academic Press, Orlando, FL, p. 383. McGarffty, G.J. and Kotani, H. (1986) Detection of cell culture mycoplasmas by a genetic probe. Exp. Cell Res. 163, 273. McGarrity, G.J., Vanamann, V. and Sarama, J. (1984) Cytogenic effects of mycoplasmal infection of cell cultures: A review. In Vitro 20, 1. McGarffty, G.J., Kotani, H. and Carson, D. (1986) Comparative studies to determine the efficiency of 6-methylpuffne desoxyriboside to detect cell culture mycoplasmas. In Vitro Cell. Dev. Biol. 22, 301.
212 Pollak-Vogelzang, A.A., Brugman, J. and Reijgers, R. (1987) Comparison of two methods for detection of mollicutes (Mycoplasma and Acholeplasma) in cell cultures in the Netherlands. Antonie Van Leeuwenhoek, J. Microbiol. 53, 107. Riethman, H.C., Boyer, M.J. and Wise, K.S. (1987) Triton X-114 phase fractionation of an integral membrane surface protein mediating monoclonal antibody killing of Mycoplasma hyorhinis. Infect. Immun. 55, 1094. Russel, W.C., Newman, C. and Williamson, D.H. (1975) A simple cytochemical technique for demonstration of DNA
in cells infected with mycoplasmas and viruses. Nature 353, 461. Schmitt, K., D~iubener, W., Bitter-Suermann, D. and Hadding, U. (1988) A safe and efficient method for elimination of cell mycoplasmas using ciprofloxacin. J. Immunol. Methods 109, 17. Towbin, H., Stachelin, T. and Cordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350.
203
JIM05641
Fast and simple procedure for the detection of cell culture mycoplasmas using a single monoclonal antibody * R o m a n Blazek, K a r l Schmitt, U t e K r a f f t a n d U l r i c h H a d d i n g Institutefor MedicalMicrobiologyand Virology,Heinrich-Heine-University,4000Diisseldorf F..R.G. (Received2 October 1989, revisedreceived16 February 1990, accepted 12 April 1990)
The detection of mycoplasmas in cell cultures is still a problem, especially in those laboratories in which the detection and identification of microorganisms is not established as a routine procedure. In our laboratory, monoclonal antibodies (mAbs) have been prepared to Acholeplasma laidlawii, Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma arginini and Mycoplasma salivarium. 30 mAbs were obtained and one of these, designated CCM-2, was shown to bind to all five mycoplasma species. It also bound to Mycoplasma fermentans, Mycoplasma hominis and to all wild types (n = 54), isolated from cell cultures submitted to our laboratory. The mAb was used in a immunofluorescent assay (IF) and the method correlated with the microbiological assay. Using this mAb immunofluorescent staining of cells is a fast and simple procedure for mycoplasma detection in cell cultures.
Key words: Cell culture mycoplasma; Mycoplasmadetection; Monoclonal antibody to mycoplasmas
Introduction Since a wide range of biological, immunological and medical research has become dependent on cells growing in vitro, good cell culture conditions are of great importance if valid and reproducible results are to be obtained. Mycoplasma infections of continuous cell lines remain a serious problem; these organisms may not be obvious with ordinary light microscopy and they may not overgrow and kill the cells as do bacteria and fungi. For that
Correspondence to: K. Schmitt, Institut fiir Medizinische Mikrobiologie und Virologie, Heinrich-Heine-Universit~it, Moorenstrasse 5, 4000 Diisseldorf, F.R.G. * This work contains essential parts of the MD thesis of Roman Blazek
reason mycoplasmas can persist undetected in cell cultures for months. Since they are able to alter a great variety of biologic, immunologic and biochemical properties of cells, mycoplasmas are a source of artifacts and false interpretation of results (McGarrity et al., 1984). Although there are several methods to detect mycoplasmas in cell cultures, most of them give unreliable results when not used by experts e.g., the detection of mycoplasmas by microbiological culture media (Chen, 1977). To be sensitive enough, some methods are dependent on an indicator cell line, such as D N A staining with diamidinophenolindol (DAPI) or Hoechst stain 33258 (Russel et al., 1975; Chen, 1977) and mycoplasma mediated cytotoxicity using 6-methylpurine desoxyriboside (6MDPR) (McGarrity et al., 1982, 1986). Thus, it may take several days to obtain results. Except for the detection of cell culture mycoplasmas by genetic probes (McGarr-
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (Biomedical Division)
204 ity et al. 1986), most of the methods take too long and are too complicated to be used in laboratories which are inexperienced in mycoplasma research. The purpose of this study was to generate mAbs to cell culture mycoplasmas and to investigate whether these could be used for the detection of such contaminants. In studies reporting the identification of mycoplasmas which were obtained from cell cultures the species Mycoplasma orale, Mycoplasma argininL Mycoplasma hyorhinis, Acholeplasma laidlawii and Mycoplasma salivarium accounted for 96% of the isolates (McGarrity et al. 1985). With this in mind we prepared mAbs to these five mycoplasma species and tested them for their potential use in the diagnosis of these mycoplasmas. The most important outcome of the present study was that one of the mAbs generated was able to bind to all of the common mycoplasma contaminants of cell cultures. This mAb was designated CCM-2 (CCM: cell culture mycoplasmas) and was used in IF tests for the organism.
Materials and methods
Mycoplasmas The following five mycoplasma species were used for immunization: A. laidlawii (NCTC 10116: from the National Collection of Type Cultures, London, U.K.), M. hyorhinis (ATCC 17981: from the American Type Culture Collection, Rockville, MD), M. orale (ATCC 23714), M. arginini (NCT C 10129) and M. salivarium (NCTC 10113). Additional species were used for subsequent assays, namely M. pneumoniae strain FH, M. fermentans (ATCC 19989), M. gallisepticum (ATCC 15302) and M. hominis (NCTC 10111). The source of these mycoplasma species and the components of the media for their propagation have been described previously (Schmitt et al., 1988). 200 ml broth cultures in logarithmic phase were centrifuged three times at 15,000 x g for 30 min, the pellets were washed in PBS and the final suspension of antigen was adjusted to a protein concentration of 1 mg/ml. Depending on the mycoplasma species the preparation, which was usually stored at - 70 o C, contained 108-109 cfu/ml.
Immunization Female BALB/c mice 3-4 months of age were immunized intraperitoneally. The initial immunization was carried out with 200 /xg of the mycoplasma protein, emulsified in complete Freund's adjuvant (CFA, Difco Laboratories, Detroit, MI, U.S.A.). The four subsequent booster injections of 100 ~tg of antigen without CFA were given every 2 weeks. Fusion and selection of hybridomas 3 days after the last immunization the spleen cells were fused with the myeloma cell line Xg3 Ag8 653 using a procedure initially described by K~Shler and Milstein (1975) and modified by Fazekas De St. Groth and Scheidegger (1980). After fusion the cells were distributed into 96-well microtiter plates (Greiner, Niirtingen, F.R.G.). The supernatant fluids of the wells with proliferating hybridomas were tested for antimycoplasma antibodies by ELISA. The hybridomas from positive wells were cloned by limiting dilution and after retesting for mAb they were expanded into bulk cultures and frozen in liquid nitrogen. The supernatants of these cultures were collected and used for further characterization of the mAbs. Ascites Ascitic fluid was produced in BALB/c mice following an intraperitoneal injection of 0.5 ml pristane (Aldrich-Chemie, Steinheim, F.R.G.). After 2 weeks the mice were injected with bulk cultures of cloned hybridomas (app. 5 x 106 cells). Ascitic fluid was withdrawn after 10-14 days. Conjugation of mAbs with biotin succinimide ester The mAbs for conjugation were purified by ammonium sulphate fractionation and FPLC ionexchange chromatography. The mAbs were conjugated as described by Goding (1983). ELISA Freeze-thaw disrupted mycoplasmas were diluted to give 20/~g/ml of protein in coating buffer (borate-buffered saline (BBS), 50 mM NanB407 + 150 mM NaC1, pH 9.0). 30/~1 volumes were then applied to each well of a microtiter plate (Falcon, Dickinson Labware, Oxnard, U.S.A.) and incubated for 60 min at room temperature (RT).
205
bodies (Dianova), (1 h for each incubation). The NC was rinsed between the steps three times with PBS. The developing solution consisted of 30 mg 4-chloro-l-naphthol in 10 ml methanol, 50 ml TBS (TBS: 50 mM Tris, 200 mM NaC1, pH 7.5) and 25 /~1 30% H202.
Free binding capacity was blocked with 1% bovine serum albumin (BSA) in PBS for 2 h. 30 /~1 of antibody solution were added per well and left to incubate for 1 h. The bound antibody was detected by goat anti-mouse peroxidase-labelled antibodies (Dianova, Hamburg, F.R.G., 1/2000, 20 /zl/well) and the substrate ABTS (Boehringer, Mannheim, F.R.G.) + H202. In order to determine the antibody class of the generated mAbs, class-specific peroxidase-labelled antibodies to IgG1, IgG2a, IgG2b and IgG3 were used (Dianova). In order to detect cross-reactivity of the mAbs with the PPLO-broth medium the ELISA was also performed with the medium as antigen at a dilution of 1/5 in BBS.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting (Western blot) The mycoplasma preparation (1 mg/ml of protein) diluted 1/2 in sample buffer (2% SDS, 5% mercaptoethanol, 12.5% glycerol, 0.5 mM Tris, pH 6.8) was heated at 100 °C for 5 min. Electrophoresis was then performed by the method of Laemmli (1970) with 9% or 12% acrylamide gels using appropriate molecular weight markers (Bio-Rad, Munich, F.R.G.). Nitrocellulose filter blots were prepared by the procedure of Towbin et al. (1979), BS was applied and the assay completed as previously described for IB.
Immunobinding assay (IB) This IB assay was modified from the procedure described by Kotani and McGarrity (1985). The assay was performed at room temperature with agitation. 5 btl of antigen suspension (app. 1 mg/ml) were applied as a spot to a nitrocellulose sheet (NC, Schleicher & Schiill, Dassel, F.R.G.) and left to dry. The NC was incubated successively in a blocking solution (BS: 5% dry nonfat milk in PBS), then with undiluted supernatants from hybridoma producing mAbs or alternatively with ascitic fluid diluted 1/1000 in BS, and finally with rabbit anti-mouse peroxidase-labelled anti-
Trypsin treatment of mycoplasma antigen A preparation of M. hominis (1 mg/ml, clinical isolate no. 25) was treated with trypsin (250/~g/ml, Sigma, Daisenhoven, F.R.G.) for time periods ranging from 1 s to 15 h. The reaction was terminated with trypsin inhibitor (750 /tg/ml, Roth, Karlsruhe, F.R.G.) and the samples prepared for subsequent immunoblotting.
TABLE I SPECIFICITY
O F 30 G E N E R A T E D
M y c o p l a s m a species as a n t i g e n
mAbs, TESTED IN ELISA
R e a c t i v i t y o f the m o n o c l o n a l a n t i b o d i e s 2219h 2220h 2222 h
22241 2226 j 2228 I
2207° 2206° 2202 o 2208 o
2209a 2210a 2211a 2214 a
2231s 2232s
22251
22271
2199° 2201 o
2198 °
22231
2205 ° 2203 o
22291
2200° 2221 h
+
+
+
-
+
+
2215 a 2216 a 2217 a M. A.
hyorhmis laidlawii
+
.
-
+
.
. -
. -
.
g.
orale
-
-
+
-
-
g.
arginini
-
-
-
+
.
M.
salivarium
.
.
.
.
mAbs were obtained from mice immunized with
.
-
+ h M.
hyorhinis,
.
+ .
. + I A.
.
.
+
-
+
+
. laidlawii,
+ o M.
. -
orale,
+
+
+
+
+
+
-
+
-
+
-
+
+
+
+
a
M.
arginini,
s M.
salivarium.
206
Triton X-114 phase partitioning of mycoplasma antigen Using a slight modification of the method of Bordier (1981) which was applied to M. hyorhinis by Riethman et al. (1987), a preparation of M. orale (1 m g / m l of protein) was separated into hydrophobic, hydrophilic and nonsoluble fractions. Three repetitions of the phase fractionation resulted in fractions of sufficient purity for subsequent SDS-PAGE and immunoblotting. IF performed with fixed cells Cell suspensions to be assayed for mycoplasmas were applied without any kind of pretreatment as a spot (50 /~1) to coverslides. The slides were dried at 50-60 ° C for 45 min. The cells were then fixed and permeabilized with ethanol (1 min in 70% ice-cold ethanol/water). In order to avoid subsequent nonspecific staining, the slides were not allowed to dry out after fixation (Goding, A M
1
2
3
4
5
6
7
M
1983). The cells were counterstained with Evans Blue (0.005% in H20, 30 min at 37°C) and then exposed to antimycoplasma antibodies for 60 min (50/~l/cover slide of either undiluted supernatant fluid or ascites 1/1000 in BS + 10% FCS or purified mAb conjugated with biotin in BS + 10% FCS). The slides were rinsed three times in PBS. Depending on whether unconjugated or biotinconjugated mAbs were used, the bound antimycoplasma antibodies were detected by either FITC-conjugated goat anti-mouse Abs or FITCconjugated streptavidin (Dianova). After three washes in PBS the coverslides were airdried and mounted on slides for microscopy with Fluoroprep (BioMrrieux, Charbanirres-lesbains, France). For the examination of the slides a Zeiss Photomicroscope-III, equipped for light fluorescence microscopy, was used. A total of 500 × (40 × 12.5) magnification was generally sufficient for the detection of mycoplasmas. In cell cultures
1
2
3
B 4
5
6
7
MW x 10 - 3
-- 200 --116
--97
--66
43
t --
31
Fig. 1. Reactivity of mAb CCM-2 with common cell culture mycoplasmas in an immunoblot. A: patterns of mycoplasma proteins separated by SDS-polyacrylamide (9%) gel electrophoresis and stained with Coomassie Blue. B: immunoblots of the separated mycoplasma components prepared from an identical gel to that in A and probed with the mAb CCM-2 (ascitic fluid 1/1000 in BS). The bound mAb was detected by goat anti-mouse peroxidase-labelled antibodies. Lanes: M, molecular weight markers; 1, M. fermentans; 2, A. laidlawii; 3, M. hyorhinis; 4, M. orale; 5, M. arginini; 6, M. salivarium; 7, M. hominis.
207
contaminated with only a few mycoplasmas, a total of 800 × (64 × 12.5) magnification was used.
IF performed with unfixed cells For this procedure mycoplasma infected and mycoplasma free AtT-20 cells (ATCC-CCL89) adherently grown on coverslides were stained with mAbs and with FITC-conjugated goat anti-mouse Abs (1 h for each incubation time). They were then fixed with 2% formaldehyde in PBS for 10 min, mounted and examined as above.
Results
A total of 30 stable hybridoma producing IgGclass antibodies to mycoplasmas were obtained after five fusions. The reactions of the mAbs generated are detailed in Table I. Out of the 30 mAbs, 19 were monospecific. The remaining 11 mAbs were able to bind to two or
more species. In contrast to a similar study by Buck et al. (1982), we decided to investigate both groups of mAbs. Ten out of the 19 mAbs, which appeared species-specific in ELISA, were selected for further studies, particularly the identification of mycoplasmas isolated from cell cultures. One mAb of IgG1 isotype described in Table I as mAb no. 2200 and later designated as CCM-2 became the main subject of the present study. As Table I shows, this mAb reacted with all five mycoplasma species. Ascitic fluid was produced and the antibody further tested by immunoblotting using two additional mycoplasma species: M. hominis and M. fermentans. The epitope recognized by the mAb CCM-2 was present on a molecule with an apparent molecular weight of 42,000-47,000, depending on the mycoplasma species. Except for M. hyorhinis and A. laidlawii, all of the mycoplasma species also showed additional components of lower molecular weight (Fig. 1). B
A M
1
2
3
4
5
6
1
2
3
4
5
6
MWxl0
-3
116
J--N 97
--66
--
43
:/!iiiill i!ii ....i:!;ii:i:i~ ii i;i!
m31 !ii!il:!!!! :~i
.... i !iiiii~ii
--22
Fig. 2. Electrophoretic patterns and immunoblot of trypsin treated and untreated M. hominis antigen. A: components of M. hominis antigen separated by SDS-PAGE (12% gel stained with Coomassie Blue). B: immunoblot from the separated components using the mAb CCM-2. Lanes: M, molecular markers; 1, untreated M. hominis antigen; 2-6, M. hominis treated with trypsin (250 # g / m l ) for 1 s (lane 2), 20 s (lane 3), 1 min (lane 4), 2.5 h (lane 5), 15 h (lane 6).
208 For further epitope analysis trypsin treatment and Triton X-114 phase fractionation of the mycoplasma antigen preparation were performed. The 44 kDa component of M. hominis antigen showed decreased staining with mAb CCM-2 after brief treatment with trypsin. This was accompanied by the appearance of a new immunoreactive band with an estimated molecular weight of 36,000. Incubation times up to 15 h resulted in significantly diminished staining of the 44 kDa component and the complete removal of the new 36 kDa band. The epitope bearing component was shown to be trypsin sensitive (Fig. 2) and in addition, to partition into the aqueous phase (Fig. 3). In order to use the mAb CCM-2 in IF, the assay was first performed on unfixed cells: AtT-20 cells were permitted to adhere to cover slides and then infected with M. orale. After 48 h the unfixed cells were subjected to the immunofluorescent
M
1
A 2
staining procedure using anti-M, orale mAb 2208 as a positive control. Since the mAb CCM-2 failed to stain unfixed mycoplasmas the epitope was assumed to be expressed inside these bacteria. Therefore subsequent IF testing was performed on ceils fixed and permeabili.zed with ethanol. For this test the mycoplasma free cell line Xg3 Ag8 653 was separately infected with six mycoplasma species: M. fermentans, M. hyorhinis, M. orale, M. arginini, M. salivarium and M. horninis. After 2-3 cell passages, immunofluorescent staining of the contaminated cells was performed. In all cases except the negative control (mycoplasma free cell line), a bright fluorescence was seen, localized either on the cells, between the cells or on the glass, depending on the contaminating mycoplasma species (Fig. 4). Previous experiments demonstrated the reactivity of mAb CCM-2 with several mycoplasma
B
3
4
1
C
2
3
1
2
3
MWxlO
-3
--116 -- 97
--
l
g
v
66
-43
--31
Fig. 3. Electrophoretic patterns and immunoblot of Triton X-114 phase partitioned M. orale antigen. A: componentsseparated with SDS-PAGE (12% gel stained with Coomassie Blue). B: immunoblot of the separated components incubated with mAb CCM-2. C: immunoblot incubated with the anti-M, orale mAb 2208 which bound to a detergent phase antigen and served as control for the phase partition experiment. Lanes: 1, untreated M. orale antigen; 2, detergent phase; 3, aqueous phase; 4, insoluble proteins.
0
0
210
species originating from the American Type Culture Collection, or from the National Collection of Type Cultures. In the following assays m A b CCM-2 was tested for binding to mycoplasmas isolated from cell cultures and clinical specimens. In view of the large number of isolates to be tested, IB was chosen as the most advantageous method. Over a period of 3 years cell cultures submitted to our laboratory were assayed for mycoplasmas by the cultivation method in PPLO broth and under anaerobic conditions on agar. 54 wild type mycoplasmas were isolated and collected to be used as antigen for lB. MAb CCM-2 produced a positive reaction with all of them.
TABLE
II
DETECTION STUDY
OF MYCOPLASMAS
ON 20 DIFFERENT
Cell lines tested
IN CELL
CELL
CULTURES,
LINES
Method used
for mycoplasmas
IF
Culture A
D
Agar
Human Cell lines B cell l i n e s : R a j i
+
+
-
+
Griese B
+
+
-
+
FH-B
.
-
+
.
.
.
.
Winkler B
+
Darius BLCL
.
MP-B
+
+
-
+
B JAB
+
+
-
+
T cell l i n e s : M o l t 4
Macrophage
. + .
+
+
-
+
CEM
+
+
-
+ +
L 428
+
+
-
Jurkat
+
+
-
+
H u t 78
+
-
+
+
B9
+
+
-
+
K 562
+
+
-
+
+
+
-
+
C o l o n i c c a r c i n o m a cell l i n e : DETA
+
+
-
+
-
+
-
+
Mouse cell lines B cell l i n e :
L 1210
+
T cell l i n e s :
3 D O 54.8
.
.
.
.
BW 5147 cell l i n e :
.
.
.
.
Mastocytoma
P 815-JEK
+
IF:
immunofluorescent
medium
Discussion
cell l i n e : U 937
broth
Another IB test was carried out with 186 mycoplasma isolates from clinical specimens, delivered to the Department of Medical Microbiology, University of Mainz. 146 of these isolates were identified by antisera as M. hominis. MAb CCM-2 reacted with all 186 isolates. When using the m A b CCM-2 for the diagnosis of cell culture m y c o p l a s m a s in the immunofluorescent assay described above, it is important that the m A b does not crossreact with uncontaminated cell cultures. For testing a great variety of cells for crossreactivity with the mAb CCM-2 in a single experiment, the IF assay was performed with h u m a n peripheral blood leucocytes (PBL) and mouse spleen cells (SC). PBL and SC were infected with M. hominis and incubated for 24 h in order to provide a positive control. Except for the positive control, no fluorescent staining of the blood cells occurred. At the next stage the m A b CCM-2 employed in the IF assay was used to diagnose mycoplasma contamination of cell cultures. Anaerobic incubation of agar and broth media, routinely used in our laboratory for the detection of mycoplasmas, was chosen as the reference method. As shown in Table II, 20 different cell lines submitted to our laboratory were assayed. All 20 cell lines originated from one laboratory, in which the cells were screened for mycoplasmas for the first time. 16 cell cultures were positive for mycoplasmas by the IF assay and this result correlated with the microbiological assay. The cells were retested 2 months later. They were still positive for mycoplasmas in both tests.
medium
+
assay with mAb
supplemented
supplemented
+
CCM-2.
with arginine.
with dextrose.
D:
A: PPLOPPLO-broth
The frequency of mycoplasma contaminated cell cultures - about 15% of cell lines are believed to be infected (McGarrity et al., 1985) - and the quick spread of the infection within the laboratory imposes a requirement for regular screening of continuous cell lines. The immunofluorescence assay described could be carried out in most cell culture laboratories. The m A b CCM-2 was shown to bind to the following mycoplasma contaminants of cell cultures: M. fermentans, A. laidlawiL M. hyorhinis, M. orale, M. argininL M. salivariurn
211
and M. hominis. Together, these account for more than 96% of the reported isolates (McGarrity et al., 1985). Furthermore, the mAb reacted with all 54 wild types isolated from contaminated cell cultures. In addition the mAb CCM-2 reacted with all tested (n = 186) clinical mycoplasma isolates, of which 146 were identified as M. hominis, a very heterogenous mycoplasma species (Andersen et al., 1987). When an immunobinding assay was performed with M. pneumoniae strain F H and M. gallisepticum (ATCC 15302) as antigen (data not shown), it became clear that the mAb CCM-2 did not bind to these two mycoplasma species. However, since M. pneumoniae and M. gallisepticum are almost never isolated from cell cultures, this result does not seriously detract from the value of this mAb for the detection of most cell culture mycoplasmas. In a 9 year study (Pollak-Vogelzang et al., 1987) only two isolates of M. pneumoniae accounted for 0.1% of the isolates and they were detected in hybridoma cultures originating from one laboratory. The antigen recognized by the mAb CCM-2 was shown to be trypsin-sensitive and to partition efficiently into the aqueous phase following Triton X-114 fractionation. Moreover, the mAb was not able to bind to native mycoplasmas in IF tests. These results suggested that the epitope is comprised of, or linked to a trypsin-sensitive polypeptide, neither exposed on the surface of the mycoplasmas, nor associated with integral membrane proteins, i.e., a cytoplasmic component. The reaction of antibodies with antigens localized within cells requires fixation of the cells in order to render them permeable to proteins (Goding, 1983). In preliminary studies ethanols fixation was found to be optimal for fluorescence staining of mycoplasmas. A troublesome technical aspect was nonspecific staining of fixed cells by the secondary antibody but this artifact was virtually eliminated if the cells were not allowed to dry after fixation and counterstained with Evans Blue. Even better results were obtained using purified and biotin-conjugated mAb CCM-2. The advantage of this was that the biotin-conjugated mAb could be used to screen hybridomas producing mouse immunoglobulin.
The IF correlated with microbiological assay, but more cell cultures should be tested. The high sensitivity of the test was due to the fact that the IF revealed even single mycoplasmas on the cell surface. N o indicator cell line was necessary and therefore the complete IF could be performed in about 5 h. Thus the major advantages of the assay are sensitivity, simplicity and rapidity.
References Andersen, H., Birkelund, S., Christiansen, G. and Freundt, E.A. (1987) Electrophoretic analysis of proteins from Mycoplasma hominis strains detected by SDS-PAGE, two dimensional gel electrophoresis and immunobinding. J. Gen. Microbiol. 133, 181. Bordier, C. (1981) Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem. 256, 1604. Buck, D.W., Kennett, H.R. and McGarrity, G. (1982) Monoclonal antibodies specific for cell culture mycoplasmas. In vitro 18, 377. Chen, T.R., (1977) In situ detection of mycoplasma contamination of cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res. 104, 255. Fazekas De St. Groth, S. and Scheidegger, D. (1980) Production of monoclonal antibodies: Strategy and tactics. J. Immunol. Methods 35, 1. Goding, J.W. (1983) Monoclonal Antibodies: Principles and Practice. Academic Press, London, p. 223. KiShler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibodies of predefined specifity. Nature 256, 495. Kotani, H. and McGarrity, G.J. (1985) Rapid and simple identification of mycoplasmas by immunobinding. J. Immunol. Methods 85, 257. Laemmfi, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. McGarrity, G.J. and Carson, D.A. (1982) Adenosine phosphorylase-mediated nucleoside toxicity. Exp. Cell Res. 139, 199. McGarrity, G.J. and Kotani, H (1985) Cell culture mycoplasmas. In: S. Razin and M.F. Baffle, (Eds.), The Mycoplasmas, IV, Mycoplasma Pathogenicity. Academic Press, Orlando, FL, p. 383. McGarffty, G.J. and Kotani, H. (1986) Detection of cell culture mycoplasmas by a genetic probe. Exp. Cell Res. 163, 273. McGarrity, G.J., Vanamann, V. and Sarama, J. (1984) Cytogenic effects of mycoplasmal infection of cell cultures: A review. In Vitro 20, 1. McGarffty, G.J., Kotani, H. and Carson, D. (1986) Comparative studies to determine the efficiency of 6-methylpuffne desoxyriboside to detect cell culture mycoplasmas. In Vitro Cell. Dev. Biol. 22, 301.
212 Pollak-Vogelzang, A.A., Brugman, J. and Reijgers, R. (1987) Comparison of two methods for detection of mollicutes (Mycoplasma and Acholeplasma) in cell cultures in the Netherlands. Antonie Van Leeuwenhoek, J. Microbiol. 53, 107. Riethman, H.C., Boyer, M.J. and Wise, K.S. (1987) Triton X-114 phase fractionation of an integral membrane surface protein mediating monoclonal antibody killing of Mycoplasma hyorhinis. Infect. Immun. 55, 1094. Russel, W.C., Newman, C. and Williamson, D.H. (1975) A simple cytochemical technique for demonstration of DNA
in cells infected with mycoplasmas and viruses. Nature 353, 461. Schmitt, K., D~iubener, W., Bitter-Suermann, D. and Hadding, U. (1988) A safe and efficient method for elimination of cell mycoplasmas using ciprofloxacin. J. Immunol. Methods 109, 17. Towbin, H., Stachelin, T. and Cordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350.
