Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
  • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/008—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions for determining co-enzymes or co-factors, e.g. NAD, ATP
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/30—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]

Definitions

• the present invention relates to assay methods and materials for detecting members of the Mollicutes family, that contaminate a test sample, such as a sample from a cell culture.
• Taxonomically the lack of cell walls has been used to separate Mollicutes from other bacteria in a class named Mollicutes (Razin et al 1998).
• the members of this class are summarised in the following table 1.
• Table 1 Major Characteristics and Taxonomy of the Class Mollicutes.
• mycoplasma is intended to embrace all members of the class Mollicutes, not just Mycoplasmatales. In fact, “mycoplasma” is the common term in the art for all of the Mollicutes.
• Mycoplasmas are widespread in nature as parasites of humans, mammals, reptiles, fish, arthropods and plants. They are the smallest and simplest prokaryotes. They lack a rigid cell wall and are incapable of peptidoglycan synthesis; they are therefore not sensitive to antibiotics, such as penicillin and its analogues.
• Mycoplasma have developed by degenerate evolution from gram-positive bacteria with a low molecular percentage guanine and cytosine content of DNA ie. the Lactobacillus, Bacillus, Streptococcus and two Clostridium species. The Mollicutes have lost, during the process of evolution, a substantial part of their genetic information. It is this limited coding capacity that has dictated the need for a parasitic way of life. Most species are facultative anaerobes, but some are obligate, and hence the similarities in their metabolism to anaerobic bacteria.
• mycoplasma infections 95% of all mycoplasma infections; these are M. orale, M. arginii, M. fermentans, M. salivarum, M. hyorhinis and A. laidlawii.
• the major cause of infection is cross contamination from other cell lines introduced into laboratories.
• tissue culture reagents such as serum products.
• Mycoplasma unlike bacterial, contamination rarely produces turbid growth or obvious cell damage.
• Viable mycoplasma can be recovered from work surfaces seven days after inoculation, and mycoplasma can also pass through bacteria-retaining filters. At their maximum population phase there can be as many as 10 8 mycoplasma/ml of supernatant, at a ratio of 5:1 with the host cells. If present, mycoplasma 'grow' to detectable concentrations in the culture medium, they are then also adsorbed onto the cell surface. It is a moot point as to whether mycoplasma enter and survive within mammalian cells in culture.
• Mycoplasma are capable of altering almost every property of an in vitro culture. They will deplete culture nutrients, in particular arginine. Infected eukaryotic cells exhibit aberrant growth, changes in metabolism and morphology. Certain biological properties have been implicated as virulence determinants; these include secretion or introduction of mycoplasmal enzymes such as phospholipases, ATPases, hemolysins, proteases and nucleases into the host cell milieu.
• Mycoplasma can be cultured on agar and in broth culture, with most mycoplasma producing microscopic colonies with a characteristic 'fried egg' appearance, growing embedded in the agar, although some colonies may not grow completely embedded.
• mycoplasma species that adsorb to host cell surfaces (Rottem and Barile 1993).
• these tests are most commonly done by mycoplasma testing service laboratories.
• DAPI 6- diamine-2-phenylindole dihydrochloride
• Hoecsht staining is considered to be the method of choice.
• Cell culture samples are taken, fixed and stained with Hoechst 33258 (bisbenzamide) and examined under UV epifluorescence (Battaglia et al 1994, Raab 1999). If there are mycoplasma associated with the cells, then the cell nuclei will appear surrounded by fluorescing structures in the cytoplasm. Negative cells are represented by just the nuclear staining of the cellular DNA. Accurate interpretation of results from DNA staining requires an experienced eye, it also needs specialist equipment i.e. a fluorescence microscope.
• Mycoplasma detection by PCR is a commonly used test by external service laboratories, and is also performed in those laboratories that have the appropriate equipment.
• the primers used in mycoplasma PCR kits anneal to conserved regions of the mycoplasma genome, allowing the detection of several species (Raab 1999).
• Most commercially available PCR kits require that the amplified products be analysed by agarose gel electrophoresis, with the resulting banding patterns determining the contaminating species present. However visualisation of banding patterns is subjective.
• the Mycoplasma PCR ELISA from Roche relies on a different system, and cannot distinguish between species.
• This kit includes digoxigenin-dUTP, and the PCR product is captured onto the surface of wells in a microtitre plate coated with anti-digoxigenin-peroxidase conjugate.
• the coloured product with tetramethylbenzidine (TMB) is visualised using a standard ELISA plate reader.
• UK patent No. 2 357 336 B describes an assay which can be used to detect mycoplasmas in cell cultures.
• the assay is based on the observation that mycoplasmas over-produce the enzyme ATPase in large amounts.
• the ATPase activity of mycoplasmas converts sufficient cellular or externally added ATP to ADP, to make the ADP detectable.
• the assay is based on detection of ADP and this is carried out by adding to the sample an enzyme containing reagent (containing a combination of pyruvate kinase and phosphoenol pyruvate; adenylate kinase; glycerol kinase, myokinase; or a combination of creative kinase and creative phosphate), which converts the ADP to ATP and detecting ATP using a bioluminescent reaction.
• an enzyme containing reagent containing a combination of pyruvate kinase and phosphoenol pyruvate; adenylate kinase; glycerol kinase, myokinase; or a combination of creative kinase and creative phosphate
• the present invention seeks to provide further means for detecting mycoplasmas in samples, such as samples from cell cultures.
• the invention provides a method of detecting the presence of contaminating mycoplasma in a test sample comprising:
• step (i) providing a test sample; (ii) detecting and or measuring the activity (B) of acetate kinase and/or carbamate kinase in the test sample, and said activity being indicative of the presence of contaminating mycoplasma; and (iii) identifying the test sample as contaminated with mycoplasma on the basis of detection and/or measurement of said activity in step (ii).
• the method further comprises the following steps performed after step (ii) but before step (iii):
• test sample is identified as contaminated with mycoplasma in step (iii) if the activity (B) detected and/or measured in the test sample in step (ii) is greater than that of the control sample (A) in step (iia), that is, the ratio -J is greater than one.
• the invention provides a method wherein detecting and/or measuring the activity (B) of acetate kinase and/or carbamate kinase in the test sample in step (ii) and/or obtaining acetate kinase and/or carbamate kinase activity information (A) in a corresponding control sample in step (iia) comprises detecting and/or measuring the appearance and/or disappearance of one or more of the substrates and/or one or more of the products of the following reactions:
• the detecting and/or measuring step comprises detecting and/or measuring ATP.
• the ATP is detected and/or measured by a light-emitting reaction, especially a bioluminescent reaction.
• Light-emitting systems have been known and isolated from many luminescent organisms, including certain bacteria, protozoa, coelenterates, molluscs, fish, millipedes, flies, fungi, worms, crustaceans, and beetles, particularly the fireflies of the genera Photinus, Photuris, and Luciola and click beetles of genus pyrophorus.
• enzymatically catalyzed oxidoreductions take place in which the free energy change is utilised to excite a molecule to a high energy state. Then, when the excited molecule spontaneously returns to the ground state, visible light is emitted. This emitted light is called "bioluminescence”.
• P. pyralis luciferase is an enzyme which appears to have no prosthetic groups or tightly bound metal ions and has 550 amino acids and a molecular weight of about 60,000 daltons; the enzyme has been available to the art in crystalline form for many years.
• firefly luciferin a polyheterocyclic organic acid, D-(-)-2-(6'-hydroxy-2'- benzothiazolyl) ⁇ 2 -thiazoline-4-carboxylic acid (herein-after referred to as "luciferin", unless otherwise indicated).
• ATP can be detected using the following bioluminescent reaction.
• Mg emission at 565 nm
• the emitted light intensity is linearly related to the ATP concentration and is measured using a luminometer.
• Luciferase has been used as a means of assaying minute concentrations of ATP; as little as 10 "16 molar ATP can be detected with high quality preparations of the enzyme.
• the luciferase-luciferin reaction is highly specific for ATP. For example, deoxy-ATP produces less than 2% of the light generated by ATP, and other nucleoside triphosphates produce less than 0.1%.
• Crystalline luciferases can be isolated directly from the light organs of beetles.
• cDNAS encoding luciferases of several beetle species including, among others, the luciferase of P.pyralis (firefly), the four luciferase isozymes of P.plagiophthalamus (click beetle), the luciferase of L.cruciata (firefly) and the luciferase of X. lateralis) (de Wet et al., 1987, Masuda et al., 1989, Wood et al., 1989, European Patent Application Publication No. 0 353 464) are available.
• cDNAs encoding luciferases of any other beetle species are readily obtainable by the skilled using known techniques (de Wet et al., 1986, Wood et al., 1989). With the cDNA encoding a beetle luciferase in hand, it is entirely straightforward to prepare large amounts of the luciferase in highly pure form by isolation from bacteria (e.g. E.coli), yeast, mammalian cells in culture, or the like, which have been transformed to express the cDNA.
• Such a mutant luciferase will have an amino acid sequence that differs from the sequence of a naturally occurring beetle luciferase at one or more positions (White et al., 1996, WO 01/31028 and WO 00/24878).
• the term "luciferase” comprehends not only the luciferases that occur naturally in beetles but also the mutants, which retain activity in providing bioluminescence by catalyzing the luciferase-luciferin reaction, of such naturally occurring luciferases.
• step (i) the sample is treated so as to lyse any mycoplasma and thereby release their cellular contents into the sample.
• lysis can be effected by a variety of methods including application of chemicals, such as detergents and mechanical methods such as sonication etc.
• the lysis is effected by treating the sample with a detergent, or other lysis method, which allows for the lysis of the Mycoplasma cell membrane but which does not affect the cell wall of any bacteria which may be present.
• a detergent or other lysis method, which allows for the lysis of the Mycoplasma cell membrane but which does not affect the cell wall of any bacteria which may be present.
• Exemplary detergent treatment includes the use of low concentrations (e.g. 0.25% v/v) of a detergent, such as Triton XI 00.
• the preferred lysis method is one that is sufficient to lyse the mycoplasmal membrane, but would be ineffective against bacterial cells.
• non-ionic detergents mainly polyethoxyethers
• bacteria For efficient lysis and total protein release, bacteria often require exposure to enzymes such as lysozyme to breach the cell wall (Pellegrini et al 1992).
• the most preferred detergent mycoplasma lysis conditions are shown hereinafter.
• mycoplasma will pass through a 0.45 ⁇ M filter used for filter sterilisation (Baseman and Tully 1997), and it is possible to distinguish between a bacterial and mycoplasmal contamination through the addition of a filtration step.
• test sample is passed through a bacterial filter in step (i).
• a bacteria filter in preferred embodiments of the invention the test sample is passed through a bacterial filter in step (i).
• the test sample is treated to remove bacteria, for example by passing it through a bacteria-retaining filter, it is not important to lyse mycoplasma selectively, i.e. without lysing bacteria.
• ADP is added to the test sample prior to the detecting and/or measuring step (ii).
• the assay can also utilise endogenous ADP.
• a mycoplasma substrate reagent is added to the test sample prior to the detecting and/or measuring step (ii), the mycoplasma substrate reagent comprising: acetyl phosphate or a precursor thereof and or carbamoyl phosphate or a precursor thereof.
• a precursor thereof we include one or more compounds from which acetyl phosphate and/or carbamoyl phosphate can be generated.
• Exemplary reactions are outlined below: (i) acetyl -CoA phosphotransace ase acetyl phosphate
• acetyl phosphate instead of adding acetyl phosphate to the mycoplasma substrate reagent, one could include a precursor, such as acetyl-CoA.
• carbamoyl phosphate instead of carbamoyl phosphate one could add a precursor, such as citrulline and ammonia to the mycoplasma substrate reagent.
• both acetyl phosphate and carbamoyl phosphate and/or precursors thereof are added to the sample prior to step (ii), in the methods of the invention.
• This enables a generic assay for mycoplasma contamination to be carried out because mycoplasmas utilise either or both substrates by means of their acetate kinase and/or carbamate kinase enzymes.
• a more specific assay can be produced by only using one of the above substrates or precursors thereof.
• Such an assay will be specific for mycoplasma which only use one of the enzymes acetate kinase or carbamate kinase.
• the following table 2 cites some examples of the members of the Mollicutes family (parasitise mammalian hosts) that utilise acetate kinase preferentially, carbamate kinase preferentially, or both.
• the "corresponding control sample” is the test sample prior to a mycoplasma lysis treatment and/or addition of a mycoplasma substrate and or a time interval (e.g. more than approximately 30 minutes). In this preferred embodiment both of the activity measurements are carried out on the same sample, the test sample.
• a first activity measurement (A) is taken either before or concurrent with a mycoplasma lysis step then, after addition of a mycoplasma substrate and/or a time interval (e.g. more than approximately 30 minutes), a second activity measurement (B) is taken. If the value of is greater than one the test sample is identified as contaminated with mycoplasma. Skilled persons will appreciate that the "corresponding control sample” could also be a predetermined negative control sample, but this is less preferred.
• control sample has been shown to be free from mycoplasma contamination. Suitable methods for doing this include PCR testing, DNA fluorescent staining or culture methods as described herein.
• corresponding control sample we mean a sample which contains substantially the same material as that contained in the test sample, but which, unlike the test sample, has been shown to be free from mycoplasma contamination. Skilled persons will appreciate that a mycoplasma uncontaminated condition can be shown by a variety of known methods. A number of suitable methods are reviewed by Rottem and Barile 1993, while an outline of testing kits and services is given in Raab et al 1999.
• test sample and/or control sample can be a cell sample, such as a cell culture sample, especially a culture of mammalian cells. Some examples are listed in the following table 3.
• Table 3 Commonly cultured cell lines that have been tested using the assay method.
• AoSMC Aortic Smooth Muscle Cells Cambrex CC-2571
• ARPE- 19 Human Retinal Pigment Epithelial Cells ATCC CRL-2302
• Wliere ECACC represents the European Collection of Animal Cell Culture
• ATCC represents the American Tissue Culture Collection
• Cambrex represents Cambrex Bio Science Wokingham, UK.
• the assays of the invention can be utilised to detect mycoplasma contamination in cultures of both adherent cells (e.g. HepG2, A549, CHO and COS cells) and cells which culture in suspension (e.g. Jurkats, U937, K562 and HL-60 Cells.)
• adherent cells e.g. HepG2, A549, CHO and COS cells
• cells which culture in suspension e.g. Jurkats, U937, K562 and HL-60 Cells.
• the sample to be tested is from the cell culture supernatant which has previously been centrifuged to remove cellular material.
• Cell-free samples can also be tested using the methods of the invention.
• the methods of the invention are particularly useful for testing samples of cell-free reagents, such as tissue culture media, and typically those containing animal-derived materials, such as serum (e.g. foetal calf serum), trypsin, and other culture supplements, etc. Examples of some commonly used media and supplements that may be tested in this manner are shown in table 4.
• Table 4 Tissue culture media and supplements that may be tested using the assay system.
• a third aspect of the invention provides a method of detecting the presence of mycoplasma in a test sample, comprising the following steps:-
• Mycoplasma lysis treatment and/or addition of mycoplasma substrate and/or a time interval occurs before step (ii).
• the "corresponding control sample” is the test sample except that it has not been subjected to Mycoplasma lysis treatment and/or addition of mycoplasma substrate and/or left for a time interval (e.g. more than approximately 30 minutes).
• both measurements are taken from the test sample.
• the control ATP and/or light output measurement is taken following addition to the sample of the mycoplasma detection reagent containing the detergent and luciferase/luciferin plus AMP, and the test ATP and/or light output measurement is taken following addition of substrates for kinase activity (or precursors thereof).
• a fourth aspect of the invention provides an in vitro process for treating a cell culture to remove mycoplasma contamination comprising:- treating a mycoplasma contaminated cell culture with an agent to remove or destroy mycoplasma; and subsequently testing a sample from the culture for mycoplasma contamination using a method of the invention; if necessary, repeating the process one or more times until mycoplasma contamination is not detected in the sample.
• the antibiotics tetracycline and ciprofloxacin are reported to have success rates of less than 80-85% (www.unc.edu/depts/tcf/mycoplasma.htm). It is therefore extremely difficult to completely irradicate mycoplasma from cultures, once a contamination has taken hold.
• mycoplasma exposed to antibiotics in eukaryotic cell culture have different profiles from the same species isolated from a human or animal source (Taylor-Robinson and Bebear 1997). While the reported success of anti- mycoplasma treatments appears highly variable, a recent study by Uphoff et al 2002, reports that 96% of leukaemia-lymphoma cell lines were rendered free of mycoplasma with at least one of the treatments tested.
• Figure 1 The kinetics of ATP generation in the presence of M.hyorhinis contamination.
• Figure 2 A Comparison between the PCR kit from Sfratagene and a preferred embodiment of the invention ratios.
• Figure 3 Treatment of contaminated cell lines with Mycoplasma Removal Agent according to a preferred embodiment of the invention.
• Figure 4 Ratio data with cells, supernatants and supernatants filtered through a 0.45 ⁇ m (FI), 0.22 ⁇ m (F2) and O.l ⁇ m (F3) filters.
• Figure 5 Effect of supernatant dilution.
• Figure 6 M. fermentans at 7900 CFUs/well, tested against the different substrates.
• Figure 7 M. orale stock at 1450 CFUs/well, tested against the different substrates.
• Figure 8 Dilution of the M.orale stock to show sensitivity of the assay.
• Figure 9 M. hyorhinis, comparison of different substrate reagents.
• FIG 10 The effect of Triton-XlOO concentrations on the detection of mycoplasma enzyme activities in K562 cells infected with M.hyorhinis (MH) and M.orale (MO).
• Figure 11 Shows the effect of increasing Triton-XlOO concentrations on K562 cells contaminated with M.hyorhinis (MH) compared to increasing numbers (1-10,000 cells/lOO ⁇ l sample) of bacterial cells (E.coli).
• the principle of the preferred assay method is to supply the appropriate substrates for mycoplasmal enzymes. If mycoplasma contamination is present, there is a conversion of ADP to ATP which can then be measured, preferably by the luciferase-luciferin reaction.
• Mycoplasma Detection Reagent is added and, after approximately 5 minutes, an initial light output reading (A) is taken, the Mycoplasma Substrate (MS) is added and any enzymatic activity is allowed to progress for approximately 10 minutes, at which point a second light reading (B) is taken. If there is mycoplasmal contamination then the second reading (B) will be higher when compared to the first reading (A), giving a ratio ⁇ of greater than 1. If the culture is negative (uncontaminated by mycoplasma), then the ratio • * will be 1 or most often less than one due to the luminescent light signal decay usually seen over time.
• Figure 1 demonstrates the kinetics of the reaction. Typically, the ratio -f seen with mycoplasma contamination is much greater than 1, for example Figure 1 shows a ratio of 114.
• a preferred assay kit of the invention comprises a Mycoplasma Detection Reagent (MDR); Mycoplasma Assay Buffer (MAB) for reconstitution of MDR and the Mycoplasma Substrate (MS).
• MDR and MS are preferably provided as lyophilised preparations.
• Mycoplasma Substrate of the invention contains substrates for one or both of these enzymatic reactions.
• ADP is a requirement for both enzymes, and is preferably supplied in excess in the Mycoplasma Detection Reagent of the invention to drive the generation of ATP formation.
• the MDR is added to a sample of culture supernatant that has previously been centrifuged to remove cellular material, although it is possible to perform the assay in the presence of cells.
• the test sample can be passed through a bacterial filter.
• the MDR contains substrates for luciferase, luciferin and other co-factors plus AMP and ADP.
• the Mycoplasma Substrate (MS) contains carbamoyl phosphate and/or acetyl phosphate or precursors thereof, required for detection of the carbamate and/or acetate kinase activities.
• a preferred sample volume is lOO ⁇ l to which lOO ⁇ l of reconstituted MDR is added. After approximately 5 minutes the first luminometric reading (A) is taken, this gives the base reading upon which the further ratio calculations ⁇ are determined.
• the assay methods of the invention have been used to investigate contamination by Acholeplasma laidlawii, M. hyorhinis, M. fermentans, M. orale, and M. genitalium and to detect a number of unknown mycoplasma contaminations.
• the inventors have compared their data to detection of mycoplasma by PCR, and have shown that there is a correlation between ratios greater than one and detection of mycoplasmal DNA. This is shown in Figure 2 where the positive PCR bands on the gel correlate with ratios of more than one.
• Example 2 use of the methods of the invention in a process for removing mycoplasma contamination from a cell culture.
• the inventors have also shown that we can detect a reduction in ratios -f as cells are treated with an exemplary Mycoplasma Removal Agent (ICN- Flow), a derivative of the quinolone family of antibiotics.
• ICN- Flow Mycoplasma Removal Agent
• Figure 3 also shows that the treatment with MRA for 10 days with COS-7 and CHO cell cultures was sufficient to remove the contaminating mycoplasma.
• Mycoplasma can form colonies as large as 600 ⁇ m in diameter, but can also exist in their life cycle as single cells as small as 0.15 ⁇ m. Due to their small size mycoplasma can pass through the 0.45 ⁇ m and 0.22 ⁇ m filters commonly used to "sterilise" tissue culture reagents.
• Figure 4 also confirms that the assay can be performed in the presence of cells, but that there is a reduced sensitivity of detection. Hence, it is preferred that the assay methods of the invention are performed on samples which are substantially cell free. This can easily be achieved by centrifugation of cell cultures and sampling of the supernatant and, optionally, filtration through a bacterial filter.
• Example 4 Sensitivity of preferred assays
• dilution of the supernatants shows the sensitivity of the assays, in that a 1 :1000 dilution of contaminated culture supernatant can still give ratios greater than 1.
• the dilution range will also vary according to the number of colony forming units in the test sample.
• Example 5 variations of the assay methods of the invention
• the assay methods of the invention will work without the external exogenous addition of carbamate and acetate kinase substrates in the form of ADP, carbamoyl phosphate and acetyl phosphate or precursors thereof.
• ADP can be generated by other externally added or cellular enzymes i.e. adenylate kinase utilising ATP and AMP.
• the two substrates could also be generated from "precursors" by utilising earlier parts of the glucose fermentation and argenine lysis pathways for example by the addition of acetyl-CoA and citrulline that could be used by mycoplasmal enzymes to synthesise acetyl phosphate and carbamoyl phosphate respectively.
• M.fermentans utilises both pathways
• M.orale utilises only the carbamate kinase pathway
• positive ratios are only observed in the single carbamate reagent or the combined reagent.
• Figure 8 shows the detection limits are as low as 14 CFU/well with M.orale.
• the inventors have investigated a mycoplasma that preferentially utilises the acetate kinase pathway, namely M.hyorhinis.
• the data are shown in Figure 9.
• the inventors have tested over 15 different cell lines (see table 3) and shown that none of the cells have sufficient background enzymatic activity to impact upon the ratios and give false positives.
• the inventors without wishing to be bound by theory, think the reason for this is that the pathways are anaerobic, and all mammalian cell cultures will generate ATP through oxidative phosphorylation.
• the methods of the invention can be varied to include an initial screening step for bacterial contamination, if necessary. This can be achieved by a variety of methods, but is preferably carried out by passing the sample through a standard bacterial filter (Baseman and Tully, 1997).
• Example 6 Preferred Reagent Components for use in the mycoplasma assay methods and kits of the invention
• MDR Mycoplasma Detection Reagent
• *RY is the name given to the recombinant luciferase supplied by Lucigen. A mixture of D and L-Luciferin has been found to give a more stable light output than D-luciferin alone.
• Ms Mycoplasma Substrate
• Preferred concentration ranges acetyl-CoA 0. ImM to 1 OOmM
• Preferred concentration ranges citrulline lmM to lOOmM ammonium bicarbonate ImM to 200mM acetate kinase iii. (sodium or potassium) acetate + ATP ⁇ acetyl phosphate +
• concentration ranges acetate (e.g. sodium or potassium) ImM to 500mM ATP O.lmM tolOOmM
• Preferred concentration ranges arnmonium bicarbonate ImM to 200mM ATP 0. ImM to lOOmM
• ADP l ⁇ M to lOOmM preferably 1 to lOO ⁇ M, more preferably 5 ⁇ M.
• Carbamoyl phosphate l ⁇ M to lOOmM preferably O.lmM to 20mM, more preferably 3mM.
• Disruption of the viable mycoplasma cell membrane to allow for the release of the enzymes into the sample is a preferred embodiment of the assay method of the invention. This allows for binding of the substrates and 0 generation of ATP. However, positive ratios indicating mycoplasma contamination can be obtained in the absence of any lysis treatment. The implication is that these enzymes can be released by viable mycoplasma The other possibility is that some non- viable organisms have released their contents through natural lysis. 5
• Figure 10 shows that it is possible to detect mycoplasmal enzymes in the absence of a detergent lysis step. It 'also shows a drop in the light output with concentrations greater than 4-5%, this is due to adverse effects of the detergent on the luciferase enzyme/reaction. However, it is still possible to detect positive activity with concentrations as high as 20% (v/v).
• the inventors have also shown that the concentrations of Triton-XlOO used in the above experiments did not result in any detectable carbamate or acetate kinase activity from the E.coli strain JM109 cells.
• Non-ionic detergents are non- denaturing and permit the solubilization of membranes without interfering with biological activity. They have principally been used for the study of protein conformations and for the separation of hydrophilic proteins from membrane spanning hydrophobic proteins.
• Anionic and cationic detergents result in greater modification of protein structure and are more effective at disrupting protein aggregation.
• Zwitterionic detergents are also low-denaturing, but are effective at disruption of protein aggregates.
• the required lysis agent is one that causes disruption of the mycoplasmal membrane and allows release of the metabolic enzymes that are required to react with the substrates.
• the chosen system does not interfere with the activity of the carbamate and/or acetate kinase, or the luciferase/luciferin/ATP reaction. It is also preferable to use a system that selectively causes the lysis of mycoplasma, with little or no effect on bacteria that may be potential contaminates of the cell cultures/samples .
• bacteria and mycoplasma The key difference between bacteria and mycoplasma is the lack of cell wall, and it is the bacterial cell wall that makes bacteria more difficult to lyse. There are a number of fairly brutal methods that can bring about total lysis, these include pressure (French Press) and sonication. Other enzyme digest methods include lysozyme followed by the addition of detergents. However, mycoplasma can be lysed with concentrations of Triton X-100 at around 1-2%.
• the lysis step would preferably cause selective lysis of mycoplasma, while having little or no effect on bacterial cells. Low concentrations of most non- ionic detergents should do this. However, a filtration step would physically remove any contaminating bacteria, and allow for the use of any detergent but preferably those that do not inhibit either the luciferase reaction or the activity of carbamate kinase and acetate kinase.
• Example 8 preferred kit contents
• LT27-216 Mycoplasma Detection Reagent Lyophilised. 1 x 10 ml vial. 2. LT27-220 Mycoplasma Assay Buffer. 1 x 20 ml bottle.
• MDR Mycoplasma Detection Reagent
• Acetyl phosphate l ⁇ M to lOOmM preferably, mM to lOmM Carbamoyl phosphate l ⁇ M to lOOmM
• the preferred embodiment of the invention provides a selective biochemical test that exploits the activity of certain mycoplasmal enzymes.
• the presence of these enzymes provides a rapid screening procedure, allowing sensitive detection of contaminating mycoplasma in a test sample.
• the viable mycoplasma are lysed and the enzymes react with the Mycoplasma Substrate catalysing the conversation of ADP to ATP.
• a ratio - can be obtained which is indicative of the presence or absence of mycoplasma. If these enzymes are not present, the second reading shows no increase over the first (A), while reaction of mycoplasmal enzymes with their specific substrates in the Mycoplasma Substrate Reagent, leads to elevated ATP levels. This increase in ATP can be detected using the following bioluminescent reaction.
• the emitted light intensity is linearly related to the ATP concentration and is measured using a luminometer.
• the assay is preferably conducted at ambient room temperature (18-22°C), the optimal temperature for luciferase activity.
• the culture supernatant be centrifuged to remove cells and, optionally, passed through a bacterial filter prior to performing the assay.
• the kit contains all the required reagents to perform the assay. lOO ⁇ l of culture supernatant is taken as the sample.
• MDR Mycoplasma Detection Reagent
• MS Mycoplasma Substrate
• MAB Mycoplasma Assay Buffer
• Mycoplasma Assay Buffer This is preferably provided ready for use. Store at 2-8°C when not in use.
• the kit requires the use of a luminometer.
• the parameters of the luminometer should be assessed and the conditions below used to produce the correct programming of the machine.
• the preferred assay of the invention has been designed for use with cuvette/tube luminometers.
• plate luminometers please see below.
• Cuvette/tube luminometers Read time 1 second (integrated).
• the ratio of Reading B to Reading A is used to determine whether a cell culture is contaminated by mycoplasma.
• kits according to the invention means that it provides a unique method for screening cultures for the presence of mycoplasma. As such it is ideally suited to routine testing of cells in culture. Frequent use of the test methods of the invention will indicate when a cell line becomes infected allowing prompt remedial action to be taken.
• the test methods of the invention can also be extended to incoming cell lines and the commonly used constituents of complete media.
• the optimal working temperature for all reagents is 22°C. If reagents have been refrigerated always allow time for them to reach room temperature (18-22°C) before use.
• the sensitivity of the assay does allow for detection of covert contamination, and if the ratio is marginally above 1 (for example up to 1.3) it is recommended that the sample be retested. Any cultures maintained in quarantine can be tested after a further 24-48 hours in culture to see if the ratios have increased.
• the assays of the invention can be performed in the presence or absence of cells. Unlike known mycoplasma detection systems, they allow for samples to be screened rapidly using cheap hand-held luminometer systems, and can give results within 15 minutes to allow for the appropriate handling of the contaminated samples.
• PCR and DAPI/Hoechst staining will bind to all DNA, be it from viable or non viable mycoplasma. Hence, if looking to treat and remove mycoplasma, you could still end up with false positives when using PCR/DNA staining even though mycoplasma have been irradicated.
• the assays can detect viable mycoplasma whereas known methods such as PCR cannot distinguish between viable and non-viable mycoplasma.
• Firefly luciferase gene structure and expression in mammalian cells

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Analytical Chemistry (AREA)
• General Engineering & Computer Science (AREA)
• Immunology (AREA)
• Microbiology (AREA)
• Molecular Biology (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Genetics & Genomics (AREA)
• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biotechnology (AREA)
• General Health & Medical Sciences (AREA)
• Toxicology (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Investigating Or Analysing Biological Materials (AREA)

Priority Applications (1)

Applications Claiming Priority (3)

Publications (1)

Family

ID=33312360

Family Applications (1)

Country Status (7)

Families Citing this family (6)

Family Cites Families (2)

• 2004
  • 2004-04-16 BR BRPI0407750A patent/BRPI0407750B8/pt active IP Right Grant
  • 2004-04-16 CA CA2512636A patent/CA2512636C/en not_active Expired - Lifetime
  • 2004-04-16 AU AU2004233290A patent/AU2004233290B2/en not_active Expired
  • 2004-04-16 EP EP04727944A patent/EP1613763A1/de not_active Withdrawn
  • 2004-04-16 WO PCT/GB2004/001647 patent/WO2004094656A1/en not_active Application Discontinuation
• 2005
  • 2005-07-05 NO NO20053276A patent/NO333474B1/no unknown
• 2011
  • 2011-03-24 HK HK11102980.5A patent/HK1148790A1/xx not_active IP Right Cessation

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events