ANALYSIS OF CELL MODIFYING SUBSTANCES

Abstract: Extracellular cell modifiers such as hormones (and autoantibodies that mimic their effects) are assayed by adding them to a culture of cells containing a cellular component the activity or quantity of which is sensitive to that cell modifier and then measuring the change in the cellular component by use of an appropriate colorimetric or chromogenic reagent. The method is conveniently operated in the wells of a microtitre plate using a colorimetric or chromogenic reagent which can be measured spectrophotometrically. The coloured product is eluted from the cells into the wells and the spectrophotometric measurements read directly in the wells with a microtitre plate reader.

Description: Analysis of cell modifying substances. This invention relates to the bioasεay of cell-modifying reagents, e.g. hormones. At present, in clinical chemistry, routine measurement of the content of a cell-modifying reagent such as a hormone in a sample, e.g. of a body fluid, is carried out by im unoaεsay. This measures the immuno-reactivity of the cell-modifier which may not, in some circumstances, accurately correlate with its actual biological activity (bioactivity) . It is therefore desirable to be able to measure the bioactivity of cell-modifying reagents such as hormones more directly. There are a wide variety of bioasεay systems and up till now these have been technically demanding and time consuming. For example some employ tedious techniques such as cell counting, and many require the use of radioactive material e.g. labelled thymidine. Many require the extraction of a cellular product which has been altered in the bioassay, e.g. cyclic AMP, and its subsequent measurement in a separate array such as a radio im unoassay. The present invention provides a novel method for the assay of cell-modifying reagents such as hormones (and autoantibodies that mimic their effect) which is capable of relatively rapid large scale automated operation and thus avoids the above-mentioned disadvantages of known me the s . The method of the present invention for the assay of an extra-cellular cell-modifying reagent (or cell modifier), e.g. a hormone (or an autoantibody that mimics its effect), comprises: (i) exposing a culture of cells containing a cellular component the activity or quantity of which is sensitive to that cell modifier to a sample containing an unknown amount of the said cell modifier for a period of time sufficient for the activity or quantity of the said component to be altered by the said cell modifier if present; (ii) adding to the cell culture a colorimetric or chromogenic reagent which interacts in a measurable way with the cellular component altered by the said cell modifier; and (iii) measuring the interaction between the said reagent and the cellular component extracellularly. As used herein, the term "colorimetric reagent" means a reagent which absorbs or generates, in a quantifiable way, electromagnetic radiation in the ultraviolet (10-400 nm), visible (400-750 nm) or infra-red (750-10 5 nm) part of the spectrum. The term "chromogenic reagent" means a reagent which is capable of being converted under the conditions of the assay into a colorimetric reagent (as defined above) under the influence of the cellular component. Cell cultures have previously been used in other types of assay. For example, Moεmann (J. Immunological Methods, 6_5 pp 55-63 1983) described a method for the colorimetric assay of cellular growth and survival. The method relied on the conversion of a tetrazolium salt (MTT) into dark blue formazan by the dehydrogenase present in living but not dead cells. The method was operated in a multiwell microtitre plate and the results were read on a microtitre plate reader. The method was used to measure the effect of extra cellular cell-modifying reagents such as proliferative lymphokineε, e.g. interleukin 2, mitogen stimulators, e.g. Concanavallin A, and complement mediated lysis. A similar method has been used in cytotoxicity assays for bacterial enterotoxins, e.g. cholera toxin, see Barer e_t al, Histochemical Journal 33 pp. 122-128 (1986) and Develop. Biol. Standard, 6_4 pp. 251-259, Karger, Basel, 1986, and Riddell e_t a_l, Fd Chem. Toxic. 2_4 No. 6/7 pp 469-471 (1986). These methods relied on the ability of living cells to bind dyes such as Naphthol Yellow S, or to bring about the converεion of a tetrazolium salt into formazan. In either case, the coloured product was eluted from the cells and then measured εpectrophotometrically. Taverne e_t a , Eur. J. Immunol. 37 pp 1855-1858 (1987) have described a method for measuring the cytotoxicity of tumor necrosis factor using the cell-line FRTL-5, a continuous strain of functional untransformed epithelial cells derived from the Fischer rat thyroid. The effect of the tumor necroεiε factor was monitored by measurement of the ability of the FRTL-5 cells exposed to the tumor necrosis factor to bind crystal violet or bring about the reduction of a tetrazolium salt to formazan. It was εhown inter alia that thyroid stimulating hormone somewhat increaεes the resistance of the cells to tumor necrosis factor. Romijn et al., The Prostate, 12_, pp.99-110 (1988) have εhown that it is possible to use this method to quantify the hormone-stimulated growth of target cells. They used the un-natural synthetic androgen R1881 and demonstrated that the increased conversion of MTT into formazan monitored the stimulation of proliferation of LNCaP cells after 4 days exposure to this drug. These prior methods rely on alterations in the number of viable cells in the cell culture, caused by the stimulating or inhibiting agent which is assayed. The present invention is based on the discovery that it is possible to monitor, in cultures of cells, changes in cellular components rather than changes in cell numbers. Such changes can occur much more rapidly, e.g. in hours rather than days, so that the new method can be much quicker to operate than the known methods. Although the method of the invention may be operated on a cell culture in which some cellular division has taken place, especially in those cases where it is the quantity rather than the activity of the cellular component which is altered, it is preferred to operate the method using a cellular component which is altered before there is any substantial alteration in the number of cells in the culture as this makes for a substantially more rapid method. The new method may be operated in the wells of a microtitre plate, and provides a rapid and convenient method for the assay of extra-cellular cell modifiers, especially when the assay is performed by the eluted stain assay method described herein. [In the following, reference is made mainly to hormones, but it will be understood that the same method can be used to assay any extracellular cell modifier, e.g. an interleukin, growth factor ( GF-1 GF-2, EGF or FGF) gamma-interferon, mitogen such as Concanavallin A, or any drug that mimics their action. Autoantibodies that mimic the effect of a particular hormone can also be assayed. In the new method, it is necessary to use a cell culture which is sensitive to the hormone to be assayed, such that a cellular component is altered in some measurable way by exposure to the hormone preferably before there is any substantial change in the number of cells in the culture. A variety of different effects is possible. The coloured or chromogenic reagent which interacts in a measurable way with a cellular component altered by the hormone, may be one which simply interacts in a quantitative way with that cellular component and thus provides a quantitative value for the total amount of that component present in the cells. For example, the reagent may be one which binds to a cellular component, e.g. a protein, and thus provides a measure of the total amount of protein present in the cell culture. Alternatively, the reagent may be a compound (or combination of compounds) which are altered by a cellular component, e.g. an enzyme, the amount or activity of which is altered by the action of the hormone on the cells. Another poεεibility is that the reagent may be a vital dye, e.g. neutral red, that is to say a dye which is taken up by unfixed living cells, especially if the cellular uptake procesε itεelf iε stimulated by the hormone. In some cases, the cellular component may be a cell constituent, e.g. fat, which is substantially only present in cells which have been exposed to the hormone being aεεayed. Another and preferred possibility is that the cellular component iε activated by the hormone without being altered in total amount. In all these cases, the interaction between the reagent and the cellular component must be measurable so as to provide a measure of the quantity of hormone in the sample. In the method of the invention, cell numbers preferably substantially do not change. The main advantages of this over assays which rely upon increases (or reductions) in cell number are: (i) Since only a short exposure time (hours as opposed to days) iε required, the assay need not be operated under sterile conditions. This greatly enhances the convenience and technical ease of the system and increases its commercial potential. (ii) A faεter asεay, e.g. same-day, aεsay is achieved. This iε important in the context of routine Clinical Chemistry, (iii) Enzymic amplification of the response iε often obtained. This system iε thereby capable of a much larger magnitude of response than that derived solely from a change in cell number. This may be demonstrated if longer exposure times are selected and a comparison iε made between increases in cell number and the increased response of the new assay system. This feature increases the precision of the bioassay. The interaction between the colorimetric reagent and the cellular component gives rise to a colour which can be measured spectrophotometrically. Such colour may either be originally present in the reagent, e.g. in the form of a dye, the amount of which bound by the cellular component depends on the amount of the cellular component which itself depends upon the amount of the hormone being assayed. Alternatively, the colour may itself be the result of the interaction between a chromogenic reagent and the cellular component, e.g. the result of the interaction between a dehydrogenase system and a tetrazolium salt. In either case, the colour is eluted from the cells of the cell culture and then measured spectrophotometrically in the elution medium. It is also possible to use a suitable chromogenic reagent capable of fluorescence or luminescence. Such a reagent can be used in a similar manner to the colorimetric reagents, and the result of the interaction between the reagent and the cell iε read with an appropriate photometer. It is especially convenient to carry out the new method in the wells of a microtitre plate. Each well contains the hormone sensitive cells either adherent to the base of the well or added aε a εuεpenεion. The hormone is added and incubated with the cellε. The cells, which will have been altered by the hormone are then subjected to the action of the reagent. Alternatively the hormone and the reagent may be added simultaneously. The colour bound by the cellε, or the colour formed by interaction between the cells and the reagent, is eluted from the cells in each of the wells of the microtitre plate and the colour in each well is then measured spectrophotometrically using for example a Molecular Devices microtitre plate reader. Although the colorimetric or chromogenic reagent is normally eluted from the cellε after it has interacted with a cellular component, it is also within the scope of the invention for the cellular component to be eluted from the cell prior to the addition of the reagent. In either case, the result of the interaction will be read in the medium outside the cell. Suitable cell lines for use in the method of the invention are generally available; see for example the catalogues of the various International Collectionε of Cell Cultureε, e.g. the European Collection of Animal Cell Cultures, published by the Public Health Laboratory Service Centre for Applied Microbiology and Research, Porton Down, U.K. For example, the 3rd Edition (1988) of the catalogue of this collection includes the stable T-cell line MOLT-4 which iε responεibe to gamma-interferon (Dadmarz e_t al , Leukemia Reεearch, JL0_, 1279-1285, 1986). The mouεe T-cell ly phoblaεt CTLL-M line which iε dependent upon Interleukin-2 (Sanderεon et al, Nature 268, 154-156, 1977) or the mouse embryo fibroblast 3T3 cell line (Swisε 3T3 cells) which responds to Epidermal Growth Factor (Rozengurt e_t aJL, Proc. Natl. Acad. Sci.) U.S.A., 7J3, 4392-4396, 1981). In addition many cell lines are responεive to hormones. For example, the cell lines known as FRTL-5 (Ambesi-Impiombato et al , Proc. Ntl . Acad. Sci, USA, 77 , 3455-3459, 1980) and SGHTL-4 (Whitely et al, Mol. Cell. Endocrinol., 5_2_, 279-283, 1987) are senεitive to thyroid simulating hormone (and the autoantibodies which mimic its effect), and FRTL-5 cellε are alεo reεponεive to human chorionic gonadotrophin (hCG) (Ealey et al , J. Endocrinol 106, 203-210, 1985; Ballabio et al, Acta Endocrinol., 116, 479-488, 1987), and inεulin-like growth factor (IGF-1). The FRTL-5 cell line has been used to assay auto-antibodies from patientε with Graveε' diεeaεe (an auto-immune thyroid disease) using conventional assays requiring the use of radioactive materials (Ambesi-Impiombato, USP 4608341). The cell lines Roε 17/2 (Majeska and Rodan, Calcif. Tissue. Int. 1 59-66, 1982) and UMR 104, 105, 106 or 108 (Partridge et al , Cancer Res. 4_3, 4308-4314, 1983) and the oposεum kidney cellε (OK cellε, Malmεtrom e_t a_l FEBS letterε 216 pp 257-260, 1987) are all εenεitive to parathyroid hormone. 3T3-F442A cells are sensitive to growth hormone (Morikawa, M. et al . , Mol. and Cellular Biology, 4_, 228-231, 1984) while rat Nb2 lympho a cells are sensitive to both growth hormone and prolactin (Tanaka et al, J. Clin. Endocrinol. Metabl. 5_6, 18-20, 1983). MA10 cells are responsive to luteinizing hormone and hCG (Ascoli, Endocrinology 108, 88-93, 1981) and Yl cells are responsive to adrenocorticotrophin (ACTH) (Kowal, Rec. Prog. Horm. Res. 2_6, 623-633 1970). In some cases, e.g. with cell-lines ROS 17/2, UMR104-108 or 3T3-F442A, the sensitivity of the cell-line is shown by redirected cell differentiation. For example, the UMR104-108 cellε and ROS17/2 show a marked reduction in alkaline phosphatase activity following exposure to parathyroid hormone, while the 3T3-F442A cells are transformed into fat cellε under the influence of growth hormone. This is accompanied by a marked increase in alpha glycerophosphate dehydrogenase activity. In εuch caεes, the reagent added to the cell culture is designed to. interact with the cell constituent which has been modified by exposure to the hormone. Further, in some caseε, the enzyme content of the cell culture may be activated by exposure to the hormone even though the total amount of the enzyme has not changed. For example, in FRTL-5 cellε the adenylate cyclaεe activity is activated almost inεtantly by expoεure of the cellε to thyroid εtimulating hormone. Here again, the reagent added to the cell culture would be choεen to reεpond to the increaεed activity of the enzyme preεent in the cell culture. With modern advanceε in cell fusion and cloning techniques, it may in some caεeε be appropriate to produce a new cell-line particularly reεponεive to a hormone which it iε desired to assay. While in most cases, the cell line used to assay the hormone iε choεen for its ability to respond to that hormone, it iε poεεible to enhance the response using certain cell stimulators. For example, the plant-derived compound forεkolin (7-beta-acetoxy-8 ,13-epoxy-l-alpha-6- beta-9-alpha-trihydroxy-labd-14-ene-ll-one ) iε known to stimulate adenylate cyclase activity in FRTL-5 cellε. Thiε can be used to potentiate the response of FRTL-5 cells to TSH ( see Fig.4) . It is also poεεible to use the new method for screening novel cell cultures for their sensitivity to a hormone. In this case, the assay method is carried out in exactly the same way except that the new cell culture is used together with the appropriate known hormone. The addition of the reagent and the measurement of the interaction between the reagent and the cellular component altered by the hormone iε carried out in exactly the same way as when the hormone itself is assayed. The reagent which interacts with the cellular component altered by the hormone is of course choεen with regard to the nature of the component which iε altered, e.g. a protein (which may be an enzyme) a lipid or a nucleic acid. Suitable dyes (stains) which interact with cellular proteins in a reproducible way include Naphthol Yellow S, Coomassie blue, dinitrofluorobenzene , light green and orange II. Staining methods for DNA include the use of the Feulgen reaction and methyl green pyronin for both DNA and RNA. Other stains may be used, including more particularly those already used in the microscopic examination of cells. Fluoreεcent probes for many cell components (e.g. as diεclosed in the catalogue of Molecular Probes Inc., Eugene, Oregon, U.S.A.) exist and can be used in the same way. Where the cellular component altered by the hormone is an enzyme, the reagent is chosen for its ability to interact with that enzyme system. The ability of MTT to interact with the cellular dehydrogenaεe system to produce dark blue formazan has already been mentioned. Other enzymes which may be present in cell cultures include peroxidaεes, acid, neutral and alkaline phoεphataseε, specific and non-specific esteraεeε, adenylate cyclaεe and ATPase. Methods for the detection of some of these enzymes and therefore reagents which interact with them, are already well known. Many of these have already been exploited for the bioasεay of hormones in the form of the Cytochemical Bioasεays devised by Chayen and co-workerε (εee Chayen e_t aJL, Cytochemical Assays in Endocrinology, in Recent Advances in Endocrinology and Metabolism 2_, pp 261-285 ed. O'Riordan, Churchill Livingεtone, 1982). In these Cytochemical Bioassays target-gland tissue segments or sections were used rather than cell cultureε. Since there iε marked heterogeneity between each tiεεue εection or εegment, it was not possible to devise a simple method of quantifying the cytochemical responses analogous to that used in the method of the present invention, which relies upon highly reproducible microcultureε. As a consequence, quantification was achieved by measuring the colorimetric reagent formed within single individual cellε. Thus whereaε with the method of the invention, the mean response of about 10 6 cellε within 96 microcultures in a microtitre plate can be monitored extremely rapidly (e.g. in about 1 second), the old Cytochemical Bioassay System relies upon single-cell measurements with a Microdensito eter, which permit the measurement of only about 20 cells in 30 minutes. However, the many different cytochemical procedures described by Chayen e_t a_l may all, in principle, be applied to the new method to obtain a parallel range of hormone bioasεays. Thus, the methodology of the present invention transforms what was widely considered the most laborious of all hormonal-bioassay systems, into one of the fastest and least technically demanding systems available. It iε possible to use two different staining methods sequentially on a single microculture. For example, the ' " assay referred to above may be performed on cellε, and, after the formazan produced has been eluted and measured, a second colorimetric assay, for example, to measure the protein or DNA content of the cellε, can be performed. The sequential εtaining technique may be useful to measure the effects of more than one hormone on the same target cell, if different cellular components (which interact with different stainε) which respond to different hormones can be identified. The techniques used in the new method are described in more detail in the following Examples. Cell Culture FRTL-5 cellε were maintained in stock cultures in Coon's modified F-12 medium supplemented with 5% newborn calf serum, 100 units penicillin/ml, 100 μq streptomycin per ml, 10 "3 units of thyroid stimulating hormone (TSH) per 1, 10 μg insulin per ml, 10 nM cortisol, 5 μg tranεferrin per ml, lOng glycyl-L-hiεtidyl-L-lysine acetate per ml and 10 ng somatostatin per ml. The cellε were plated out in wellε of a microtitre plate at a density of 5 X 10 4 cells/ml (150 / 1/well) in the above medium. In order to minimise plating density errors, a repeater Eppendorf pipette was uεed to dispense the cellε, which were mixed at frequent intervalε to prevent the cells settling to the bottom of the container from which they were being withdrawn. After 3 days in this medium, it was removed and replaced by a medium containing all the above constituents apart from the TSH in order to allow the cells to come to a resting state. At least 7 days were allowed for this to happen. The medium was changed every 3-4 days. Care was taken whenever the culture medium was removed not to cause any loss of the adherent cellε from the wellε. In order to achieve thiε, the medium was carefully tipped from the entire plate with a flick of the wrist, and then the plate was blotted on a sterile wad of tissue. In this way errors due to differences in cell density from well to well were reduced. Thiε cell culture and manipulation technique may be used with other strainε of cellε which adhere to wellε. Rat lymphoma cellε Nb2, which grow in suspension culture were grown in RPMI medium containing 10 ~4 M 2-mercaptoethanol , 50 units penicillin/ml, 50 μg streptomycin/ml, 2x 10 ~3 M glutamine, 10% foetal calf serum and 10% horse serum. Exposure to Hormone (i) Adherent Cellε The medium in the wellε waε replaced with medium containing the sample to be assayed and appropriate controls for 2 to 48 hours. When the asεay of hormoneε iε undertaken on a routine basis it iε poεεible to maintain a conεtant supply in the incubator of microtitre plates with cellε attached thereto in order to minimise delays between receiving and assaying samples of hormones. (ii) Cells in Suεpenεion The hormone was added to a suspension of cells in the wellε of the microtitre plates. A εtock εupply of cellε in suspension can be maintained in the incubator for routine use, by culture in tisεue culture flaεkε from which they can be dispensed into the microtitre plates. The reagent which reveals the effect of the hormone may then be added in accordance with one of the following procedures: 1. COLORIMETRIC DYE STAINING PROCEDURE In this Example, Naphthol Yellow S (NYS) was used, but other εtainε can be used instead. (I) Reagents (a) Fixation 2% glutaraldehyde in Dulbecco's phoεphate buffered saline (PBS) pH7.3. Dulbecco'ε phosphate buffered saline consists of 8g NaCl/1; 200mg KCl/1; 1.15g Na 2 HP0 4 /l , 132mg CaCl 2 .2H 2 0/1; 200mg KH 2 P0 4 /I and lOO g MgCl 2 .6H 2 0/1. 1ml 25% glutaraldehyde . was added to 11.5ml PBS to make the fixer solution. This may be stored at 4° C for up to 2 weeks. (b) Staining solution A 1% (w/v) εtock solution of NYS in 1% (w/v) acetic acid was made up. It was diluted 1:10 in 1% acetic acid just before use to make the 0.1% NYS staining solution. ( II ) Fixation and Staining procedure (a) Fixation 50 μl of fixer solution was added to each microtitre well which already contained 150 μl of culture medium, and left to fix for lh at room temperature. The medium was tipped off with a εharp flick of the wriεt and the plate was blotted on a paper towel to remove as much liquid as poεεible from the wellε. Each well waε filled with double diεtilled water and emptied aε before. Thiε wash procedure was repeated twice more. The plate was inverted and allowed to drain to dryneεε. It waε stored in the dark until required. (b) Staining 100 μl of 0.1% NYS solution was added to each well and left for 1 hour at room temperature. The staining εolution waε removed by tipping and blotting as described above. The plate waε immersed in 1% (w/v) acetic acid for 30 seconds taking care to remove air bubbles from the wells by tapping the plate. The plate waε removed, tipped and blotted. The plate waε then immersed in fresh 1% acetic acid for 5 minutes. The plate was again removed, tipped and blotted. The plate was immersed in a final fresh solution of 1% acetic acid for 30 minutes at room temperature. These washing steps remove non specific staining, for example, to the plastic of the plates. The plate was then inverted and allowed to dry and stored in the dark until required. (Ill) Measurement 100 μl 0.25M sodium hydroxide waε added to each well and mixed gently on a plate εhaker for approximately 30 seconds to elute the stain from the cellε. The optical density of the liquid in each well was measured using a microtitre plate reader at a teεt wavelength of 410 nm and a reference wavelength of 630 nm. ENZYME ACTIVATED STAINING PROCEDURE The cell culture waε as before. ( I) Reagents Staining Solution 5 mg/ml of 3-[4,5-dimethylthiazol-2-yl ] -2, 5- diphenyl-tetrazolium bromide (MTT) in phosphate buffered saline (PBS, as deεcribed above) . ( II ) Staining Procedure - Adherent Cells 15 μl of MTT at 50 mg/ml was added to the 150 μl culture medium already preεent in the microtitre wellε. The final concentration of MTT was thus 0.5 mg/ml. The contents of the wells were mixed gently by shaking the plate for approximately 10 seconds. The cells were incubated at 37° C in a dry incubator for 15 minutes. At the end of 15 minutes, the plate waε tipped and blotted aε deεcribed above. (Ill) Meaεurement - Adherent Cellε 100 μl of isopropanol containing 0.014M HCl was added to each well to elute the stain from the cells. The plate was shaken gently for 30 seconds. The optical density of the liquid in the microtitre wells was measured immediately to avoid evaporation, using a microtitre plate reader at a test wavelength of 570 nm and a reference wavelength of 630 nm. ( IV) Staining and Measurement - Non-adherent cells The cells were plated out at the required density in lOOμl of medium per well of a microtitre plate. After a suitable period of growth of the cells, their dehydrogenase activity was assayed by adding 10 μl of MTT solution at 5 mg/ml. After 40 minutes 150 l of 10% Triton-X in 0.5 M HCl waε addded to each well. The plate waε shaken gently on a plate shaker for 1 hour to release the formazan into the medium, which waε meaεured using a microtitre plate reader at a test wavelength of 570 nm and a reference wavelength of 630 nm. RESULTS The results show: (1) that cell stimulators such aε the anterior pituitary polypeptide hormones, e.g. thyroid stimulating hormone (TSH) induce early enzymic activation, which may be detected by the eluted stain system; (2) that this activation is independent of increases in cell number; and (3) that improved bioassays for such hormones may be based upon thiε observation. In Fig. 1 of the accompaning drawings, the responses of the two eluted stain systems are compared at selected time intervals subsequent to the addition of TSH to resting FRTL-5 cellε. Clearly the dehydrogenase activity, aε measured by cleavage of MTT, iε enhanced by the earliest reading taken (6 hours) at which time there has been no detectable increase in the NYS staining which reflects overall cell protein. This demonstrates that it iε possible to detect specific changes in enzyme activation, stimulated by the trophic hormone (TSH (100 mU/L ) , which occur before an increase in the number of cells. The metaphase index waε determined for a parallel series of microcultureε treated in the same way. The metaphase index iε the number of cells exhibiting metaphaεe figureε expreεεed aε a % of the total cells in a given culture (Ealey et al, J. Immunol. Methods 3 1, 117-123, 1988). This demonstrates that a wave of mitoεiε paεsed through the cultures after 20 hours exposure to the hormone. Thiε iε conεiεtent with a doubling time of 30 hourε for FRTL-5 cellε, and supports the observation that the bioasεay εyste can measure hormonal activation of the enzymic system. An additional noteworthy feature of these results iε that the two eluted stain assays were carried out sequentially. First, the dehydrogenase reaction was carried to completion; then the eluted formazan was removed and after fixation of the cellε, they were stained with NYS. Having observed the early enzymic activation, this was exploited for doεe reεponεe curveε after 4 hourε exposure of the microcultureε to TSH (Fig. 2) or the International Reference Standard for Thyroid Stimulating Antibodies, known aε Long Acting Thyroid Stimulator B (LATS-B) (Fig. 3). Doεe related increases in O.D. were obtained, and clearly these form the baεiε of 4 hour bioaεεayε for theεe stimulators. It is noteworthy that the use of the short 4 hour exposure time enhanced the senεitivity of both aεεayε. For example in Fig. 2, the detection limit to TSH waε <0.5 mU TSH/L whereaε it was ~5 mU TSH/L for aεεayε incubated with TSH for 48 hourε (See Fig. 4, which alεo εhowε that the addition of a low dose of Forskolin can potentiate the responses to TSH). Moreover the response to LATS-B plateaued at 10 U LATS-B/1 (Fig.3). This is the detection limit for the only comparable commercially available measurement εyεtem for thyroid εtimulating antibodies, which iε therefore clearly leεε sensitive. The old method moreover, measures only the ability of an antibody to displace TSH bound to its receptor, and not its potency aε a stimulator of thyroid cellε, as is measured by the new more sensitive bioaεsay εystem of the present invention. Figure 5 εhowε the reεultε of growth hormone (GH) εtimulation of Nb2 cellε. The doεe-related response in terms of an increase in cell number iε compared with the increaεe in dehydrogenase activity as detected with the eluted stain system. Thiε shows that the latter syεtem measures a ~30 fold increase in response, when the cell number which was measured with a Coulter Counter has increaεed by approximately only 4 fold. Clearly the enzymic activation, detected with the eluted εtain εystem, has produced conεiderable amplification of the reεponεe of these cellε to growth hormone.

Claims: CLAIMS 1. A method for the asεay of an extracellular cell-modifier which comprises: (i) exposing a culture of cellε containing a cellular component the activity or quantity of which iε sensitive to that cell modifier to a sample containing an unknown amount of the said cell modifier for a period of time sufficient for the activity or quantity of the said component to be altered by the said cell modifier if present; (ii) adding to the cell culture a colorimetric or chromogenic reagent which interacts in a measurable way with the cellular component altered by the said cell modifier; and (iii) measuring the interaction between the said reagent and the cellular component extracellularly. 2. A method according to claim 1 wherein the said cellular component iε protein, lipid, or nucleic acid and the said reagent is a dye or fluorescent or luminescent agent that binds to the said cellular component. 3. A method according to claim 2 wherein the said reagent is Naphthol Yellow S, Coomasεie blue, dinitrofluorobenzene, light green, orange II, Schiffε reagent as used in the Feulgen method, methyl green pyronin or neutral red. 4. A method according to claim 1 wherein the εaid cellular component iε an enzyme and the εaid reagent iε a chromogenic subεtrate which undergoeε a measurable change under the action of that enzyme. 5. A method according to claim 4 wherein the enzyme iε a dehydrogenaεe, peroxidaεe, acid or alkaline phoεphataεe, eεterase, or ATP-aεe. 6. A method according to claim 4 wherein the enzyme is a dehydrogenase and the said reagent is a tetrazolium salt e.g. 3-( 4 , 5-dimethylthiazol-2-yl )- 2, 5-diphenyltetrazolium bromide. 7. A method according to any one of claims 1 to 6 wherein the cell modifier assayed iε a hormone or autoantibody which mimics the effect of a hormone. 8. A method according to claim 7 wherein the hormone assayed iε thyroid-stimulating hormone, parathyroid hormone, growth hormone, luteinizing hormone, human chorionic gonadotrophin, prolactin, adrenocorticotrophin, or insulin-like growth factor. 9. A method according to claim 7 wherein the cell modifier assayed is an autoantibody which mimics the effect of thyroid εtimulating hormone. 10. A method according to any one of claimε 1 to 9 wherein the culture of cellε is adherent to the εurface of a well of a microtitre plate. 11. A method according to any one of claims 1 to 9 wherein the culture of cellε iε in εuεpenεion. 12. A method according to any one of claimε 1 to 11 wherein the nteraction between the said reagent and the cellular component iε measured by measuring with a spectrophotometer a colour the depth of which iε dependent on εaid interaction. 13. A teεt kit for the aεεay of an extra cellular cell modifier by the method of any of the preceding claimε which comprises: (a) a culture of cells containing a component the activity or quantity of which is sensitive to the cell modifier; (b) a colorimetric or chromogenic reagent which interacts in a meaεurable way with the cellular component altered by the said cell modifier; and (c) control samples of the εaid cell modifier. 14. A teεt kit according to claim 13 which also comprises a veεεel for the cell culture. 15. A test kit according to claim 13 or 14 which also comprises means for measuring the interaction between the εaid reagent and the cellular component. 16. A method for testing a cell culture for the presence therein of a cellular component the activity or quantity of which is sensitive to a cell modifier which comprises: (i) exposing the cell culture to the said cell modifier; (ii) adding to the cell culture a colorimetric or chromogenic reagent which interacts in a measurable way with a cellular component altered by the said cell modifier; and (iii) measuring the interaction between the said reagent and the cellular component extracellularly.

Inventors: EALEY PATRICIA A, HOLT STANLEY J, MARSHALL NICHOLAS J, EALEY, PATRICIA, A.;, HOLT, STANLEY, J., MARSHALL, NICHOLAS, J.;
Applicants: UNIV LONDON, UNIVERSITY COLLEGE LONDON
Application year: 1989
Main classification: C12Q1
IPC codes: C12Q1, G01N21, G01N33