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Detection of mycoplasma contamination in lymphoblastoid cell lines by monoclonal antibodies
Detection of Mycoplasma Contamination in Lymphoblastoid Cell Lines by Monoclonal Antibodies Susan F. Radka, Dana M. Hester, Anna A. Polak-Vogelzang, and Reinder L.H. Bolhuis
A B S T R A C T : We have produced two monoc/onal antibodies. SFR l-Myco 1 and SFR 1 -Myco 2. that detect Mycoplasma fermentans Jound to contaminate/ymphoblastoid cell lines (LCL). The specifici(y of these monodonal antibodies against the M. fermentans was determined by indirect immunofluorescence by demonstrating the reactivity of the monoc/onal antibodies with known re]erence strains of mycoplasma grown on soft agar. The reactivity of these antibodies against LCL in a number of immunoassays correlates completely with the presence of mycop/asma in these ce/i~ as determined by a standard mycoplasma detection assa). Because of the potential for ,'idespread contamination of B [ymphoblastoid cell lines tranfformed with Epstein-Barr virus-containing supernatants obtained from marmoset call lines contaminated with M. fermentans, these monoc/ona/ antibodies have value as screening reagents .fi)r this no'cop/asma species in LCL. ABBREVIATIONS
LCL HAT BLCL EBV
lymphoblastoid cell line hypoxanthine-aminopterinthymidine B lymphoblastoid cell line Epstein-Barr virus
PBA FACS
phosphate buffered saline, pH v.4, 2'7~ bovine serum albumin, 0.02(7{ sodium azide fluorescence-activated cell sorter
INTRODUCTION The existence of mycoplasma contamination in the majority of lymphoblastoid cell lines (LCL) currently used for research purposes has plagued investigators interested in using these lines for somatic cell hybridization experiments. Many of the mycoplasma strains which commonly contaminate LCL are capable of depleting thymidine from HAT*-selective medium, resulting in toxicity to hybrid cells, and thus often preventing successful fusions [ 1 ]. Mycoplasma-contaminated LCL often grow at a much slower rate than LCL free of mycoplasma. In the process o f developing a novel fusion protocol for the production of monoclonal antibodies to human Ia antigens, we used a mycoplasma-contaminated BLCL* (Swei) as immunogen, and produced a number of monoclonal antibodies
From the Department of Mierobiolo£1' and lmmuno/og3. Duke Uniz'ersitr Medical Center. Durham. North Carolina. National Institute fi)r Public Health. Bi/thoren. The Nether/andr: and Radio/o~hal Institute. TNO. Rijswyk. The Netherlanda. Address requeatsfor reprints to Susan F. Radka. Ph.D.. Amerkan Red Cro~.
111
112
S.F. Radka et al. directed against various cell surface antigens. When the Swei cell line was eliminated of mycoplasma by an eradication protocol recently described [2], two antibodies, SFR1-Myco 1 and SFR1-Myco 2, were no longer reactive. We describe here the reactivity of these antibodies with both mycoplasma-positive and -negative cell lines by a variety of assays. Isolation of Mycoplasmafermentans from a contaminated BLCL reactive with SFRl-myco 1 and SFRl-myco 2, and their reactivity with only this mycoplasma species confirmed the anti-mycoplasma specificity of these antibodies.
METHODS
Cell lines. The Swei cell line was the generous gift of Dr. John Hansen (Seattle, WA). LG-10 was obtained from Dr. Jack Silver (East Lansing, MI), GM3104 from the Mutant Cell Repository (Camden, NJ), and PB-4 from Dr. Nancy Collins (New York, NY). The JY cell line was obtained from Dr. Dean Mann (Bethesda, MD), while the Pala and A2M cell lines were transformed at Duke University from the peripheral blood of two different individuals. The cell line SB is an EBV-transformed BLCL described previously [3] while Raji is a BLCL obtained from a patient with Burkitt's lymphoma.
Immunization and fusion. Adult male K G H rats obtained from Dr. Thomas Gill and Dr. Heinz Kunz of the University of Pittsburgh, were immunized in the hind footpads with a 0.2 ml suspension of 107 Swei cells emulsified in Freund's Complete Adjuvant. Two weeks later the animals were boosted in the hind footpads with 107 Swei cells emulsified in 0.2 ml of Incomplete Freund's Adjuvant. Three days after boosting, the popliteal lymph nodes were removed and the lymph node suspension was fused with the SP2/O-Ag14 murine myeloma at a 10:1 lymph node to myeloma ratio. The fusing agent was polyethylene glycol (1000 MW, Fisher, Pittsburgh, PA) at a 35% concentration. Fusion products were plated into each of two 24 well tissue culture plates (Costar) containing 105 irradiated (1800 rads) murine peritoneal exudate cells. Growing hybrids were screened for antibody production by the Amos modification of the standard N I H microcytotoxicity test [4]. Positive hybrids were cloned in semisolid agar.
Radioimmunoassay. The cellular radioimmunoassay used for detection of antibody binding is a modification of the method described by Mason and Williams [5]. Briefly, 105 lymphoblastoid cells and 4 x 105 glutaraldehyde-fixed sheep red blood cells were incubated with 100 gl of tissue culture supernatant for 1 hr at 4°C in round-bottomed microtiter plates (Linbro, McLean, VA). After 3 washes with phosphate buffered saline, pH 7.4, 2% bovine serum albumin, 0.02% sodium azide (PBA*), 2 x 105 cpm of affinity-purified 125I-F(ab)'2 rabbit antimouse Ig were added to each well, and the plates were incubated at 4°C for 45 min. Wells were washed 3 times with PBA, after which the contents from each well were transferred to 6 x 50 mm test tubes for gamma counting.
Mycoplasma screening test. The assay for detection of mycoplasma is based on that described by Chen [6]. Briefly, a mouse fibroblast cell line used as indicator cells was incubated in 15 x 60 mm tissue culture dishes containing cover slips for 3 to 5 days with spent medium from the cell lines under investigation for mycoplasma contamination. Subsequently, the cells which adhered to the cover slip were fixed with Carnoy's solution and dried. A 0.05 g/ml solution of Hoechst 33258 stain in Hank's balanced salt solution, pH 7, was incubated with the fixed cells for 10 min at room temperature. After two washes with distilled water, the
Anti-mycoplasma Monoclonal Antibodies
113
slides were air dried. Coverslips were mounted in 0.1 M acetate-acetic acid buffer, p H 5.5. One hour after mounting, the slides were examined by fluorescence microscopy. A positive test was indicated by cytoplasmic and/or membrane (both nuclear and plasma) fluorescence, depending upon the degree of mycoplasma contamination.
Isolation of mycoplasma from BLCL. The procedure for isolation of mycoplasma from BLCL was described previously [7]. Briefly, the BLCL were incubated under anaerobic conditions on Difco PPLO medium (Difco) with 20U heat-inactivated horse serum and 10% (W/V) acid yeast extract for 5 days.
Flow cytometry. Cells to be analyzed were washed with PBA. Tissue culture ( 100 btl) supernatant containing monoclonal antibody was incubated with the pelleted cells for 1 hr at 25°C. The cells were washed twice with PBA, after which fluoresceinated goat anti-mouse Ig (Meloy, Springfield, VA) was added. After 45 min at 4°C, the cells were washed, resuspended in PBA, and analyzed for fluorescence with an Ortho Cytofluorograph model 50-15 equipped with an argon laser exciting at 488 nm.
RESULTS Tissue culture supernatants from lymphoblastoid cell lines to be tested for mycoplasma contamination by the Chert test were incubated for 4 days with the mouse fibroblast cell line, after which the remainder of the Chen protocol was followed. Concordantly, aliquots of cells, obtained from the same flasks as the supernatants for the Chen assay, were investigated for binding of the putative anti-mycoplasma monoclonal antibodies by three different assays: radioimmunoassay, FACS* analysis, and examination of individual samples under the fluorescence microscope. In all experiments, a monoclonal antibody detecting monomorphic determinants on human class II antigens was used as a positive control, and gave appropriate positive results. Results comparing ten different BLCL for mycoplasma contamination by the Chen assay and radioimmunoassay are summarized in Table 1. There is complete agreement between the results obtained by the Chen assay and the binding of the two monoclonal antibodies. In fact, the relative binding levels of the antibodies on various cell lines suggests that quantitative differences in amounts of mycoplasma expressed on the cell surface (therefore presumably the degree of mycoplasma contamination) can be detected by this assay. An examination of the data obtained by FACS analysis of these cell lines with the monoclonal antibodies (Figure 1) verifies that quantitative differences in degree of contamination exist among three mycoplasma contaminated cell lines. Figure l(a) depicts the lack of reactivity of SFR1-Myco 1 and SFR1-Myco 2 with mycoplasma-free Swei. In Figure l(b), Swei cells which had been contaminated intentionally with mycoplasma a week prior to assay show a pattern of low fluorescence intensity relative to the positive control. The Pala cell line [Figure l(c)], which has been contaminated for several years, exhibits a fluorescence profile with the anti-mycoplasma monoclonal antibodies which is much more intense than that for the newly contaminated Swei. The cell line LG10 [Figure l(d)] shows a fluorescence pattern intermediate to that of the other contaminated cell lines. These results correlate well with results obtained by radioimmunoassay. Although not shown here, the data obtained by examination of individual cells by fluorescence microscopy are in complete agreement with the results obtained by radioimmunoassay and FACS analysis.
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TABLE
1
C o m p a r i s o n o f results o f m y c o p l a s m a s c r e e n i n g by C h e n m y c o p l a s m a test vs. r a d i o i m m u n o a s s a y with S F R 1 - M y c o 1 and SFR1 M y c o 2 Binding of anti-mycoplasma antibodies b
Cell line
Chen test"
SFR1-Myco 1
SFR1-Myco 2
Swei (Myco +) Swei (Myco - ) JY Pala PB-4 SB GM 3104 A2M LG-10 Raji. 5
+ + + + +
3.4 0 0 8.4 6.3 0 0 7.1 4.0
2.5 0 0 6.6 5.2 0 0 6.8 3.7
-
0
0
"Determination of + or - is based on presence or absence of fluorescence of a cell suspension prepared as described in "Methods". bResults listed are ratios of binding of SFR1-Myco 1 and SFR1-Myco 2 relativeto the binding of a negativecontrol antibody.
F r o m the Pala cell line, m y c o p l a s m a s c o ul d be isolated on D i f c o - P P L O m e d i u m after a n a e r o b i c i n c u b a t i o n at 37°C for 5 days [7]. T h e S F R 1 - M y c o 1 and SFR1 M y c o 2 a n t i b o d i e s w e r e t e s t e d in the i n d i r e c t i m m u n o f l u o r e s c e n c e test o n unfixed agar c o l o n i e s [8] o f r e f e r e n c e strains o f M. arginini, M. fermentans ( r e f e r e n c e strain and that f r o m the Pala cell line), M. hyorhinis, and M. Orale, using a s h e e p a n t i m o u s e I g G - F I T C c o n j u g a t e . T h e y r e act ed only with c o l o n i e s o f the M. fermentans strains, with each a n t i b o d y reacting up to a d i l u t i o n o f 1:8. Since M. fermentans is t h o u g h t to be a fairly rare tissue c u l t u r e c o n t a m i n a n t [9], it is highly p o s s i b l e that the m a r m o s e t cell line used for E B V * t r a n s f o r m a t i o n o f p e r i p h e r a l
a
50-
~"
',"~
1 Fluorescence-activated cell sorter analyses of the reactivity of four monoclonal antibodies on different BLCL. ( ' ' ") indicates reactivity of negative control antibody on each cell line. ( - - - ) is monomorphic anti-human class II monoclonal antibody. (--) is SFR1-Myco 1. ( - ' - ) is SFR1-Myco 2. Panel (A) indicates fluorescence pattern of these antibodies on mycoplasma-free Swei; panel (B) is mycoplasma-contaminated Swei; panel (C) is the Pala BLCL; and panel (D) is LG-10. FIGURE
50 ~
/'4
,~50 k
~,, y,,,',,
'~
J 50 k
,
f',%r,, 2
INCREASING
FLUORESCENCE INTENSITY
Anti-mycoplasma Monoclonal Antibodies
l 15
blood B cells into BLCL was the source of mycoplasma contamination in the Swei cell line. Therefore, a sample of the B95-8 marmoset line which was used for transformation of the Swei cell line was obtained from Dr. J o h n Hansen and examined for reactivity with SFR1-Myco 1 and SFR1-Myco 2. It was also found to be contamined with 34. fermentans, based on reactivity with the monoclonal antibodies (the cell line was not tested for other mycoplasma strains). DISCUSSION The detection and elimination of mycoplasma contamination in lymphoblastoid cell lines has b e c o m e a major concern of researchers who use these cellular reagents for various investigations. Recently, a procedure for mycoplasma elimination from lymphoblastoid cell lines was described which results in reasonable success [2]. O u r discovery of the anti-mycoplasma monoclonal antibodies SFR1Myco 1 and SFR1-Myco 2 are the direct results of mycoplasma elimination from the immunizing cell line. By examining the supernatants from a BLCL with which the antibodies reacted, we have determined the specificity of the monoclonal antibodies and the strain of mycoplasma contaminating the cell line. O f four different strains of mycoplasma (M. arginini, M. hyorhin#, 34. orale, and M. fermentans) for which the supernatant and monoclonal antibodies were screenecl, only 34. jbrmentans was positive. Since this strain is thought to be the least c o m m o n tissue culture contaminant of the four strains and yet all the mycoplasma-contaminated BLCLs we tested carried 34. fermentans, we attempted to determine whether some of the marmoset cell lines used to transform peripheral blood B cells into cell lines were contaminated with 34. fermentans. A sample of the B95-8 line used to transform Swei, which was the source of the antigen against which these monoclonal antibodies were produced was indeed found to be contaminated with 34. fermentans. H o w e v e r , since it is not certain that the marmoset cell line was contaminated with 34. fermentans at the time supernatant was taken for transformation, we are not certain of the source of contamination of the Swei. We do know that until the M C U V - 5 marmoset cell line which we routinely use for transforming peripheral blood B cells was eliminated of mycoplasma, it was contaminated with M. fermentans, based on the reactivity of these antibodies. Therefore, until recently, any BLCL we have generated with supernatants from MCUV-5 are likely to be contaminated with 34. fermentans. We suspect that this may be the case for many BLCL which have been generated in a number of laboratories. Because of the possible widespread contamination with 34. fermentans of BLCL generated by contaminated EBV-containing supernatant, these antibodies are useful as screening reagents for detecting contamination by any of a n u m b e r of immunoassays which are simpler and more rapid than the standard assays used for mycoplasma detection. These results obviously indicate the dangers of using cell lines known or suspected to be mycoplasma contaminated as immunogens for the production of monoclonal antibodies. REFERENCES i. Shin SI, van Diggelen OP: Phenotypic alterations in mammalian cells after mycoplasma infection. In: GJ McGarrity, DG Murphy, WW Nichols, Eds. Mycoplasma Infection of Cell Cultures. New York, Plenum Press, 1978, p. 191. 2. Howell DN, Machamer CE, Cresswell P: Elimination of mycoplasma from human Blymphoblastoid cell lines. Hum Immunol 5:233, 1982.
116
S.F. Radka et al. 3. Adams RA: Formal discussion: the role of transplantation in the experimental investigation of human leukemia and lymphoma. Cancer Res 27:2479, 1967. 4. Amos DB, Pool P, Grier J: HLA-A, HLA-B, HLA-C, and HLA-DR. In: NR Rose, H Friedman, Eds. Manual of Clinical Immunology. Washington, DC, American Society for Microbiology, 1980, pp. 978-986. 5. Mason DW, Williams AF: The kinetics of antibody binding to membrane antigens in solution and at the cell surface. Biochem J 187:1, 1980. 6. Chen TR: In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp Cell Res 104:225, 1977. 7. Polak-Vogelzang AA, Reygers R, Hekens FEN: Isolation of Mycoplasma hyorhinis and Mycoplasma fermentans from cell cultures. J Biol Stand 8:243, 1980. 8. Rosendal S, Black FT: Direct and indirect immunofluorescence of unfixed and fixed mycoplasma colonies. Acta Path Microbiol Scand B 80:615, 1972. 9. Barile MF: Mycoplasma-tissue cell interactions. In: JG Tully, RF Whitcomb, Eds. The Mycoplasmas. Vol. II, San Francisco, Academic Press, 1979, p. 425.
A B S T R A C T : We have produced two monoc/onal antibodies. SFR l-Myco 1 and SFR 1 -Myco 2. that detect Mycoplasma fermentans Jound to contaminate/ymphoblastoid cell lines (LCL). The specifici(y of these monodonal antibodies against the M. fermentans was determined by indirect immunofluorescence by demonstrating the reactivity of the monoc/onal antibodies with known re]erence strains of mycoplasma grown on soft agar. The reactivity of these antibodies against LCL in a number of immunoassays correlates completely with the presence of mycop/asma in these ce/i~ as determined by a standard mycoplasma detection assa). Because of the potential for ,'idespread contamination of B [ymphoblastoid cell lines tranfformed with Epstein-Barr virus-containing supernatants obtained from marmoset call lines contaminated with M. fermentans, these monoc/ona/ antibodies have value as screening reagents .fi)r this no'cop/asma species in LCL. ABBREVIATIONS
LCL HAT BLCL EBV
lymphoblastoid cell line hypoxanthine-aminopterinthymidine B lymphoblastoid cell line Epstein-Barr virus
PBA FACS
phosphate buffered saline, pH v.4, 2'7~ bovine serum albumin, 0.02(7{ sodium azide fluorescence-activated cell sorter
INTRODUCTION The existence of mycoplasma contamination in the majority of lymphoblastoid cell lines (LCL) currently used for research purposes has plagued investigators interested in using these lines for somatic cell hybridization experiments. Many of the mycoplasma strains which commonly contaminate LCL are capable of depleting thymidine from HAT*-selective medium, resulting in toxicity to hybrid cells, and thus often preventing successful fusions [ 1 ]. Mycoplasma-contaminated LCL often grow at a much slower rate than LCL free of mycoplasma. In the process o f developing a novel fusion protocol for the production of monoclonal antibodies to human Ia antigens, we used a mycoplasma-contaminated BLCL* (Swei) as immunogen, and produced a number of monoclonal antibodies
From the Department of Mierobiolo£1' and lmmuno/og3. Duke Uniz'ersitr Medical Center. Durham. North Carolina. National Institute fi)r Public Health. Bi/thoren. The Nether/andr: and Radio/o~hal Institute. TNO. Rijswyk. The Netherlanda. Address requeatsfor reprints to Susan F. Radka. Ph.D.. Amerkan Red Cro~.
111
112
S.F. Radka et al. directed against various cell surface antigens. When the Swei cell line was eliminated of mycoplasma by an eradication protocol recently described [2], two antibodies, SFR1-Myco 1 and SFR1-Myco 2, were no longer reactive. We describe here the reactivity of these antibodies with both mycoplasma-positive and -negative cell lines by a variety of assays. Isolation of Mycoplasmafermentans from a contaminated BLCL reactive with SFRl-myco 1 and SFRl-myco 2, and their reactivity with only this mycoplasma species confirmed the anti-mycoplasma specificity of these antibodies.
METHODS
Cell lines. The Swei cell line was the generous gift of Dr. John Hansen (Seattle, WA). LG-10 was obtained from Dr. Jack Silver (East Lansing, MI), GM3104 from the Mutant Cell Repository (Camden, NJ), and PB-4 from Dr. Nancy Collins (New York, NY). The JY cell line was obtained from Dr. Dean Mann (Bethesda, MD), while the Pala and A2M cell lines were transformed at Duke University from the peripheral blood of two different individuals. The cell line SB is an EBV-transformed BLCL described previously [3] while Raji is a BLCL obtained from a patient with Burkitt's lymphoma.
Immunization and fusion. Adult male K G H rats obtained from Dr. Thomas Gill and Dr. Heinz Kunz of the University of Pittsburgh, were immunized in the hind footpads with a 0.2 ml suspension of 107 Swei cells emulsified in Freund's Complete Adjuvant. Two weeks later the animals were boosted in the hind footpads with 107 Swei cells emulsified in 0.2 ml of Incomplete Freund's Adjuvant. Three days after boosting, the popliteal lymph nodes were removed and the lymph node suspension was fused with the SP2/O-Ag14 murine myeloma at a 10:1 lymph node to myeloma ratio. The fusing agent was polyethylene glycol (1000 MW, Fisher, Pittsburgh, PA) at a 35% concentration. Fusion products were plated into each of two 24 well tissue culture plates (Costar) containing 105 irradiated (1800 rads) murine peritoneal exudate cells. Growing hybrids were screened for antibody production by the Amos modification of the standard N I H microcytotoxicity test [4]. Positive hybrids were cloned in semisolid agar.
Radioimmunoassay. The cellular radioimmunoassay used for detection of antibody binding is a modification of the method described by Mason and Williams [5]. Briefly, 105 lymphoblastoid cells and 4 x 105 glutaraldehyde-fixed sheep red blood cells were incubated with 100 gl of tissue culture supernatant for 1 hr at 4°C in round-bottomed microtiter plates (Linbro, McLean, VA). After 3 washes with phosphate buffered saline, pH 7.4, 2% bovine serum albumin, 0.02% sodium azide (PBA*), 2 x 105 cpm of affinity-purified 125I-F(ab)'2 rabbit antimouse Ig were added to each well, and the plates were incubated at 4°C for 45 min. Wells were washed 3 times with PBA, after which the contents from each well were transferred to 6 x 50 mm test tubes for gamma counting.
Mycoplasma screening test. The assay for detection of mycoplasma is based on that described by Chen [6]. Briefly, a mouse fibroblast cell line used as indicator cells was incubated in 15 x 60 mm tissue culture dishes containing cover slips for 3 to 5 days with spent medium from the cell lines under investigation for mycoplasma contamination. Subsequently, the cells which adhered to the cover slip were fixed with Carnoy's solution and dried. A 0.05 g/ml solution of Hoechst 33258 stain in Hank's balanced salt solution, pH 7, was incubated with the fixed cells for 10 min at room temperature. After two washes with distilled water, the
Anti-mycoplasma Monoclonal Antibodies
113
slides were air dried. Coverslips were mounted in 0.1 M acetate-acetic acid buffer, p H 5.5. One hour after mounting, the slides were examined by fluorescence microscopy. A positive test was indicated by cytoplasmic and/or membrane (both nuclear and plasma) fluorescence, depending upon the degree of mycoplasma contamination.
Isolation of mycoplasma from BLCL. The procedure for isolation of mycoplasma from BLCL was described previously [7]. Briefly, the BLCL were incubated under anaerobic conditions on Difco PPLO medium (Difco) with 20U heat-inactivated horse serum and 10% (W/V) acid yeast extract for 5 days.
Flow cytometry. Cells to be analyzed were washed with PBA. Tissue culture ( 100 btl) supernatant containing monoclonal antibody was incubated with the pelleted cells for 1 hr at 25°C. The cells were washed twice with PBA, after which fluoresceinated goat anti-mouse Ig (Meloy, Springfield, VA) was added. After 45 min at 4°C, the cells were washed, resuspended in PBA, and analyzed for fluorescence with an Ortho Cytofluorograph model 50-15 equipped with an argon laser exciting at 488 nm.
RESULTS Tissue culture supernatants from lymphoblastoid cell lines to be tested for mycoplasma contamination by the Chert test were incubated for 4 days with the mouse fibroblast cell line, after which the remainder of the Chen protocol was followed. Concordantly, aliquots of cells, obtained from the same flasks as the supernatants for the Chen assay, were investigated for binding of the putative anti-mycoplasma monoclonal antibodies by three different assays: radioimmunoassay, FACS* analysis, and examination of individual samples under the fluorescence microscope. In all experiments, a monoclonal antibody detecting monomorphic determinants on human class II antigens was used as a positive control, and gave appropriate positive results. Results comparing ten different BLCL for mycoplasma contamination by the Chen assay and radioimmunoassay are summarized in Table 1. There is complete agreement between the results obtained by the Chen assay and the binding of the two monoclonal antibodies. In fact, the relative binding levels of the antibodies on various cell lines suggests that quantitative differences in amounts of mycoplasma expressed on the cell surface (therefore presumably the degree of mycoplasma contamination) can be detected by this assay. An examination of the data obtained by FACS analysis of these cell lines with the monoclonal antibodies (Figure 1) verifies that quantitative differences in degree of contamination exist among three mycoplasma contaminated cell lines. Figure l(a) depicts the lack of reactivity of SFR1-Myco 1 and SFR1-Myco 2 with mycoplasma-free Swei. In Figure l(b), Swei cells which had been contaminated intentionally with mycoplasma a week prior to assay show a pattern of low fluorescence intensity relative to the positive control. The Pala cell line [Figure l(c)], which has been contaminated for several years, exhibits a fluorescence profile with the anti-mycoplasma monoclonal antibodies which is much more intense than that for the newly contaminated Swei. The cell line LG10 [Figure l(d)] shows a fluorescence pattern intermediate to that of the other contaminated cell lines. These results correlate well with results obtained by radioimmunoassay. Although not shown here, the data obtained by examination of individual cells by fluorescence microscopy are in complete agreement with the results obtained by radioimmunoassay and FACS analysis.
114
S.F. Radka et al.
TABLE
1
C o m p a r i s o n o f results o f m y c o p l a s m a s c r e e n i n g by C h e n m y c o p l a s m a test vs. r a d i o i m m u n o a s s a y with S F R 1 - M y c o 1 and SFR1 M y c o 2 Binding of anti-mycoplasma antibodies b
Cell line
Chen test"
SFR1-Myco 1
SFR1-Myco 2
Swei (Myco +) Swei (Myco - ) JY Pala PB-4 SB GM 3104 A2M LG-10 Raji. 5
+ + + + +
3.4 0 0 8.4 6.3 0 0 7.1 4.0
2.5 0 0 6.6 5.2 0 0 6.8 3.7
-
0
0
"Determination of + or - is based on presence or absence of fluorescence of a cell suspension prepared as described in "Methods". bResults listed are ratios of binding of SFR1-Myco 1 and SFR1-Myco 2 relativeto the binding of a negativecontrol antibody.
F r o m the Pala cell line, m y c o p l a s m a s c o ul d be isolated on D i f c o - P P L O m e d i u m after a n a e r o b i c i n c u b a t i o n at 37°C for 5 days [7]. T h e S F R 1 - M y c o 1 and SFR1 M y c o 2 a n t i b o d i e s w e r e t e s t e d in the i n d i r e c t i m m u n o f l u o r e s c e n c e test o n unfixed agar c o l o n i e s [8] o f r e f e r e n c e strains o f M. arginini, M. fermentans ( r e f e r e n c e strain and that f r o m the Pala cell line), M. hyorhinis, and M. Orale, using a s h e e p a n t i m o u s e I g G - F I T C c o n j u g a t e . T h e y r e act ed only with c o l o n i e s o f the M. fermentans strains, with each a n t i b o d y reacting up to a d i l u t i o n o f 1:8. Since M. fermentans is t h o u g h t to be a fairly rare tissue c u l t u r e c o n t a m i n a n t [9], it is highly p o s s i b l e that the m a r m o s e t cell line used for E B V * t r a n s f o r m a t i o n o f p e r i p h e r a l
a
50-
~"
',"~
1 Fluorescence-activated cell sorter analyses of the reactivity of four monoclonal antibodies on different BLCL. ( ' ' ") indicates reactivity of negative control antibody on each cell line. ( - - - ) is monomorphic anti-human class II monoclonal antibody. (--) is SFR1-Myco 1. ( - ' - ) is SFR1-Myco 2. Panel (A) indicates fluorescence pattern of these antibodies on mycoplasma-free Swei; panel (B) is mycoplasma-contaminated Swei; panel (C) is the Pala BLCL; and panel (D) is LG-10. FIGURE
50 ~
/'4
,~50 k
~,, y,,,',,
'~
J 50 k
,
f',%r,, 2
INCREASING
FLUORESCENCE INTENSITY
Anti-mycoplasma Monoclonal Antibodies
l 15
blood B cells into BLCL was the source of mycoplasma contamination in the Swei cell line. Therefore, a sample of the B95-8 marmoset line which was used for transformation of the Swei cell line was obtained from Dr. J o h n Hansen and examined for reactivity with SFR1-Myco 1 and SFR1-Myco 2. It was also found to be contamined with 34. fermentans, based on reactivity with the monoclonal antibodies (the cell line was not tested for other mycoplasma strains). DISCUSSION The detection and elimination of mycoplasma contamination in lymphoblastoid cell lines has b e c o m e a major concern of researchers who use these cellular reagents for various investigations. Recently, a procedure for mycoplasma elimination from lymphoblastoid cell lines was described which results in reasonable success [2]. O u r discovery of the anti-mycoplasma monoclonal antibodies SFR1Myco 1 and SFR1-Myco 2 are the direct results of mycoplasma elimination from the immunizing cell line. By examining the supernatants from a BLCL with which the antibodies reacted, we have determined the specificity of the monoclonal antibodies and the strain of mycoplasma contaminating the cell line. O f four different strains of mycoplasma (M. arginini, M. hyorhin#, 34. orale, and M. fermentans) for which the supernatant and monoclonal antibodies were screenecl, only 34. jbrmentans was positive. Since this strain is thought to be the least c o m m o n tissue culture contaminant of the four strains and yet all the mycoplasma-contaminated BLCLs we tested carried 34. fermentans, we attempted to determine whether some of the marmoset cell lines used to transform peripheral blood B cells into cell lines were contaminated with 34. fermentans. A sample of the B95-8 line used to transform Swei, which was the source of the antigen against which these monoclonal antibodies were produced was indeed found to be contaminated with 34. fermentans. H o w e v e r , since it is not certain that the marmoset cell line was contaminated with 34. fermentans at the time supernatant was taken for transformation, we are not certain of the source of contamination of the Swei. We do know that until the M C U V - 5 marmoset cell line which we routinely use for transforming peripheral blood B cells was eliminated of mycoplasma, it was contaminated with M. fermentans, based on the reactivity of these antibodies. Therefore, until recently, any BLCL we have generated with supernatants from MCUV-5 are likely to be contaminated with 34. fermentans. We suspect that this may be the case for many BLCL which have been generated in a number of laboratories. Because of the possible widespread contamination with 34. fermentans of BLCL generated by contaminated EBV-containing supernatant, these antibodies are useful as screening reagents for detecting contamination by any of a n u m b e r of immunoassays which are simpler and more rapid than the standard assays used for mycoplasma detection. These results obviously indicate the dangers of using cell lines known or suspected to be mycoplasma contaminated as immunogens for the production of monoclonal antibodies. REFERENCES i. Shin SI, van Diggelen OP: Phenotypic alterations in mammalian cells after mycoplasma infection. In: GJ McGarrity, DG Murphy, WW Nichols, Eds. Mycoplasma Infection of Cell Cultures. New York, Plenum Press, 1978, p. 191. 2. Howell DN, Machamer CE, Cresswell P: Elimination of mycoplasma from human Blymphoblastoid cell lines. Hum Immunol 5:233, 1982.
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S.F. Radka et al. 3. Adams RA: Formal discussion: the role of transplantation in the experimental investigation of human leukemia and lymphoma. Cancer Res 27:2479, 1967. 4. Amos DB, Pool P, Grier J: HLA-A, HLA-B, HLA-C, and HLA-DR. In: NR Rose, H Friedman, Eds. Manual of Clinical Immunology. Washington, DC, American Society for Microbiology, 1980, pp. 978-986. 5. Mason DW, Williams AF: The kinetics of antibody binding to membrane antigens in solution and at the cell surface. Biochem J 187:1, 1980. 6. Chen TR: In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp Cell Res 104:225, 1977. 7. Polak-Vogelzang AA, Reygers R, Hekens FEN: Isolation of Mycoplasma hyorhinis and Mycoplasma fermentans from cell cultures. J Biol Stand 8:243, 1980. 8. Rosendal S, Black FT: Direct and indirect immunofluorescence of unfixed and fixed mycoplasma colonies. Acta Path Microbiol Scand B 80:615, 1972. 9. Barile MF: Mycoplasma-tissue cell interactions. In: JG Tully, RF Whitcomb, Eds. The Mycoplasmas. Vol. II, San Francisco, Academic Press, 1979, p. 425.