A rapid method for detection of mycoplasmas in mammalian cell cultures and comparison with other routine techniques

A rapid method for detection of mycoplasmas in mammalian cell cultures and comparison with other routine techniques Ulrich Valley,* Klaus Scharfenberg,” Katja Miller,* Thomas Ryll,*** and Roland Wagner* *Gesellschaft fiir Biotechnologische Forschung mbH, Cell Culture Techniques Department, Braunschweig, Germany ‘Present address: Genentech, Inc., Cell Culture and Fermentation Research & Development, South San Francisco, California

A .simple and rapid method for the detection of mycoplasmas in mammalian cell cultures has been delleloped on the basis of the enzymatic activity of adenosine phosphorylase. This enzyme. which has been found in high activity in mycoplasmas in contrast to mammalian cells, catalyzes the transformation of 6meth_vlpurine deoxyriboside (6-MPDR) into the two cytoto.xic products 6methvlpurine and 6-methvlpurine riboside and is the basis of an established indirect cytotoxicity test for mycoplasmas. In this study estimation of parasitic incidence relies on the direct visualization of the enzymatic conversion of 6-MPDR by isocratic ion-pair reversed-phase highperformance liquid chromatography. The final result of the test is available after 3 to 24 h of incubation of the c,ells together with the indicator metabolite 6-MPDR and depends on the adenosine phosphotyvlase actiGty of the respective mycoplasma species. The direct detection of enzymatic activity results in a high sensitivity, allowing the observation of infections that could not be found by the indirect cytoto.xiciQ test. Comparison of the direct adenosine phosphorvlase activity test with other commonly used methods reveals distinct cost and time advantages.

Keywords:

Mycoplasma

detection;

adenosine

phosphorylase;

ion-pair reversed-phase

Introduction Mycoplasmas

and cell culture

Mycoplasmas are the smallest prokaryotic organisms that propagate independently. Their variable morphology enables them to pass sterilizing filters of a pore size of 0.22 pm. Despite the improvement in sterilization techniques and quality control, animal cell cultures cannot be totally protected from infection with mycoplasmas. It has been

Address reprint requests to Dr. Wagner, Abteilung Zellkulturtechnik. Gesellschaft fiir Biotechnologische Forschung mbH, Mascheroder Weg 1, D-38124 Braunschweig, Germany This article is dedicated to the sixty-fifth birthday of Prof. Dr. Fritz Wagner Received 1ZJanuary 1994; revised 20 September 1994; accepted 3 October 1994

Enzyme and Microbial Technology 17:391-400, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

HPLC: mammalian

cell culture

estimated that between 1 and 92% of cell cultures are currently contaminated,‘,’ and that the extent of contamination is increasing. 3-6 In particular. continuous cell lines, which are used for the production of pharmaceuticals, have a higher incidence of infection than primary lines. Barile et al.’ detected mycoplasmas in 16% of continuous cell lines investigated. In contrast, only 1% incidence was found in primary cells listed in the same report. Four species from human, bovine, and porcine sources account for more than 98% of the isolates: Mycoplasma orale (human source), M. hyorhinis (porcine), M. arginini (bovine), and Acholeplasma laidlawii (bovine). 4.8 Mycoplasma contamination can have serious consequences, because many cellular processes can be altered by the infection.’ Acholeplasma laidlawii and M. hyorhinis are glucose-fermenting organisms. whereas M. arginini and M. orale convert medium arginine via arginine deiminase to omithine and ammonia,” resulting in the production of acid and alkaline shifts, respec-

0141-0229/95/$10.00 SSDI 0141-0229(94)00075-3

Papers tively. Moreover, glucose fermentation, arginine deprivation, and the production of ammonia may arrest growth of the eukaryotic cell line.

Elimination

rzk

6-MP (Ade)

Rib-l-P

Pi

6-MPR (Ado)

Figure 1 Biochemical transformation of 6-MPDR to 6-MP and 6-MPR, catalyzed by adenosine phosphorylase. The natural substrates are given in parentheses. Ade, Adenine; Ado, adenosine; dAdo, deoxyadenosine; Rib-l-P, ribose l-phosphate; dRib-l-P, deoxyribose l-phosphate

lies on the detection by high-performance liquid chromatography (HPLC) of enzymatic degradation products derived from 6-methylpurindeoxyriboside (6-MPDR). The method requires a minimum effort and time expenditure in comparison to all other routinely used methods.

of mycoplasmas

Owing to the potential risk of mycoplasma contamination a continuous monitoring of cell cultures is required. The small size of mycoplasmas allows them to evade detection in a light microscope, which has led to the development of alternative direct and indirect methods of detection. However, these differ drastically in their sensitivity, rapidity, and unambiguity. 3.5S23Effective methods include microbial culture techniques and fluorescence staining by application of fluorochromes such as 4’ ,6-diamidino-2-phenylindole(Hoechst dihydrochloride (DAPI)24 or bisbenzimide Alzani et a1.‘7 have reported a 33258). 25,26 Additionally, sensitive proliferation assay based on the stimulation or inhibition of thymidine incorporation by unstimulated splenocytes. Tests based on DNA-DNA and DNA-RNA hybridization and polymerase chain reaction (PCR) have been developed that have several times higher sensitivity and reliability than classic methods.28 These methods rely on the construction and application of special robes for the P detection of mycoplasmas in cell cultures.29- 3

Adenosine phosphorylase

activity

Another frequently used mycoplasma detection procedure is based on the biochemical difference between mammalian cells and mycoplasmas with res ect to the enzyme adenoB4 (see Figure I). This test sine phosphorylase (AdoPase)‘. is commercially available as a detection kit but does not always give definitive results. In addition, the common disadvantage of this and most other tests is a time-consuming procedure that results in a considerable delay before reliable results are obtained. However, early knowledge of contamination is an absolute necessity for cost-intensive biotechnological production processes based on mammalian cell cultivation. In this article we present a rapid method for the detection of adenosine phosphorylase-positive mycoplasmas that re-

392

Pi

t/-

dRib-1 -P

of mycoplasmas

The high frequency of infection has resulted in the development of many effective elimination methods,4.” including passage in athymic mice” or cocultivation with mouse macro hages,13 growth of cells in rabbit or guinea pig semm, ’ juse of nucleic acid analogs,15 antibiotic treatment,16 supplementation of culture medium with specific antisera and many other techniques. Treatagainst mycoplasmas,‘7 ment with antibiotics seems to represent the most effective and simplest alternative for elimination of mycoplasma from contaminated cell lines.” Highly efficient mycoplasma elimination kits based on mixtures of several different antibiotics, such as BM-cyclin, are commercially available. ’ 8-20 However, they are often cytotoxic to the eukaryotic cell line, which restricts their current and long-term use.‘l New fluoroquinolone antibiotics, such as ciprofloxacin, have been reported to be successful in decontaminating mycoplasma-infected cell cultures.6~‘6~‘8,2’~22

Detection

6-MPDR (dAdo)

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Materials and methods Cell lines and culture conditions The following cell lines were tested for AdoPase activity and/or for mycoplasma infection: BHK 21 C-13 (ATCC CCL-lo), a mouse embryo cell line NIHi3T3 (ATCC-CRL 1658). the human kidney cell line 293 (ATCC CRL-1573), the monkey kidney cell line COS-1 (ATCC CRL 1650), a transfected CHO-Kl cell line (CHO-Kl pMDIIIGPTR’S). and CHO DUKX-B 1 lj6; the murine hybridoma cell line MAXl6H5 (kindly provided by F. Emmrich. Institute for Clinical Immunology and Rheumatology, University of Erlangen-Ntirnberg. Niimberg, Germany): the mouse connective tissue cell line L-929 (ATCC CCL-l, NCTC clone 929); a mycoplasma-infected human promyelocytic leukemia cell line HL-60 (ATCC CCL-240). and two mycoplasma-infected human leukemia cell lines named CTV- 1 (derived from acute monocytic leukemia, DSM ACC 13) and L-428 (derived from Hodgkin’s lymphoma and kindly provided by the DSM-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). The latter two cell lines have been described in previous studies.23 Additionally, a mycoplasma infected murine embryo fibroblast cell line PC13 M (kindly provided by the Department of Gene Regulation and Differentiation, GBF, Braunschweig, Germany) based on a PG13 (ATCC CRL-10685) cell line was used as positive control. Cells were cultivated in standard cell culture flasks (Nunc, Roskilde. Denmark) at 37°C. 7.5% CO,, and 90% humidity. All cell lines except CHO grew in serum-free DIF medium according to Jgger er ~1.“’ or in a 10% fetal calf serum (FCS)containing medium based on IF (Iscove’s-Ham’s F2, 1:l) or in Dulbecco’s modified Eagle’s (DME) basal medium as described earlier.38 CHO cells were cultivated in a special protein-free medium formulation (SMIF). Mycoplasmafermentuns was cultivated as described.‘” Cell counting. Cell were counted by means of a hemocytometer. The number of dead cells was estimated by trypan blue exclusion.

Protein determination. Protein concentration the bicinchoninic acid method.”

was quantified

by

Mycoplasma Mycoplasma

detection methods

Direct fluorescent DNA staining. This method is based on cytochemical staining with fluorescence dyes DAPI or Hoechst 33258, which stain any DNA present in the preparation.24.‘5.4’ Cells to be tested and mycoplasma-infected cells (positive control) at a density of 2 . 10’ ml - ’ were cultured in chamber slides (Nunc Lab Tek Tissue Culture Chamber-Slide; Nunc). After 12-24 h the slide was washed with sterile phosphate-buffered saline (PBS) and stained for 15 min at 37°C in DAPI-methanol solution (Sigma, St. Louis, MO). After washing with water and drying with air the preparation was mounted in Mcllvain’s citrate buffer (pH 5.5): glycerin (2: 1) and sealed with nail polish.23 The slides were analyzed under a fluorescence microscope (Zeiss Axiovert 100 [Zeiss. Oberkochen, Germany] lamp HBO 50 and ultraviolet filter set; excitation BP 450-490. beam splitter FI 510, barrier filter LP 520).

ELISA An enzyme-linked immunosorbent assay (ELISA) is commercially available (Mycoplasma detection kit; Boehringer GmbH, Mannheim, Germany) that contains polyclonal antibodies directed toward antigens of the most frequent mycoplasma/acholeplasma species in cell cultures (M. arginini, M. hyorhinis, A. luidluwii, and M. orde). Antibodies for detection were conjugated with biotin and visualized by a streptavidin-alkaline phosphatase assay based on enzymatic hydrolysis of 4-nitropheno! phosphate to a yellow nitrophenol product, which was quantified macroscopically or by use of a microplate reader at a wavelength of 405 nm (SLT 340 ATTC; SLT Laboratory Instruments, Griidig, Austria).

DNA-based polymerase amplification test

chain reaction

Mycoplasmas were detected using species-specific DNA oligonucleotide primer sequences. The test is based on a double-step PCR analysis that employs primers that anneal to gene sequences encoding the evolutionarily conserved 16s rRNA of mycoplasma species most commonly found in cell cultures.” Sterility of all material used must be guaranteed. Samples were prepared as follows: 5 . 10’ test cells were sedimented for 1 min in a centrifuge at 13,000 ,q. The pellet was resuspended in 250 ~1 of water. One hundred microliters of this suspension was added to a pellet obtained by a IO-min centrifugation of 1 ml of supematant from the cell culture to be tested and pellet and supematant were resuspended. The sample was incubated at 95°C for 3 min and the precipitated protein was discarded by centrifugation for 1 min. The supernatant was harvested and stored at 4°C. Subsequently, PCR analysis was performed according to Hoper? et ~1.~~One microliter of this test DNA solution is included in a 50-k! reaction mix for the first PCR. Ten microliters of the first PCR round was used for the second round. Cycle programs were started with a heat step of 5 min at 94°C (DNA thermal cycler 480; Perkin Elmer Instrument Division, Norwalk, CT). Reaction products were separated by agarose gel electrophoresis (AGEP), stained with ethidium bromide and visualized under ultraviolet light, and photographed.

Biochemical 6-MPDR cytotoxicity AdoPase test)

detection

comparison:

U. Valley et al.

(6.MPR) (Figure I). An AdoPase kit is commercially available (MycoTect; GIBCO-BRL, Eggenstein. Germany). Tests were performed with mycoplasma-free 3T6 cells (ATCC CCL-96) as indicator cell line. A total of 2 10J cells in I .5 ml of mycoplasmaand antibiotic-free Dulbecco‘s modified Eagle’s medium (DMEM) with 10% FCS was inoculated in each well of a 24-well plate and incubated at 37°C in an air atmosphere with 5% CO,. After 3 h of incubation 200 ~1 of the supernatant from each culture to be tested was placed into each of two wells. On the next day, 25 and 50 p.1 of 6-MPDR (1 mmol I-‘) were added to well B and well C. respectively ( 15 kmol l- ’ well B and 30 pmol I- ’ well C end concentration; see Figure 2). One well filled with AdoPase instead of a sample but with 6-MPDR served as a positive control (P.C. ). Negative controls were performed by adding medium alone (N.C. - ) and medium together with 6-MPDR (true negative. N.C. + ). respectively. After incubation for 3 to 4 days (confluence of the untreated cells) viability was determined by staining with 0.2% crystal violet (E. Merck AC, Darmstadt. Germany) in 10% (v/v) formaldehyde solution (Riedel de Haen. Seelze. Germany )

6-MPDR conversion detected b! HPLC (direct AdoPase test) The direct AdoPase test is based on the direct detection of 6-MP synthesized from 6-MPDR by action of AdoPase. No indicator cell line is used during this assay. A stock solution of 6-MPDR was prepared as followed: 5 mg of lyophilized 6-MPDR (Boehringer GmbH) was mixed with 2 ml of sterile PBS. One hundredmicroliter samples of this 10 mmol-1~ ’ solution were frozen at - 20°C until used in the test. Two milliliters of cell suspension with a maximum density of 5 IO’ ml was added to 40 CL!of a I mmol-! _ ’ 6-MPDR work solution dissolved in PBS in a petri dish (Nunclon Delta. 35.mm diameter; Nunc). After different incubation times 300-p,! samples were taken from the petri dishes containing test and control cultures. Samples were purified from major proteins and other high molecular weight components by ultrafiltration using a membrane cutoff of 10.000 (Nanospin NMWCO 10000: Gelman. Ann Arbor. MI). The supematant was

123456

A B

C D

assay (indirect

Identification of mycoplasmas is based on the different biochemical constitution of the extracellular parasitic organism and mammalian cells with respect to the enzyme adenosine phosphorylase.’ AdoPase is able to transform 6-MPDR, a nontoxic analog of adenosine, into the two toxic products 6-methylpurine (6-MP) and, in the presence of ribose l-phosphate, to 6-methylpurine riboside

Figure 2 Cytotoxicity test for the detection of mvcoplasma infections. Wells 1 B and C, true negative control (NC. T 1; column 2 (B-D), BHK-21 C-13 without mvconlasma; column 3 (B-D). Cl%-1 infected with M. arginini; c&umn 4 (B-D), L-428 infected with M. orale; column 5 (B-D), L-929 infected with an unknown mycoplasma species; column 6 (ED), positive control (P.C.); row A (l-6) and well 1 D. negative control (N.C. - )

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Papers centrifuged for 15 min at 10,000 g (Hettich Microliter; Hettich, Tuttlingen, Germany) and the clarified ultrafiltrate was stored at - 70°C or directly loaded onto the HPLC column. When the direct test was compared with the indirect cytotoxicity method, two 24well microplates were prepared as described for the indirect AdoPase assay. Samples for HPLC analysis were taken from 1 of the parallel incubated 24-well microplates. Standard deviation of 6-MPDR analysis by HPLC was 0.4 for 20 mm01 1~ ’ (calculated from 10 independent HPLC runs). The confidence interval was calculated to 0.8 mm01 l- ’ (99%). A decline > 1.6 mmol I - ’ was set as a threshold value for a significant AdoPase activity.

Ion-pair reversed-phase high-performance liquid chromatography Separation and quantification of 6-MP and 6-MPR formed and 6-MPDR consumed were performed by an ion-pair reversed-phase HPLC system based on a method described earlier by Ryll and Wagner.42 The total elution time took 25 min. The basic solution for mobile phases consisted of KH,PO,/K,HPO, buffer (100 mm01 l-, pH 6.0). Buffer A consisted of basic solution supplemented with tetrabutyl ammonium hydrogen sulfate (8 mmol 1- ’ , pH 5.3). Buffer B contained 70% buffer A plus 30% methanol, pH 5.9. The gradient was as follows: 3.0 min, 100% buffer A; 12 min, MO% buffer B; 0.5 min. 40-100% buffer B; 6 min. 100% buffer B; 0.5 min, 100-O% buffer B, followed by an equilibration phase of 100% buffer A for 8 min. The flow rate was adjusted to 1.5 ml min-‘. Temperature was set to 26°C. For a rapid detection of mycoplasmas in cell culture this gradient procedure has been simplified to an isocratic process characterized by the following elution parameters: KH,PO,/K,HPO, (100 mm01 l- ‘, pH 5.6) supplemented with tetrabutyl ammonium hydrogen sulfate (8 mmol I- ‘) dissolved in water plus 15% methanol were used as elution buffer at a flow rate of 1.5 ml min ’ Total elution time from sample to sample takes only 10 min. Reference substances were purchased from Sigma. Peaks were identified by comparison of retention times, spiked samples, calculation of ratio between absorbance at 254 and 280 nm, and on-line scanning of ultraviolet (UV) spectra.

Identification of mycoplasma species The identification of mycoplasmas in the infected leukemia cell lines (CTV-1, L-428) was performed by Uphoff et al.‘” The CTV-1 cell line was infected with M. arginini, the L-428 culture contained M. orale. The HL-60 cell line infected with M. fermentans was kindly provided by P. Miihlradt (CJBF).~~ The hybridoma cell line MAXl6H5 was infected with A. laidlawii as detected by the ELISA. Infection of L-929 cell line was determined by direct DAPI staining. Mycoplasmas of PG13 were detected by PCR. Mycoplasma species of these latter two cell lines were unknown.

anol or perchloric extraction

acid (PCA) as described earlier for the of nucleotides.42 These methods have been cho-

sen to inactivate directly the mycoplasmas and mammalian cells and to separate quantitatively the proteins from the supernatant.42 However, both procedures resulted in a cleavage of the substrate 6-MPDR. Therefore we replaced precipitation by ultrafiltration through a Nanospin filtration unit that contains a low protein-binding membrane. Filtrate was obtained by centrifugation of the filtration units at 10,000 g. During 15 min of centrifugation 250 ~1 of filtrate was produced from 300 ~1 of culture supematant. The average recovery for all three substances was 100% for standard solutions, indicating that no significant adsorption to the membrane occurred. Substances that interfered with 6-MPDR, 6-MPR, and 6-MP during separation by highresolution gradient HPLC were removed by ultrafiltration (Figure 3~). Using the more advantageous rapid analysis by isocratic HPLC, a clear separation of 6-MP and 6-MPR from medium and cell-derived compounds could not be obtained (Figure 36). However, 6-MPDR alone could be reliably quantified because no interfering substances could be detected. Hence, the kinetic decline in 6-MPDR was used for estimation of the enzymatic activity when the rapid isocratic method was selected. Separation and identification of a standard mixture of the three substances 6-MPDR, 6-MPR, and 6-MP with isocratic HPLC is shown in Figure 3~. 6-MPDR was eluted after 3.8 min whereas 6-MP and 6-MPR appeared after 2.5 and 3.5 min, respectively. Quantification is possible in the range between 1 and 50 kmol l_ ’ The use of an ultrafiltration unit allows removal of most of the proteins and other high molecular weight compounds that interfered in the elution profile. All other remaining substances were eluted within the first 2 min of the HPLC elution and thus did not interfere with the assay. The entire procedure was finished within 10 min. In conclusion, the whole extraction procedure, from taking the sample to the final result. requires 30 min. This test is therefore able to quantify AdoPase activity by detection of the transformation of 6-MPDR. In addition, 6-MPR could not be synthesized from 6-MPDR by mycoplasma-infected cell cultures because no ribose l-phosphate is present. This result could also be confirmed by gradient ion-pair RP-HPLC (Figure 3c), which gave a higher resolution of the antimetabolites (data not shown). For isocratic HPLC it was not necessary to separate 6-MPR from the coeluting tryptophan, as shown in Figure 3b for a mycoplasma-infected MAX16H5 hybridoma cell line, hence the kinetic decline in 6-MPDR should be sufficient for quantification.

Elimination of mycoplasmas Mycoplasma-positive hybridoma MAX16H5 cells were treated with ciprofloxacin-supplemented serum-free DIF medium for 3 weeks according to Schmitt et aZ.18 Cells were exposed to Ciprobay (Ciprobay 100, 10 mg 1-l; Bayer AG, Leverkusen, Germany) in standard cell culture flasks.

Results Extraction and identification of methylpurines Extraction of methylpurines from culture supematant first performed by precipitation of proteins with either

394

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Detection of AdoPase activity by HPLC Different cell lines were tested for 6-MPDR conversion by AdoPase activity from mycoplasmas (Figure 4). Two parallel 24-well microplates filled with 3T6 cells serving as sensitive cell line were used to compare the direct and indirect AdoPase tests. Samples for HPLC analysis were taken from the microplates after 22, 48, and 72 h. Twentytwo hours after the start of incubation with 6-MPDR the conversion was obvious for L-929 and the positive control (P.C.), using the direct HPLC technique. The decrease in 6-MPDR is shown in Figure 4~. One day after the begin-

Mycoplasma

a

b

detection

comparison:

U. Valley et al.

Elution Time /min

Elution Time /min

Elution Time /min

Figure 3 Separation of 6-MPDR, 6-MPR, and 6-MP by isocratic (a and b) and gradient (c) ion-pair reversed-phase HPLC. Substances at a concentration of 20 kmol I-’ were used and a 100~t.d mixture of the three substances was loaded onto the column. Separation of standard substances in water (a) and mycoplasma test for MAX16H5 after a 3-h incubation of 6-MPDR (20 pmol I ‘) (b). One hundred microliters of a 20-pmol I-’ mixture of 6-MP, 6-MPR, and 6-MPDR in medium supplemented with 10% FCS after clarification through the ultrafiltration unit (c). HPLC separation takes 5 min for isocratic (a and b) and 13 min for gradient elution (cl

ning of the test 6-MPDR was quantitatively transformed into 6-MP by culture supematant derived from the L-929 cell line, indicating a high incidence of infection. This mycoplasma contamination has already been clearly visualized by direct DAPI staining. No 6-MPR was produced by this cell line, as expected from the absence of ribose l-phosphate (data not shown). The true negative control with 3T6 cells supplemented with 6-MPDR (N.C. +) did not produce significant amounts of 6-MPR and/or 6-MP. The two leukemia cell lines CTV-1 and L-428, which were infected with known mycoplasma strains, have already been used to compare various mycoplasma detection methods for their efficiency.23 The CTV-1 cell line was contaminated with M. arginini whereas L-428 cells were infected with M. or-ale. In both cases a mycoplasma contamination could not be detected by the commonly used cytotoxicity test. Uphoff et ul. concluded that these mycoplasma species do not contain AdoPase active enough to exceed the sensitivity level

of the test kit. However. even low enzymatic activity should be visualized by direct observation of the 6-MPDR conversion with HPLC. Indeed, the conversion of 6-MPDR into 6-MP monitored by gradient HPLC indicated a small but significant mycoplasma contamination of both cell lines (Figure 4). In the L-929 sample 6-MPDR was quantitatively transformed after 22 h. The positive control (P.C. ) with the pure enzyme alone converted the substrate completely only after 72 h, suggesting either a damaged or unstable enzyme. Supematant of the noninfected BHK 21 C-13 cell culture (negative) and the negative control (N.C. + ) did not show any significant conversion of 6-MPDR. In conclusion, the kinetic analysis of the formation of 6-MP allows characterization of the incidence of infection from the enzymatic activity of AdoPase alone. An enzymatic activity of more than 1200 nmol 1.. ’ h ~-’ was found for the infected L-929 cell line. For comparison the lightly infected CTV-1 sample showed an activity of only Enzyme

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395

--

0

a

2o

4o

”

Incubation time I h

so

40

60

60

b” 0

‘,=10

1:. 20

I

I

30

40

49 nmol 1-i h- ’ (data calculated from the average slope of 6-MP formation during 72 h). In all following direct AdoPase tests the mycoplasmasensitive 3T6 cell line was omitted. To check the mammalian cell-derived conversion of 6-MPDR, different cell lines were tested for their cellular AdoPase activity (Figure 5). For all cell cultures investigated no significant AdoPase activity could be determined within 72 h of incubation. In addition, reliability of this method was verified by analyzing the intracellular enzyme activity released from 5 . 10’ lysed cells ml - ’ (Figure 5b). No AdoPase activity in mammalian cells could be detected within the time period used for the test. Moreover, BHK 21 cells were infected with four different amounts from a pure culture of M. fermentans39 corresponding to 31, 125, 250, and 2,000 ng of pr$in ml - ’ (a pp.roximately 10 kg of mycoplasma protein was found m the supematant of a strongly infected HL-60 cell line). Cell culture infection with the highest dose was detectable after 3 h whereas light contamination was detected after 24 h of incubation with 6-MPDR (Figure 5b).

396

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\

.J=m

I

60

50

Incubation time / Figure 4 Transformation of 6-MPDR as a result of adenosine phosphorylase activity in different cell culture samples (a) and production of 6MP (b). Samples (200 ml) consisting of culture supernatant and approximately 1,000 cells were added to mycoplasma-free 3T6 cells for comparison with the cytotoxicity test (Figure 2). Subsequently, 50 ~1 of a 6-MPDR-containing solution (1 mmol I-‘) was added to the wells of a 24-well plate. After 24 h the total 6-MPDR has been converted by cultures of the L-929 cell line. The cell lines CTV-1, L-428, and L-929 were infected with mycoplasmas; BHK cells were mycoplasma-free. P.C., Positive control with AdoPase; NC. +, a true negative control only with 3T6 cells and 6-MPDR; N.C. -, a negative control without 6-MPDR

NIH3T3 293 CHO DUKX-Bll BHK 21 cos-1 L-929

+~

Incubation time /h

100

b ’

20

t

1::. 70

h

Figure 5 Detection by the direct HPLC method of AdoPase activity in six different cell lines. (a) Normal cell-derived enzyme activity in the culture broth. (b) Intracellular AdoPase activity of noninfected BHK 21 and CHO DUKX-Bll cells. In addition, four profiles for BHK 21 cells, which were infected with different mycoplasma concentrations, are presented. BHK cells were infected with different mycoplasma concentrations expressed in nanograms of mycoplasma protein; low, 31 ng ml-‘; medium, 125 ng ml-‘; high, 250 ng ml-‘; very high, 2,000 ng ml-’

AdoPase is stable toward freezing such that samples can be stored frozen for later testing (data not shown).

Detection of AdoPase activity by the cytotoxicity

test

Results from the first experiment obtained by HPLC measurements (Figure 4) were checked by the commercial cytotoxicity test based on the same enzymatic activity (Figure 2). Wells 1B and C show the true negative control (N.C. + ) and revealed a confluent cell layer of the 3T6 control cell line. In contrast, cells growing in wells of row A (1-6) and ID responded with reduced growth although this row represents the negative control without 6-MPDR (N.C. - ). These cells had formed multilayers and were sheared off from the surface after staining. The cells growing in column 2 (B-D) were treated with culture supematant from noninfected BHK-2 1 C- 13. Confluent layers were found for cells in wells 2B and 2C, indicating no mycoplasma infection and confirming the data obtained by the direct HPLC technique. Cells growing in wells of column 3 and particularly 4 (B-D) also responded with good growth, which is evidence of a mycoplasma-free culture. However, both test

Mycoplasma cell lines, CTV-1 and L-428, were infected with M. arginini and M. orule, respectively, such that reduced growth had been expected for cells in columns 3 and 4. Only the test for infection of L-929 cells (column 5), which showed the highest enzymatic activity (Figure 4), gave clear results and responded with reduced cell growth. In conclusion, the cytotoxicity test often did not give clear results. Sometimes even the positive control gave no growth inhibition response. This problem could be explained by a damaged enzyme or an instability during storage. In addition, in many cases a bad staining of the negative control occurs, although best growth should have been achieved. Moreover, a low enzymatic activity in this test, as demonstrated for the L-428 cell culture by HPLC (see above), resulted in only small amounts of 6-MP being produced, which is too low for a clear cytotoxic effect on the indicator cell line.

Detection of rnycoplasmas by polymerase reaction and direct AdoPase test

chain

The direct AdoPase test for the detection of mycoplasma infection was compared to the sensitive PCR test. Figure 6 shows an AGEP including the first (lanes 2-6) and second PCR (lanes 7-l 1) for cultures of PG13 M (lanes 2 and 7, true positive), MAX16H5 (lanes 4 and 9, negative; 5 and 10, positive) and CHO-K 1 pMDIIIGPTR (6 and 11, negative). The first round of PCR resulted in oligonucleotides of approximately 520 bp (lanes 2 and 5) and the second round of PCR revealed a main band at 330 bp (lanes 7 and IO). The PG 13 M true positive control (lane 2 and 7) consisted of an unspecified mycoplasma infection. Infection of hybridoma and CHO cells has been identified by ELISA as A. laidlawii, which was probably introduced by supplementation of the culture media with serum components. MAX16H5 and the CHO cell line were also investigated by

detection

comparison:

U. Valley et al.

the HPLC technique. A high enzymatic activity of 3.3 pmol 1~ ’ h- ’ were found for the hybridoma cell line (Figure 7), whereas no significant decline in 6-MPDR could be detected for the recombinant CHO cells (data not shown).

Elimination

of mycoplasmas

with ciprojloxacin

The hybridoma cell line MAX16H5, which was contaminated with A. fuidlawii (ELISA and PCR test), was treated with ciprofloxacin for 3 weeks. Figure 7 shows the transformation of 6-MPDR before and after treatment with ciprofloxacin. 6-MPDR was totally converted within 3 h by the mycoplasma-infected culture. The enzymatic activity in the cell-containing sample was twice as high as in the centrifuged supematant (3 min, 190 g). which was free of mammalian cells but contained mycoplasmas. No AdoPase activity could be detected in the ciprofloxacin-treated culture, indicating that the cells were successfully cured of mycoplasmas.

Discussion Direct detection of AdoPase A direct detection of reaction products of the adenosine phosphorylase-catalyzed phosphorolytic cleavage of the substrate 6-MPDR by mycoplasmas has been developed on the basis of a simple and rapid isocratic reversed-phase HPLC method. The extent of contamination could be estimated by calculation of the enzymatic activity based on educt (isocratic method) and/or product (gradient method) quantification (Figure 2). The test requires a minimum of experimental expenditure as only the indicator metabolite must be added for preparation of the test cell culture. The HPLC analysis provides reliable results after an incubation time of 3-24 h (Figures 3 and 6). No 6-MPR was formed when media without ribose were used. Although it is possible to detect the natural substrates of AdoPase, the use of

bp

20

1631

517 396 298 220 154 75

Figure 6 Agarose gel stained with ethidium bromide containing the reaction products following PCR amplification. Lanes 2-6, first round of amplification with 30 cycles resulting in products of 500-520 bp according to the mycoplasma species; lanes 7-11, second round of amplification using the inner primers together with the oligonucleotides obtained from the first round resulting in reaction products of 310-330 bp; lane 1, pBR322 (cut with Hinfl) as size marker (in bp: 1631, [517, 5061, 396, 344, 298, [221, 2201, 154, 75); lane 2, cell line PG13 M (control, true positive); lane 3, water (control, true negative); lane 4, MAX16H5 (negative); lane 5, MAX16H5 (positive); lane 6, CHO-Kl pMDlllGPTR (negative); lanes 7-l 1, second PCR corresponding to lanes 2-6

0

5

20

25

Figure 7 Elimination by ciprofloxacin of mycoplasmas from MAX16H5 hybridoma cells. Detection has been performed by the direct isocratic HPLC method. After 3 weeks of continuous antibiotic treatment no AdoPase activity has been found. (0) Sample from cured cell culture; (A) sample from mycoplasmacontaminated supernatant; (0) sample from mycoplasmacontaminated cells

Enzyme

Microb.

Technol.,

1995, vol.

17, May

397

Papers 6-MPDR is suggested because separation and quantification of deoxyadenosine and adenosine are impeded by medium components. Retention times of adenine and deoxyadenosine by the gradient HPLC method have been determined as 3.2 and 8.1 min, respectively (data not shown).

Comparison

of AdoPase

detection methods

Direct estimation of AdoPase activity based on HPLC was compared with the conventional indirect cytotoxicity test, which relies on the same enzyme. The cytotoxicity test requires a sensitive test cell line that must be free of mycoplasmas. Furthermore, it is influenced by growth differences resulting from the variable microenvironments in the wells of the microplate. The direct determination of antimetabolites by HPLC does not have these disadvantages. In addition, sensitivity of the indirect AdoPase test is lower than the direct method because the indicator cell line used for cytotoxicity responds only when the concentration of the toxic reaction products exceeds the cell-specific threshold value or when the incubation time is extremely long. The use of a sample with a low cell concentration and its dilution with the culture broth of the sensitive cell line also contributes to the lower sensitivity of the cytotoxicity test. On the other hand, McGarrity’ found that 6-MPDR (10 p,mol l- ‘) caused a cytotoxic effect on the indicator cell line in 96.6% of cases of mycoplasma-infected cell cultures, indicating that most of the commonly found mycoplasma species have enough AdoPase activity to synthesize cytotoxic amounts of 6-MP. Moreover, McGarrity and Carson4” reported that 6-MP and/or 6-MPR ( I pm011 - ‘) significantly reduced cell viability after 4 days of incubation. 6-MPR is considered to be more toxic than 6-MP although in most cases 6-MPR is not produced. The cytotoxicity test often gave ambiguous results by showing divergent positive detection rates. Yoshida et a1.44 reported a detection rate of 56% whereas other authors determined 76,5%45 and 97-100%.32 This variation in the detection rates is most likely explained by the different activity of AdoPase in the different mycoplasma species and strains as shown by the mycoplasmas present in L-929, CTV- 1, and L-428 cultures. The higher sensitivity of the HPLC method allows a much quicker analysis compared with the cytotoxicity test. This indirect test requires a minimum incubation time of 3 to 4 days, which is too long for Table 1

Time required

by various mycoplasma

a fast routine control. In contrast, ion-pair reversed-phase HPLC is able to detect heavy infections after a 3-h incubation time, and incubation times can be prolonged as necessary to increase the sensitivity in the case of lighter mycoplasma infections or diluted samples (Figure Sb). Table 1 summarizes the various times for sample preparation and the total time necessary to obtain the final result for both procedures and for other standard test methods currently being used.

Comparison with other mycoplasma detection methods A further strategy for the detection of mycoplasmas in cell cultures is based on the presence of the enzyme arginine deiminase, which is a constitutive part of the arginine dihydrolase pathway necessary for energy supply in many mycoplasma species. ‘OThis enzyme has been proposed for the detection of contaminating nonpathogenic mycoplasma species (M. hominis, M. fermentans, M. salivarium, and M. gallinarum) whereas most pathogenic species (M. pneumoniae, M. gallise ticum, M. neurolyticum, and M. hyorhinis) POThe concentration of nutrients such as were. negative. amino acids is often monitored in mammalian cell cultures. Amino acid analysis, performed by either gas chromatography or HPLC.46,47 can be used to detect omithine formed by the enzymatic conversion of arginine via arginine deiminase. This enzymatic activity is generally low in mammalian cells, such that the presence of larger amounts of ornithine is a good indication of the presence of mycoplasmas. The experimental expenditure for this method is similar to that of the other HPLC technique for the detection of methylpurines. The serious disadvantage of this test is the limited spectrum of detectable species. The DNA-based PCR amplification test is the most sensitive detection method available, but the procedure is time consuming. Special precautions are needed to avoid falsepositive results. Moreover, primer synthesis and enzymes are expensive. Fluorescence microscopy after cell staining with DAPI is the least expensive detection method but only heavy infections can be clearly detected. ELISA also allow the determination of contaminating mycoplasma species, but detection is limited to only four species. Table 1 summarizes the time necessary for the five standard mycoplasma tests in comparison to the direct AdoPase

detection methods Time (hr) required Procedure

Direct DAPI

ELISA

ArgDase

Cytotoxicity

Sample preparation Preparation and procedure Incubation Detection and visualization

0.5 0.5 24 0.3

3.5 8.3 12 0.2

0.5 1 24 1

0.5 1 70-93 1.5

Total time required:

25

24

26.5

72-96

Step

Abbreviation:

398

ArgDase,

Arginine

Enzyme Microb.

deiminase

Technol.,

1995, vol. 17, May

PCR 0.5 8 6 1.1 lo-12

HPLC 0.5 1 3-24 0.1 4-25

Mycoplasma Table 2 detection

Comparison tests

of sample

costs (DEM)

Assay

1.90

costs for different

Sample volume (ml)

Agar-broth” Direct DAPI

0.10

0.4 0.1

Indirect

2.60

0.1

RNA hybridizationa ELISA

26.30 17.45a

1.5 2.0

Cytotoxicity

1 5.40a

0.4

DAPI”

4.70

0.1

14.75 14.10

0.1 1.0

3.95

0.3

aTaken from Uphoff et aLz3 One dollar is approximately

1.5 DEM.

MAb” ArgDase PCR Direct AdoPase

4 5

Fluorescence microscope Fluorescence microscope p Counter Optional microplate reader Price reduced when TestKit is replaced by 6-MPDR” Fluorescence microscope Amino acid analysis Thermal cycler, AGEP HPLC

test by HPLC. The cytotoxicity test is three to four times more time consuming than the other procedures. In contrast, data become available after 1 day using the ELlSA and after 12 h when applying the PCR. The direct AdoPase test is the fastest detection procedure, giving results after 3 h of incubation (Figures 3 and 7) if a heavy contamination is present. The sensitivity of the direct quantification of AdoPase activity can be increased by elongation of the incubation time (normally up to 24 h). This is an important prerequisite for the control of a successful mycoplasma elimination procedure. Twenty-four hours of incubation, as shown in Figure 7, is necessary to verify complete clearance of contaminating organisms. Current costs of one sample are in the range of 4 DEM, which is four times lower than the cytotoxicity test (Table 2) provided that standard laboratory and cell culture equipment are present.

6

7

8

9

10

II

I2

I3

IS

I6

I9

20

22

M. F. Mycoplasma-tissue cell interactions In: The MycoII. (Tully, J. G. and Rasin, S., Eds.). Academic Press, New York. 1979, pp. 425414 McGarrity, G. I. Detection of mycoplasmal infection of cell cultures. In: Advances in Cell Culfure. Vol. 2 (Maramorosch. K.. Ed. ). Academic Press. New York, 1982. pp. 99-131 Del Giudice, R. A. and Gardella, R. S. Mycoplasma infection of cell culture: Effects, incidence, and detection. In: Uses and Sran-

23

plasmus

2

3

Jeanason. S. and Brorson, J. E. Ehmindtion of mycoplasmas from cell cultures utilizing hyperimmune hera. Erp, Cell Res. 1985. 161, 181-188 Uphoff. C. C.. Gignac. S. M.. and Drexler, H. G. Mycoplasma contamination in human leukemia cell lines. II. Elimination with various antibiotics. J. Immunol. Merhods 1992. 149, 55-62 Branch. D. R. and Guilbert, L. J. Practical in virro assay systems for the measurement of hematopoietic growth factors. J. Tissue Culture Methods 1986. 10. lOl&lOR Ravaoarinoro, M. and Lacomte. J. Evaluation of three methods for curing hydridomas from mycoplasma contamination. Hybridoma 1988. 7, 79-86

?I

References Barile.

1980, 285, 661-662

Nair. C. N. Elimination of mycoplasma contaminants from cell cultures with ammal serum. Prw .Soc t’vp. Biol. Med. 1985. 179, 25&25X Marcus. M.. Lavi. U.. Nattenberg. A. Rottem, S.. and Markowitz. 0. Selective killing of mycoplasmas from contaminated mammalian cella in cell culture. Narurr (London) 1080. 285, 659-661 Schmitt, K.. DBubener. W.. Bitter-Suermann, D.. and Hadding. U. A safe and efficient method for elimination of cell culture mycoplasmas using ciprofloxacin. J. Immwwl. Methods 1988. 109, l72

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U. Valley et al.

1984, pp. 104-I I5 Hay, R. J.. Macy. M. L.. and Chen, T R. Mycoplasma infection of cultured cells. Nature (London) 1989. 339, 487-488 McGarrity, G. J. and Kontani. H. Cell culture mycoplasmas: Mycoplasma pathogenicity. In: The Mycoplasma Vol. IV (Razin. S. and Barile. M. F.. Eds.). Academic Press. New York. 1985. pp. 353390 Mowles, J. M. The use of ciprofloxacin for the elimination of mycoplasma from naturally infected cell line\. C\torechno/ogy 1988. 1, 355-358 Barile, M. F., Hopps. H. E.. and Grabowski. M. W. Incidence and sources of mycoplasma contamination: A brief review. In: Mvcoplusmu Infection ofCrllCultures (McGarrity. G. J.. Murphy. D. G., and Nichols. W. W.. Eds. I. Plenum Press. New York, 1978. pp. 35-45 McGarrity. G. J.. Vanaman. V.. and Sarama, J. Factors influencing microbial detection of mycoplaamas in cell culture. In Vitro 1979. 15, 73-81 Schoenfeld. Y. and Schwartz, R. S. The production of monoclonal antibodies by human-human hybridomas: Their application to studies of autoimmune diseases. In: Human Hybridomas and Monoclono/ Antibodies (Engleman. E. G.. Foung, S. K. H., Lanick. J.. ef ul.. Eds. 1. Plenum Press. New York. 1085. pp. 247-262 Banle. M. F.. Schimke. R. T.. and Riggs. D. B. Presence of the arginine dihydrolase pathway in Mwop/u.vmu. J Bucteriol. 1966, 91. 189-192 Schmidt. J. and Erfle. V. Elimination of mycoplasmas from cell cultures and establishment of mycoplasma-free cell lines. Ex~. Cell Rrs. 1984. 152, 565-570 van Diggelen. 0. P.. Shin. S.. and Phillips, D. M. Reduction in cellular tumorigenicity after mycoplasma infection and elimination of mycoplasma from infected cultures by passage in nude mice. Ctmcrr Re.s. 1977. 31, 3680-2687 Sctummelpfeng. L.. Langenberg. I:.. and Peters. J. H. Macrophages overcome mycoplasma infections of cells in virro. Nurrtre (London)

I4

Acknowledgments We thank Dr. Hansjorg Hauser (Department of Gene Regulation and Differentiation), Dr. Manfred Wirth (Department of Gene Expression), and their colleagues of both departments of the GBF and Dr. H. Drexler and C. Uphoff of the DSM for helpful technical support and preparation of the primer DNA for PCR analysis. We are grateful to Dr. Volker JBger and Herbert Krafft for elimination of mycoplasmas from the hybridoma cell line.

comparison:

dardirarion of Vertebrate Cell Cultures. Tissue Culture Association. In Vitro, Monograph 5 (Levine. E. M.. Ed. 1. Gaithersburg. MD,

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Somasundaram, C.. Nicklas, W., and Matzku, S. Use of ciprofloxacm and BM-cyclin in mycoplasma decontamination. In Vitro Cell. Drv. Biol. 1992. 28A, 708-710 Gignac. S. M.. Brauer. S.. Hlne. B.. Quentenmier, H.. and Drexler, H. G. Elimination of mycoplasma from infected leukemia cell lines. Le&emra 1991. 5, 162-165 Uphoff. C. C.. Gignac. S. M.. and Drexler. H. G. Mycoplasma contamination in human leukemia cell lines. 1. Comparison of various detection methods. J. Immunol. Methods 1992. 149, 43-53 Russel. W. C., Newman, C.. and Williamson. D. H. A simple cytochemical technique for demonstration of DNA in cells infected with mycoplasmas and viruses. .Ynrure (London) 1975, 253, 461461

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cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res. 1977, 104, 255-262 Hessling, J. J., Miller. S. E.. and Levy. N. L. A direct comparison of procedures for the detection of mycoplasma in tissue culture. J. Immunol. Methods 1980, 38, 315-324 Alzani, R., Rossi, R., Cozzi, E., Trizio, D., and Marcucci, F. Detection of mycoplasma contamination through modulation (stimulation or inhibition) of thymidine incorporation by unstimulated mouse spleen cells. J. Immunol. Methods 1992, 152, 35-42 Hopert, A., Uphoff. C. C., Wirth. M.. Hauser. H., and Drexler. H. G. Specificity and sensitivity of polymerase chain reaction (PCR) in comparison with other methods for the detection of mycoplasma contamination in cell lines. J. Immunol. Merhods 1993, 164,91-100 Gobel. U. B. and Stanbridge, E. Cloned mycoplasma ribosomal RNA genes for the detection of mycoplasma contamination in tissue culture. Science 1984, 226, 1211-1213 Johannson, K. E. and Bolske, G. Evaluation and practical aspects of the use of a commercial DNA probe for detection of mycoplasma infections in cell cultures. J. Eiomed. Biophys. Methods 1989, 19, 158-200 Johannson, K. E., Johannson. I., and Gobel. U. B. Evaluation of different hybridization procedures for the detection of mycoplasma contamination in cell cultures. Mol. Cell. Probes 1990, 4, 33-42 McGarrity, G. J.. Kontani, H., and Carson, D. Comparative studies to determine the efficiency of 6-methylpurine deoxyribose to detect cell culture mycoplasmas. In Vitro Cell De\,. Biol. 1986. 22, 301304 Razin. S.. Hyman. H. C., Nur. I., and Yogev, D. DNA probes for detection and identification of mycoplasma (Mollicutes). Isr. J. Med. Sri. 1987. 23, 735-741 Finch, L. R. and Mitchell, A. Source of nucleotides. In: Mycoplasmas: Molecular Biology and Pathogenesis. (Maniloff. J.. McElhaney, R. N.. Finch, L. R., and Baseman. J. B.. Eds.) American Society for Microbiology, Washington, D.C.. 1992. pp. 21 l-230 Motz, M.. Deby, G., and Wolf, H. Truncated versions of the two major Epstein-Barr viral glycoproteins (gp2501350) are secreted by recombinant Chinese hamster ovary cells. Gene 1987, 58, 149-154 Urlaub. G. and Chasin. L. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc. Nat/. Acad. Sci. U.S.A. 1980, 77. 421CG-4220

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Jlger, V., Lehmann. J., and Friedl, P. Serum-free growth medium for the cultivation of a wide spectrum of mammalian cells in stirred bioreactors. Cytotechnology 1988, 1, 31%329 Lucki-Lange. M. and Wagner, R. Conditions for the production of recombinant IL-2 in stirred suspension culture using a protein-free medium. In: Production of Biologicals from Animal Cells in Culture (Spier, R. E.. Griffiths, J. B., and Meignier, B., Eds.). Butterworth-Heinemann. Oxford, 1991, pp. 180-186 Ruschmeyer, D., Thude, H., and Muhlradt, P. F. MDHM, a macrophage-activating product of Mycoplasma fermentans. stimulates murine macrophages to synthesize nitric oxide and become tumoritidal. FEMS Immunol. Med. Microbial. 1993, I. 223-230 Smith, P. K., Krohn, R. I., Hermanson, G. T.. Mallia. A. K.. Gartner, F. H.. Provenzano. M. D.. Fujimoto. E. K., Goeke, N. M.. Olson. B. J.. and Klenk, D. C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150, 7685 Hay, R. J. Preservation and characterization. In: Animal Cell Cullure: A Practical Approach (Freshney. R. I., Ed.). IRL Press. Oxford. 1986. pp. 71-112 Ryll. T. and Wagner R. An improved ion-pair HPLC method for the quantification of a wide variety of nucleotides and sugar-nucleotides in animal cells. J. Chromarogr. 1991, 570, 77-88 McGarrity, G. J. and Carson, D. A. Adenosine phosphorylasemediated nucleoside toxicity. Exp. Cell Res. 1982, 139, 199-205 Yoshida. T.. Kawase. M.. Sasaki, K.. Mizusawa. H., and Ishidate. M. Comparative studies on the sensitivity of three methods for detection mycoplasmal contamination in cell cultures. Bull. JFCC 1988, 4, 9-15 Douma, R.. Dular, R.. Brodeur. B. R.. and Kasatiya. S. S. Development and application of monoclonal antibodies for the detection of cell infecting mycoplasmas. J. Immunol. Methods 1989, 124, 197203 Larsen, B. R. and West, F. G. A method for quantitative amino acid analysis using precolumn o-phthalaldehyde derivation and high performance liquid chromatography. J. Chromatogr. Sci. 1981. 19, 259-265 Ryll, T.. Lucki-Lange, M.. Jlger. V.. and Wagner, R. Production of recombinant human interleukin-2 with BHK cells in a hollow fibre and a stirred tank reactor with protein-free medium. J. Biorechnol 1990, 14, 3777392