WO1992003734A1 - A method for measuring t-lymphocyte responses by chemiluminescent assays - Google Patents

A method for measuring t-lymphocyte responses by chemiluminescent assays Download PDF

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Publication number
WO1992003734A1
WO1992003734A1 PCT/EP1991/001569 EP9101569W WO9203734A1 WO 1992003734 A1 WO1992003734 A1 WO 1992003734A1 EP 9101569 W EP9101569 W EP 9101569W WO 9203734 A1 WO9203734 A1 WO 9203734A1
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samples
sample
signals
photoconductors
carrier
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PCT/EP1991/001569
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French (fr)
Inventor
Alain De Weck
Friedrich-Ernst Maly
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Alain De Weck
Maly Friedrich Ernst
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Publication of WO1992003734A1 publication Critical patent/WO1992003734A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G01N21/763—Bioluminescence

Definitions

  • the present invention concerns a method for mea ⁇ suring T-lymphocyte responses by chemiluminescent assays, especially of T-lymphocyte responses in whole blood.
  • the registering and evaluating of the chemiluminescent assays is carried out by a computer system.
  • chemiluminescent assays for de ⁇ tecting serological immunological reactions has been known for at least 15 years (K. Van Dyke, Bioluminescence and che iluminescence: Instruments and applications. CRC Press, 1985). Basically such assays are very similar to those based on enzyme labelled techniques (ELISA) in which antigens and antibodies are covalently coupled with an enzyme.
  • ELISA enzyme labelled techniques
  • the substrate to be used is not a chromogen deliv ⁇ ering some color in the visible range but a substrate such as luminol, luciferin or lucigenin and becomes lumi ⁇ nescent when reacting with a reactive oxygen species pro- cuted by the enzyme (e.g. peroxidase) .
  • Chemiluminescent assays have over the classical and widespread ELISA assays the advantage of being markedly more sensitive (10-100 fold). They have the dis ⁇ advantage of requiring more sensitive instruments for de- tection (luminometer instead of color densitometer) which are more difficult to produce and calibrate and also require more stringent optical conditions. This is probably the main reason why chemiluminescent assays have not become so widely used as ELISA or radioimmuno assays (RIA); the automated or semiautomated instrumentation re ⁇ quired for simultaneous analysis of a large number of samples has not been developed and/or is rather expen ⁇ sive. Beside classical antigen-antibody reactions in fluid phase or on solid phase (e.g.
  • chemiluminescent reactions can also be used to assess the reactivity of a wide variety of blood cells, in particular neutrophils and monocytes, which produce large amounts of oxygen radicals when properly stimu ⁇ lated.
  • neutrophils and monocytes which produce large amounts of oxygen radicals when properly stimu ⁇ lated.
  • lymphokines capable of di ⁇ rectly stimulating chemiluminescence reaction in target cells or of preparing such cells ( "priming") for more ef ⁇ ficient reaction following interaction with a chemilumi ⁇ nescent stimulant under quantitative standardized condi ⁇ tions.
  • This use of chemiluminescent cellular reactions is still in its infancy (Fritsche R. , de Week A. L. Detec- tion of Chemiluminescence in single cells. Eur. J. Im ⁇ munol.,18, 817-820, 1988), but the potential of this type of technology for assessing cellular reactions, in par ⁇ ticular the reactivity of T lymphocytes which play a key role in many immunological situations, is considerable.
  • the Chemiluminescence Multiwell Analyzer developed on the basis of an existing Hamamatsu videoimaging system obviates these disadvantages and per ⁇ mits simultaneous quantitative evaluation of a very large number of samples, paving the way for widespread and rou ⁇ tine analytical application of serological and chemilumi- nescent techniques (P.-E. Maly et al, J. Immunol. Meth., 122 (1989) 91-96).
  • the used instrumentation for this method comprises a black box, a special video camera (Vidicon camera with photon enhancing device), an image processor and an image controller and a personal computer with videomonitor and printer.
  • the chemi ⁇ luminescence signals are recorded as a picture which is converted into a series of digital signals corresponding to the chemiluminesce emission of the samples. It was possible to seize and print 192 results in about 3 min ⁇ utes.
  • ex vivo tests assessments of the spontaneous luminescence emitted by cells taken from blood and tissues (so called ex vivo tests) enables evaluation of the degree of cell activation which has occurred in vivo and thereby the intensity of the inflammatory process going on during the course of a disease. Such tests may be useful in order to monitor the spontaneous evolution of the effect of treatment in chronic inflammatory diseases.
  • the addition of well known doses of cell activators to cells in vitro enables quantitative assessment of the reactivity of such inflammatory cells taken from a diseased organism.
  • the cells may be less reactive than those of a normal healthy individual, which then indicates an immune deficiency or toxic effects on cells participating in immune defences.
  • the cells may be hyperreactive because they have been primed in vivo for better reactivity to inflammatory mediators.
  • the subject-matter of the present invention is a process according to the definition of claim 1 and a de ⁇ vice according to claim 6 for carrying out this process.
  • chemi ⁇ luminescence signals of antigen-T cell interaction in presence of luminescence agents are simultaneously de ⁇ tected and .evaluated for measuring T-lymphocytes re- sponses to different activators, as mitogens and anti ⁇ gens.
  • the T-lymphocytes are brought for example into con ⁇ tact with antigens and a luminescent substrate is added for registering and evaluating the occurring chemilumi ⁇ nescence.
  • the process according to the invention has substantially the following marked advantages over cur ⁇ rent radioimmuno assays and enzyme-labelled immunoassays:
  • the simultaneous and continu ⁇ ous monitoring of the parallel photon emission of samples on microtiter plates, for the first time allows the study and quantitative evaluation over a prolonged period of time of various activation phenomena of T lymphocytes, with proper controls at the same time.
  • This permits the development of diagnostic assays hitherto not available, the principles and some examples of which are disclosed here.
  • the only other technical way of achieving a similar goal is the use of a videoimaging system (Maly et al, J. of Immunol. Meth. 122, (1989) 91-96; Anal. Biochem. (1988) 168, 462.)
  • the gain in sensitivity obtained by continuous recording and integration of photon emission is a deci ⁇ sive advantage over conventional immunoassay techniques. This permits: a) performance of immunoassays with stable reagents in instances where otherwise only expensive, un ⁇ stable and ecologically objectionable radioimmunological techniques are required; b) reduction of the amounts of fluids required for analysis (e.g whole blood, cell sus- pension etc.) thereby making diagnostic tests more prac ⁇ ticable or at all possible.
  • the device for carrying out the process according to the invention comprises a receiving unit for the chemiluminescence emissions, a detector unit and a data evaluation unit, wherein the receiving unit for the chemiluminescent samples comprises a cover for covering multiple-sample-carriers or a cassette for holding multi ⁇ ple-sample-carriers wherein a multiplicity of photocon- ductors proceed from this receiver and lead to the detec- tor unit.
  • the number of photoconductors corresponds to the foreseen number of samples on the carrier to be mea ⁇ sured.
  • the inlets of the photoconductors are disposed as near as possible to the foreseen position.
  • the detector unit is capable of intensifying the signals for each pho- toconductor individually and of converting them into electrical signals which can be processed by a computer system for evaluating the series of signals.
  • the detector unit normally comprises a position resolving photomultiplier or a position resolving mi- crochannel plate.
  • the receiving unit for the chemiluminescent sam ⁇ ples is a light tight black box comprising preferably a drawer for the carrier of the standardized carrier of the chemiluminescent samples.
  • the carrier is normally a mi- crotiter plate having 96 or 192 wells for receiving the samples. If necessary the receiving unit can be adapted to another type of carrier having another number of wells. Of course the number of photoconductors must cor ⁇ respond to the number of wells on the carrier.
  • the sur- face of the carrier, especially the inner surface of the wells, is preferably coated with a layer of a material reflecting the emission of the samples.
  • Fig. 1 is a view of a means comprising photoconductors for covering a carrier comprising multi ⁇ ple samples
  • Fig. 2 is a diagrammatic view of a device for carrying out the process according to the invention.
  • Fig 3 shows the detection of increasing T cell dependent chemiluminescence in human blood cultured fol ⁇ lowing stimulation by polyclonal mitogen (anti-T cell OKT3 antibody) or by antigen (Candida albicans) (example 1),
  • Fig. 4A to 4D show a comparison of chemilumines ⁇ cence assay in MTP-Reader Multi Channel according to the invention with H- ⁇ -Thymidine Incorporation Assay (example 2),
  • Fig 5 shows the correlation between the reaction to Candida and to Rabies antigens in immunized and non immunized individuals (rabies vaccine),
  • Fig 6 shows a CL Test with supernatant of human mononuclear cells stimulated by anti-T cell OKT3 antibody for 48 hours (Example 3),
  • Fig.l and 2 give a schematic overview of an ar ⁇ rangement of instruments forming a device according to the invention.
  • a microtiter plate 3 hav- ing wells 4 for carrying the samples 5.
  • the drawer carrying the microtiter plate 3 is introduced into the opening 5 of a light-proof sample re ⁇ ceiving unit 1.
  • a cover 6 is moved in direction of the arrows 8 close to the surface of the microtiter plate 3 carrying the light emitting samples 5.
  • the microtiter plate is moved close to the cover 6 where technically required.
  • the microtiter plate is preferably coated with a light reflecting layer, at least in the wells 4.
  • inlets of light conductors 9 are disposed, in such a manner that during the measurement the samples are positioned as close as possible to the inlet of the corresponding light conduc- tors 9.
  • the light conductors leaving the cover 6 are bun ⁇ dled together forming the track 10 leading to the detec ⁇ tor unit 12 comprising a position resolving photomulti- plier or a position resolving microchannel plate.
  • the optical signals are converted into digitalized electronic signals which are sent by the conductor 13 to the computer system 14 being connected with a plotter 15.
  • T lymphocytes are key cells in immune responses. Upon non specific activation, e.g. by various mitogens or lymphokines, they produce themselves various lymphokines and cell mediators which transmit messages to other cells and initiate further amplification reactions, such as re ⁇ quired for the build up of inflammatory cellular infil ⁇ trates, for the growth and development of blood cells (hemopoiesis) or for various types of tissue reactions (graft rejection, wound healing, tissue fibrosis). T lym ⁇ phocytes are also the prime carriers of specific immune reactions, in which either foreign or autoantigens are recognized and react primarily with specific T cell receptors. Such specific antigen-T cell interactions lead also to cellular activation and to the production of the above mentioned lymphokines and cellular mediators.
  • T lymphocytes Due to the key function of T lymphocytes, diagnostic tests to assess their reactivity either fol ⁇ lowing specific or non specific stimulation have a great potential in human and veterinary medicine in such fields as allergy, diagnostic and monitoring of infectious dis ⁇ eases, diagnostic and monitoring of autoimmune or chronic inflammatory diseases, evaluation of immune deficiencies, evaluation of immunotherapy, organ and tissue transplan ⁇ tation and many others.
  • current tests to assess T cell reactivity are rather cumbersome and/or in ⁇ sensitive, n addition, they require rather sophisti ⁇ cated equipment and techniques, which make them possible only in a few highly specialized laboratories.
  • lymphocyte proliferation or transformation assay
  • DNA synthesis of activated lymphocytes set in culture is evaluated aftar a period of at least 3 - 5 days, from the incorporation into DNA of a radiolabelled nucleotide, such as tritium-labelled thymidine.
  • a radiolabelled nucleotide such as tritium-labelled thymidine.
  • An additional disad ⁇ vantage of these tests is that in the lymphocyte prolif- eration assay, isolated and cultured lymphocytes must be set in culture and put in contact with non specific or specific stimulants soon after, i.e. within a few hours of having been collected from the organism, e.g. by draw ⁇ ing blood. Accordingly, blood taken from patients has to be processed near to the laboratory, which also limits the practical usefulness of the method.
  • T lymphocytes An alternative methodology for assessing specific or non specific reactivity of T lymphocytes is the object of the present invention and has been made possible by the chemiluminescence multiwell analyzer described here.
  • This methodology is based on the fact that very soon fol ⁇ lowing contact between activator (mitogen or antigen) and T lymphocytes, activation of these cells takes place and leads to the synthesis of various mediators, such as lym ⁇ phokines, which themselves are capable of activating other blood* cells present in the same blood or cell sus- pension.
  • mediators such as lym ⁇ phokines, which themselves are capable of activating other blood* cells present in the same blood or cell sus- pension.
  • monocytes and neutrophils produce a potent and prolonged oxydative burst, which may be appropriately translated into chemiluminescent signals.
  • Luminescence Assays Perspectives in Endocrinology and Clinical Chemistry, Eds. Serio,M. and M.Pazzagli, Raven Press, New York, p 229-241) on the one hand, and the variable reactivity state of potential target cells for lymphokines, on the other hand, would seem to pre- elude the development of reliable and reproducible chemi ⁇ luminescent tests of T cell activation.
  • whole blood is slightly diluted in a suitable culture medium to which appropriate amounts of non specific stimulant (e.g. mito- gen such as anti-CD 3 antibody) or specific stimulant (e.g. antigen, allogeneic cells, tumor cells, etc.) have been added.
  • non specific stimulant e.g. mito- gen such as anti-CD 3 antibody
  • specific stimulant e.g. antigen, allogeneic cells, tumor cells, etc.
  • the blood cells are separated by gradient centrifugation and analyzed in the chemiluminescence multiwell analyzer.
  • the chemiluminescent signals ob ⁇ served in whole blood do not originate, at least not in significant manner, from the activated T lymphocytes themselves but from bystander cells in the reaction, such as monocytes or neutrophils, secondarily activated by lymphokines and possibly other mediators produced by the activated T lymphocytes.
  • Another way to perform the chemiluminescent assays for T cell activation consists in first culturing isolated lymphocytes together with non specific mitogens or specific allergens, then harvesting culture supematants and setting up the supematants to ⁇ gether with a chemiluminescent substrate system and an appropriate target cell population generating chemilumi ⁇ nescent signals.
  • this mode of operation may have several advantages. First, it enables more precise analysis of the factors and mediators produced by acti ⁇ vated T cells. Second, it enables some standardization of the assay by better definition and reproducibility of the target cell population generating the chemilumines ⁇ cent signals. For example, this may be a neutrophil or monocyte pool, or a cell line cultured under standardized conditions. In this way, one of the variables in the as ⁇ say, namely an autologous target cell population, may be eliminated.
  • chemiluminescent (CL) T cell activation test for realization of a chemiluminescent (CL) T cell activation test in whole blood, a set of sterile flasks with rubber seal are prepared, containing RPMI 1640 medium supplemented with 10% pooled human serum, or any other suitable culture medium, as controls.
  • RPMI 1640 medium supplemented with 10% pooled human serum, or any other suitable culture medium, as controls.
  • non specific mitogens e.g. 0.1 - 10 ⁇ g of monoclonal anti-CD 3 antibody
  • specific anti- gens e.g. 50 microliters of a 1 - 20 ⁇ g/ml Candida anti ⁇ gen solution
  • the experimenter adds 5 milliliters of freshly drawn venous blood under sterile conditions. After thorough agitation, the cul ⁇ ture flasks are kept (or transported to the laboratory) at ambient temperature (15 - 37°C) for periods of up to 24 hours. After 24 hours, the culture flasks are opened and culture continued for another 24-48 hours in a tissue culture incubator set up at 37°C in an atmosphere of 5% CO2• At the end of the culture period, the cultured blood is centrifuged over suitable gradient (e.g.
  • Ficoll- Hypaque Ficoll- Hypaque
  • a suitable medium e.g. TO 38/BSA
  • the cells at a concentra ⁇ tion of 1 x 10 6 mononuclear cells/ml are distributed in white microstrip wells (e.g. DYNATECH) . 120 micromoles / liter lucigenin are added to each microwell and the re ⁇ sulting photon emission followed for 30 minutes up to several hours in the chemiluminescence multiwell analyzer described here.
  • Fig. 3 The-results of such an experiment, including non stimulated control cells and experimental cells stimu- lated by various doses of mitogen and antigen are shown in Fig. 3. It must be emphasized that the general proce ⁇ dure outlined here can be slightly modified. For exam ⁇ ple, the original cells cultured in whole blood for up to 24 hours may be then immediately centrifuged upon arrival in the analyzing laboratory and then transferred to ordi ⁇ nary cell culture conditions, with or without supplemen ⁇ tation with mediators such as lymphokines (e.g. inter- leukin 2) known to potentiate T cell functions, and with or without the addition of standardized target cells, such as neutrophil or monocyte pools, or suitable chemi ⁇ luminescent signal producing cell lines.
  • lymphokines e.g. inter- leukin 2
  • target cells such as neutrophil or monocyte pools, or suitable chemi ⁇ luminescent signal producing cell lines.
  • Mononuclear cells are isolated from venous pe ⁇ ripheral blood by Ficoll-Hypaque gradient centrifugation and resuspended in RPMI 1640 culture medium supplemented with 10% human AB serum pool. Cultures are set in white microstrip wells (DYNATECH) at the concentration of 1 x 10 6 cells / 250 microliters. In experimental wells, Can ⁇ dida antigen prepared as indicated above is added at var- ious concentrations (1 - 20 ⁇ g/ml) or anti-CD3 antibody (0.1 - 10 ⁇ g/ml) as control of polyclonal activation. In order to demonstrate the antigen specificity of the CL T cell activation test, and in addition to the ubiquitous antigen Candida, two groups of individual cultures were also set up with rabies vaccine antigen (Lyssavec,
  • Cultures were set in a tissue culture incubator at 37°C and 5% C0 2 for periods varying between 24 and 72 hours, following which 25 microliters of a 12 mmol/1 lu- cigenin solution are added to each well.
  • Parallel cul ⁇ tures were set up to as ⁇ sess lymphocyte proliferation by radiolabelled thymidine incorporation.
  • Fig. 4A and 4B show Candida and Lyssavac chemiluminescence stimulation indices SImax, detected during the culture interval from 24 h to 72 h and Fig 4C and 4D show the Candida and Lyssavac induced 3 H-thymidine incorporation stimulation indices SI after a culture in- tervall of 72h.
  • Fig 5 shows the correlation between Candida and Lyssavac induced chemiluminescence.
  • Mononuclear cells are isolated from venous peripheral blood and set in cultures with various stimu ⁇ lants as indicated above, for periods varying between 24 and 72 hours. At the end of the culture periods, the cells are centrifuged and discarded. The harvested su ⁇ pematants (100 microliters) are then given to target cells distributed in white microstrips at concentrations varying between 10 5 and 10 6 / ml. As target cells, a pool of monocytes or neutrophils isolated by Ficoll-Hy- paque gradient centrifugation, or a suitable cell line, such as HL-60 or U 937 may be used.

Abstract

The response T lymphocytes to different activators is detected by simultaneously registering and evaluating the chemiluminescence emission of specific reactions in multiple samples (5) on a multiple-sample-carrier (3). After the contact with an activator (mitogen or antigen), T lymphocytes are activated and various mediators are produced, being capable of activating other blood cells being present in the same blood or cell suspension. They can produce an oxydative burst, which can be, together with a chemiluminescent agent, translated into chemiluminescent signals. The samples (5) to be tested and the reagents required for the chemiluminscence reaction are placed at the foreseen locations (4) into the multiple-sample-carrier (30). This carrier is light-proof sealed with a suitable cover plate (6) or cassette having several photoconductors (9). The connections (7) of the photoconductors to the cover plate (6) or the cassette correspond to the samples placed on the multiple-sample-carrier (3) with regard to location and number. The photons emitted by the samples are led by the photoconductor (9, 10) to a detector system (12) which converts the signals of each sample individually into signals which can be detected by a computer. These converted signals are directed to a computer evaluation system (14) which evaluates the signals emitted by the samples (5).

Description

A method for measuring T-lymphocyte responses by chemilu¬ minescent assays
The present invention concerns a method for mea¬ suring T-lymphocyte responses by chemiluminescent assays, especially of T-lymphocyte responses in whole blood. The registering and evaluating of the chemiluminescent assays is carried out by a computer system.
The principle of chemiluminescent assays for de¬ tecting serological immunological reactions has been known for at least 15 years (K. Van Dyke, Bioluminescence and che iluminescence: Instruments and applications. CRC Press, 1985). Basically such assays are very similar to those based on enzyme labelled techniques (ELISA) in which antigens and antibodies are covalently coupled with an enzyme. However, in the case of chemiluminescent as¬ says, the substrate to be used is not a chromogen deliv¬ ering some color in the visible range but a substrate such as luminol, luciferin or lucigenin and becomes lumi¬ nescent when reacting with a reactive oxygen species pro- duced by the enzyme (e.g. peroxidase) .
Chemiluminescent assays have over the classical and widespread ELISA assays the advantage of being markedly more sensitive (10-100 fold). They have the dis¬ advantage of requiring more sensitive instruments for de- tection (luminometer instead of color densitometer) which are more difficult to produce and calibrate and also require more stringent optical conditions. This is probably the main reason why chemiluminescent assays have not become so widely used as ELISA or radioimmuno assays (RIA); the automated or semiautomated instrumentation re¬ quired for simultaneous analysis of a large number of samples has not been developed and/or is rather expen¬ sive. Beside classical antigen-antibody reactions in fluid phase or on solid phase (e.g. in ELISA type micro- wells), chemiluminescent reactions can also be used to assess the reactivity of a wide variety of blood cells, in particular neutrophils and monocytes, which produce large amounts of oxygen radicals when properly stimu¬ lated. Although much research on cellular reactions has been devoted up to now to the direct study of cellular activation itself and to the production of oxygen radi- cals, it has become clear that the chemiluminescence phe¬ nomena could be used to evaluate indirectly the function of a large variety of other cells, which upon stimulation may produce factors (e.g. lymphokines) capable of di¬ rectly stimulating chemiluminescence reaction in target cells or of preparing such cells ( "priming") for more ef¬ ficient reaction following interaction with a chemilumi¬ nescent stimulant under quantitative standardized condi¬ tions. This use of chemiluminescent cellular reactions is still in its infancy (Fritsche R. , de Week A. L. Detec- tion of Chemiluminescence in single cells. Eur. J. Im¬ munol.,18, 817-820, 1988), but the potential of this type of technology for assessing cellular reactions, in par¬ ticular the reactivity of T lymphocytes which play a key role in many immunological situations, is considerable.
The major drawback for the use of chemilumines¬ cent reactions in diagnostic analysis has been on the one hand the relative complexity and expense of the equipment required for chemiluminescence measurement and on the other hand the fact that the kinetics of such reactions may be quite variable: from a few seconds for serolo'gical reactions to several hours for some cellular reactions. Accordingly, instruments possessing only one or a limited number of analytical channels may be quite limited in their performance of multiple analysis.
The Chemiluminescence Multiwell Analyzer (CMA) developed on the basis of an existing Hamamatsu videoimaging system obviates these disadvantages and per¬ mits simultaneous quantitative evaluation of a very large number of samples, paving the way for widespread and rou¬ tine analytical application of serological and chemilumi- nescent techniques (P.-E. Maly et al, J. Immunol. Meth., 122 (1989) 91-96). The used instrumentation for this method comprises a black box, a special video camera (Vidicon camera with photon enhancing device), an image processor and an image controller and a personal computer with videomonitor and printer. In this method the chemi¬ luminescence signals are recorded as a picture which is converted into a series of digital signals corresponding to the chemiluminesce emission of the samples. It was possible to seize and print 192 results in about 3 min¬ utes.
However, this method requires very expensive equipment, even if trimmed to the bare essential; it can be run economically only in a qualified laboratory. There is a need for a method using instrumentation which is less expensive and which can be readily used in a small laboratory e. g. in a laboratory of a small hospital.
In US-A-4 349 510 (Kolehmainen et al.) a method and an apparatus for measurement of chemiluminescence are disclosed. In this method the simultaneous measurement of 2-20 samples or more is possible. The samples are de¬ posited in depressions of a light-reflecting tape. In an embodiment it is proposed to use a bundle of optical fibers and a single photo ultiplier detector to measure a whole row of samples. In this system the fiber bundle will move from one sample position to another, until all wells which are in a row are measured sequentially. After the scanning of one row, the tape will move to place the next row of samples in the measuring position. The method allows the measurement of up to 500 samples per hour. In comparison to the above mentioned method of Maly et al. this procedure is relatively slow because only a limited number of samples can be measured and evaluated simulta¬ neously.
It s an object of the present invention to cre¬ ate a more efficient and more economical method for si- multaneously registering and evaluating the chemilumines¬ cence emission of specific reactions in many samples on a multi-sample carrier for measuring T lymphocyte responses to different antigens, especially in whole blood. It solves the problem of how to carry out the method without using the expensive videoimaging system, but with the same efficiency. The suggested method is free of the above-mentioned drawbacks.
Assessment of the spontaneous luminescence emitted by cells taken from blood and tissues (so called ex vivo tests) enables evaluation of the degree of cell activation which has occurred in vivo and thereby the intensity of the inflammatory process going on during the course of a disease. Such tests may be useful in order to monitor the spontaneous evolution of the effect of treatment in chronic inflammatory diseases. The addition of well known doses of cell activators to cells in vitro, enables quantitative assessment of the reactivity of such inflammatory cells taken from a diseased organism. In some cases, the cells may be less reactive than those of a normal healthy individual, which then indicates an immune deficiency or toxic effects on cells participating in immune defences. In other cases, on the contrary, the cells may be hyperreactive because they have been primed in vivo for better reactivity to inflammatory mediators.
For all such analyses, which are becoming of increasing diagnostic significance in clinical medicine, it is essential that together with the cells being analyzed and activated by various agents in vitro or in vivo, appropriate controls be performed. These controls usually consist of cells from the same individual not mixed with chemiluminescent substrates (detection system background) and of non activated cells (activation background) . Since the biological state of the activated cells, in particular their viability and functionality when taken from the organism and kept in vitro, rapidly varies, it would be of great advantage for precision and reproducibility of the assays, if the control and the cells to be assayed could be investigated simultaneously. This has not been possible up to now with chemilumines- cence detecting instruments possessing a single or a few parallel detecting channels. Accordingly, a chemilu¬ minescence detecting instrument possessing multiple detecting channels, such as the one described here, has decisive advantages over detectors with single or few channels.
This advantage is made even more decisive in practice by the slow kinetic of cellular chemilumines¬ cence reactions. The chemiluminescent signals produced by the cells under investigation usually take several minutes, and in some cases several hours to reach their peak or plateau intensity. Accordingly, a single channel detector will necessitate, for investigating a given number of experimental and control samples, a total time obtained by multiplying the required observation/detec- tion time (e.g. 60 minutes per sample) by the number of samples to be analyzed. This procedure rapidly becomes prohibitive and also has the great disadvantage that experimental and control cell populations are analyzed at very different times and therefore in different bio- logical conditions (see above). One possible way of obviating this inconvenience is to multiply the number of detecting channels operating in parallel but this leads to an expensive construction and has both technical and economic limitations. For these reasons, the only multi- channel chemiluminescence detector currently available possesses 6 parallel detecting channels (e.g. Berthold LB 9505) . Another possible way to achieve reading of multiple samples within a reasonable operating time is to reduce the detection time for each sample to a few seconds or microseconds, shifting mechanically and successively the samples to be analyzed in front of a single detector. For analysis of biological processes lasting several minutes or hours, the detection procedure may be repeated at various times, using an appropriate mechanical device. This is the procedure currently applied in at least two commercially available Microtiter Plate Chemiluminescence Analyzers (Amerlite, Flow) . This, however, has the inconvenience of not enabling simultaneous analysis of experimental and control samples at the same time. Furthermore, since chemiluminescence detection time of individual samples is arbitrarily reduced, only a small portion of the photons emitted by each individual sample is actually recorded. In the detecting instrument described here, all photons emitted by each sample are continuously recorded and the total number of photons emitted over a period of time, which may be freely chosen and varied by the investigator, can be integrated.
This leads to a considerable increase in sensitivity (Maly, F.-E., et al. 1989, A dual micro- titerplate luminometer employing computer-aided single- photon imaging applicable to cellular luminescence and luminescence immunoassay. J.Immunol. eth. 122 : 91-96). A major practical advantage in such an increase in sensitivity is the possibility of performing chemiluminescence assays with a much lower amount of cells thereby permitting such tests to be performed on infants or on limited cell numbers available from inflammatory exudates.
The subject-matter of the present invention is a process according to the definition of claim 1 and a de¬ vice according to claim 6 for carrying out this process. In the method according to the invention chemi¬ luminescence signals of antigen-T cell interaction in presence of luminescence agents are simultaneously de¬ tected and .evaluated for measuring T-lymphocytes re- sponses to different activators, as mitogens and anti¬ gens. The T-lymphocytes are brought for example into con¬ tact with antigens and a luminescent substrate is added for registering and evaluating the occurring chemilumi¬ nescence.
The process according to the invention has substantially the following marked advantages over cur¬ rent radioimmuno assays and enzyme-labelled immunoassays:
In some instances, the simultaneous and continu¬ ous monitoring of the parallel photon emission of samples on microtiter plates, for the first time, allows the study and quantitative evaluation over a prolonged period of time of various activation phenomena of T lymphocytes, with proper controls at the same time. This permits the development of diagnostic assays hitherto not available, the principles and some examples of which are disclosed here. The only other technical way of achieving a similar goal is the use of a videoimaging system (Maly et al, J. of Immunol. Meth. 122, (1989) 91-96; Anal. Biochem. (1988) 168, 462.)
However the use of a detector unit technology as described here enables achievement of superior results at a fraction of the cost. Under consideration of the cur¬ rent state of the art it was surprising that a low-cost instrument of the required sensitivity could be made without using the videoimaging technology.
The gain in sensitivity obtained by continuous recording and integration of photon emission is a deci¬ sive advantage over conventional immunoassay techniques. This permits: a) performance of immunoassays with stable reagents in instances where otherwise only expensive, un¬ stable and ecologically objectionable radioimmunological techniques are required; b) reduction of the amounts of fluids required for analysis (e.g whole blood, cell sus- pension etc.) thereby making diagnostic tests more prac¬ ticable or at all possible.
The device for carrying out the process according to the invention comprises a receiving unit for the chemiluminescence emissions, a detector unit and a data evaluation unit, wherein the receiving unit for the chemiluminescent samples comprises a cover for covering multiple-sample-carriers or a cassette for holding multi¬ ple-sample-carriers wherein a multiplicity of photocon- ductors proceed from this receiver and lead to the detec- tor unit. The number of photoconductors corresponds to the foreseen number of samples on the carrier to be mea¬ sured. The inlets of the photoconductors are disposed as near as possible to the foreseen position. The detector unit is capable of intensifying the signals for each pho- toconductor individually and of converting them into electrical signals which can be processed by a computer system for evaluating the series of signals.
The detector unit normally comprises a position resolving photomultiplier or a position resolving mi- crochannel plate.
The receiving unit for the chemiluminescent sam¬ ples is a light tight black box comprising preferably a drawer for the carrier of the standardized carrier of the chemiluminescent samples. The carrier is normally a mi- crotiter plate having 96 or 192 wells for receiving the samples. If necessary the receiving unit can be adapted to another type of carrier having another number of wells. Of course the number of photoconductors must cor¬ respond to the number of wells on the carrier. The sur- face of the carrier, especially the inner surface of the wells, is preferably coated with a layer of a material reflecting the emission of the samples. The invention is described in detail below with reference to the attached figures, which illustrate specific embodiments of the in- vention, in which:
Fig. 1 is a view of a means comprising photoconductors for covering a carrier comprising multi¬ ple samples,
Fig. 2 is a diagrammatic view of a device for carrying out the process according to the invention, and
Fig 3 shows the detection of increasing T cell dependent chemiluminescence in human blood cultured fol¬ lowing stimulation by polyclonal mitogen (anti-T cell OKT3 antibody) or by antigen (Candida albicans) (example 1),
Fig. 4A to 4D show a comparison of chemilumines¬ cence assay in MTP-Reader Multi Channel according to the invention with H-^-Thymidine Incorporation Assay (example 2),
Fig 5 shows the correlation between the reaction to Candida and to Rabies antigens in immunized and non immunized individuals (rabies vaccine),
Fig 6 shows a CL Test with supernatant of human mononuclear cells stimulated by anti-T cell OKT3 antibody for 48 hours (Example 3),
Fig.l and 2 give a schematic overview of an ar¬ rangement of instruments forming a device according to the invention.
In a drawer 2 there is a microtiter plate 3 hav- ing wells 4 for carrying the samples 5. For measuring the samples, the drawer carrying the microtiter plate 3 is introduced into the opening 5 of a light-proof sample re¬ ceiving unit 1. As soon as the microtiter plate is in the correct poaition a cover 6 is moved in direction of the arrows 8 close to the surface of the microtiter plate 3 carrying the light emitting samples 5. Alternatively, it is also possible that the microtiter plate is moved close to the cover 6 where technically required. The microtiter plate is preferably coated with a light reflecting layer, at least in the wells 4. At positions corresponding to the sites of the wells 4 at the cover 6 inlets of light conductors 9 are disposed, in such a manner that during the measurement the samples are positioned as close as possible to the inlet of the corresponding light conduc- tors 9. The light conductors leaving the cover 6 are bun¬ dled together forming the track 10 leading to the detec¬ tor unit 12 comprising a position resolving photomulti- plier or a position resolving microchannel plate. In this unit the optical signals are converted into digitalized electronic signals which are sent by the conductor 13 to the computer system 14 being connected with a plotter 15.
T lymphocytes are key cells in immune responses. Upon non specific activation, e.g. by various mitogens or lymphokines, they produce themselves various lymphokines and cell mediators which transmit messages to other cells and initiate further amplification reactions, such as re¬ quired for the build up of inflammatory cellular infil¬ trates, for the growth and development of blood cells (hemopoiesis) or for various types of tissue reactions (graft rejection, wound healing, tissue fibrosis). T lym¬ phocytes are also the prime carriers of specific immune reactions, in which either foreign or autoantigens are recognized and react primarily with specific T cell receptors. Such specific antigen-T cell interactions lead also to cellular activation and to the production of the above mentioned lymphokines and cellular mediators. Due to the key function of T lymphocytes, diagnostic tests to assess their reactivity either fol¬ lowing specific or non specific stimulation have a great potential in human and veterinary medicine in such fields as allergy, diagnostic and monitoring of infectious dis¬ eases, diagnostic and monitoring of autoimmune or chronic inflammatory diseases, evaluation of immune deficiencies, evaluation of immunotherapy, organ and tissue transplan¬ tation and many others. Unfortunately, current tests to assess T cell reactivity are rather cumbersome and/or in¬ sensitive, n addition, they require rather sophisti¬ cated equipment and techniques, which make them possible only in a few highly specialized laboratories. The most widely used test for T cell reactivity is the lymphocyte proliferation (or transformation assay), in which the DNA synthesis of activated lymphocytes set in culture is evaluated aftar a period of at least 3 - 5 days, from the incorporation into DNA of a radiolabelled nucleotide, such as tritium-labelled thymidine. Such assays are very cumbersome, complicated and expensive. This explains why in many fields of potential application and interest (e.g. allergy, infectious diseases, transplantation) they have not become a routine feature. An additional disad¬ vantage of these tests is that in the lymphocyte prolif- eration assay, isolated and cultured lymphocytes must be set in culture and put in contact with non specific or specific stimulants soon after, i.e. within a few hours of having been collected from the organism, e.g. by draw¬ ing blood. Accordingly, blood taken from patients has to be processed near to the laboratory, which also limits the practical usefulness of the method.
An alternative methodology for assessing specific or non specific reactivity of T lymphocytes is the object of the present invention and has been made possible by the chemiluminescence multiwell analyzer described here. This methodology is based on the fact that very soon fol¬ lowing contact between activator (mitogen or antigen) and T lymphocytes, activation of these cells takes place and leads to the synthesis of various mediators, such as lym¬ phokines, which themselves are capable of activating other blood* cells present in the same blood or cell sus- pension. Upon activation, such cells, in particular monocytes and neutrophils, produce a potent and prolonged oxydative burst, which may be appropriately translated into chemiluminescent signals.
Although the early production of various lym- phokines by activated T cells has been known before, partly as result of our own investigations, (de Week A.L. et al Springer, Sem. Immunopathol. 7, 273-289, 1984), it was not to be foreseen that chemiluminescence-inducing lymphokines would be produced by activated T cells in free form and in sufficient amounts as to permit the ex¬ pression of chemiluminescent signals in whole blood. Quenching of chemiluminescence by various blood compo¬ nents (Fischer,H. et al.,1982, Chemiluminescence assays in the diagnosis of immune and hematological diseases. In: Luminescence Assays : Perspectives in Endocrinology and Clinical Chemistry, Eds. Serio,M. and M.Pazzagli, Raven Press, New York, p 229-241) on the one hand, and the variable reactivity state of potential target cells for lymphokines, on the other hand, would seem to pre- elude the development of reliable and reproducible chemi¬ luminescent tests of T cell activation.
As shown in the present invention, however, the discovery of adequate conditions for performance of the tests and in particular the use of the very sensitive multiwell chemiluminescence analyzer, have made possible new diagnostic tests of T cell reactivity, which have hitherto not been described. Such tests may be performed in several ways:
a) In whole blood. In this case, whole blood is slightly diluted in a suitable culture medium to which appropriate amounts of non specific stimulant (e.g. mito- gen such as anti-CD 3 antibody) or specific stimulant (e.g. antigen, allogeneic cells, tumor cells, etc.) have been added. Following a period of incubation in culture varying between 24 and 72 hours, preferably 48 hours, the blood cells are separated by gradient centrifugation and analyzed in the chemiluminescence multiwell analyzer.
Whole blood cells to which no stimulants have been added are used as control.
This technique has the great practical advantage of enabling for the first time such cellular tests to be initiated also by doctors in their own practice or by pe¬ ripheral hospitals, far away from the analyzing labora¬ tory. We have surprisingly found that T cell activation and production of the chemiluminescent signals by the target cells may also occur in whole blood, under appro- priate conditions, at room temperature. This makes transport of blood by ordinary means between the location where blood is drawn from the patient and a distant ana¬ lyzing laboratory possible, thereby opening entirely new practical possibilities to perform cellular immunological tests restricted hitherto to some sophisticated centers. The high sensitivity and possibility of analyzing simul¬ taneously cellular controls offered by the multiwell chemiluminescence analyzer have been essential for this new development.
b) In isolated mononuclear cell suspension. For large scale experiments and in order to analyze precisely the respective roles of the cell populations involved in the development of chemiluminescent signals by non spe¬ cific mitogens or specific antigens, it may be advanta- geous to use isolated cell fractions, such as mononuclear cells (Lymphocytes + monocytes), or neutrophils. This is usually achieved by Ficoll-Hypaque gradient centrifuga- tion. Following isolation, the cells are cultured to¬ gether with mitogen or antigen for 24-72 hours. To the experimental and to control cells, a chemiluminescent substrate system is added and chemiluminescent signals followed and integrated in the chemiluminescence multi¬ well analyzer described in this invention, for periods which may vary from 30 minutes to several hours.
c) In supematants from lymphocyte cultures.
Through experiments with isolated cell populations, it has been found out that the chemiluminescent signals ob¬ served in whole blood do not originate, at least not in significant manner, from the activated T lymphocytes themselves but from bystander cells in the reaction, such as monocytes or neutrophils, secondarily activated by lymphokines and possibly other mediators produced by the activated T lymphocytes.
Accordingly, another way to perform the chemiluminescent assays for T cell activation consists in first culturing isolated lymphocytes together with non specific mitogens or specific allergens, then harvesting culture supematants and setting up the supematants to¬ gether with a chemiluminescent substrate system and an appropriate target cell population generating chemilumi¬ nescent signals.
According to the clinical indications and pur¬ poses of the investigation, this mode of operation may have several advantages. First, it enables more precise analysis of the factors and mediators produced by acti¬ vated T cells. Second, it enables some standardization of the assay by better definition and reproducibility of the target cell population generating the chemilumines¬ cent signals. For example, this may be a neutrophil or monocyte pool, or a cell line cultured under standardized conditions. In this way, one of the variables in the as¬ say, namely an autologous target cell population, may be eliminated.
The following examples are used for illustrating the present invention without limiting the scope of pro¬ tection of the appended claims.
Example 1:
For realization of a chemiluminescent (CL) T cell activation test in whole blood, a set of sterile flasks with rubber seal are prepared, containing RPMI 1640 medium supplemented with 10% pooled human serum, or any other suitable culture medium, as controls. In experi¬ mental flasks, either non specific mitogens (e.g. 0.1 - 10 μg of monoclonal anti-CD 3 antibody) or specific anti- gens (e.g. 50 microliters of a 1 - 20 μg/ml Candida anti¬ gen solution) are added. To these ready to use culture flasks, which may be kept either at 4°C for up to one year or for longer times lyophilized, the experimenter adds 5 milliliters of freshly drawn venous blood under sterile conditions. After thorough agitation, the cul¬ ture flasks are kept (or transported to the laboratory) at ambient temperature (15 - 37°C) for periods of up to 24 hours. After 24 hours, the culture flasks are opened and culture continued for another 24-48 hours in a tissue culture incubator set up at 37°C in an atmosphere of 5% CO2• At the end of the culture period, the cultured blood is centrifuged over suitable gradient (e.g. Ficoll- Hypaque) and the white cell population resuspended in a suitable medium (e.g. TO 38/BSA) for carrying the CL as- say itself. For the CL assay, the cells, at a concentra¬ tion of 1 x 106 mononuclear cells/ml are distributed in white microstrip wells (e.g. DYNATECH) . 120 micromoles / liter lucigenin are added to each microwell and the re¬ sulting photon emission followed for 30 minutes up to several hours in the chemiluminescence multiwell analyzer described here.
The-results of such an experiment, including non stimulated control cells and experimental cells stimu- lated by various doses of mitogen and antigen are shown in Fig. 3. It must be emphasized that the general proce¬ dure outlined here can be slightly modified. For exam¬ ple, the original cells cultured in whole blood for up to 24 hours may be then immediately centrifuged upon arrival in the analyzing laboratory and then transferred to ordi¬ nary cell culture conditions, with or without supplemen¬ tation with mediators such as lymphokines (e.g. inter- leukin 2) known to potentiate T cell functions, and with or without the addition of standardized target cells, such as neutrophil or monocyte pools, or suitable chemi¬ luminescent signal producing cell lines.
Example 2.
Mononuclear cells are isolated from venous pe¬ ripheral blood by Ficoll-Hypaque gradient centrifugation and resuspended in RPMI 1640 culture medium supplemented with 10% human AB serum pool. Cultures are set in white microstrip wells (DYNATECH) at the concentration of 1 x 106 cells / 250 microliters. In experimental wells, Can¬ dida antigen prepared as indicated above is added at var- ious concentrations (1 - 20 μg/ml) or anti-CD3 antibody (0.1 - 10 μg/ml) as control of polyclonal activation. In order to demonstrate the antigen specificity of the CL T cell activation test, and in addition to the ubiquitous antigen Candida, two groups of individual cultures were also set up with rabies vaccine antigen (Lyssavec,
Berna) . One group of blood donors were non immune while the other had been vaccinated with the rabies vaccine.
Cultures were set in a tissue culture incubator at 37°C and 5% C02 for periods varying between 24 and 72 hours, following which 25 microliters of a 12 mmol/1 lu- cigenin solution are added to each well. Parallel cul¬ tures, for the purpose of comparison, were set up to as¬ sess lymphocyte proliferation by radiolabelled thymidine incorporation.
The resulting photon emission is followed in the chemiluminescence multiwell analyzer described here for periods of 30 minutes up to several hours and the total integrated simultaneously from all samples, as made uniquely possible by the instrument. An example of such an analysis is given in Figs. 4. and 5. It can be seen from these experiments that: a) a state of specific cel¬ lular immunity is detected more efficiently by our CL T cell activation technique than by the classical lympho- cyte proliferation test; b) the CL T cell activation technique allows immunized from non immunized individuals to a particular antigen to be distinguished.
Fig. 4A and 4B show Candida and Lyssavac chemiluminescence stimulation indices SImax, detected during the culture interval from 24 h to 72 h and Fig 4C and 4D show the Candida and Lyssavac induced 3H-thymidine incorporation stimulation indices SI after a culture in- tervall of 72h.
Fig 5 shows the correlation between Candida and Lyssavac induced chemiluminescence.
Example 3.
Mononuclear cells are isolated from venous peripheral blood and set in cultures with various stimu¬ lants as indicated above, for periods varying between 24 and 72 hours. At the end of the culture periods, the cells are centrifuged and discarded. The harvested su¬ pematants (100 microliters) are then given to target cells distributed in white microstrips at concentrations varying between 105 and 106 / ml. As target cells, a pool of monocytes or neutrophils isolated by Ficoll-Hy- paque gradient centrifugation, or a suitable cell line, such as HL-60 or U 937 may be used. After addition of 20 microliters to each culture of a 12 mmol/1 lucigenin so¬ lution, the emission of photons is followed and inte¬ grated at various times, as described above. The results of such an experiment are shown as examples in Fig. 6.

Claims

Claims
1. A process for measuring T lymphocyte re¬ sponses to different activators, characterized in that samples comprising T lymphocytes are brought into contact with an activator and a luminescent substrate, and any chemiluminescence which occurs is simultaneously regis¬ tered and evaluated in the samples (5) on a multiple sam¬ ple-carrier (3), where the samples (5) to be tested, the activators and the luminescent substrates required for the chemiluminescence reaction are placed at the foreseen locations (4), in that this carrier is light-proof sealed with a suitable cover plate (6) or cassette having sev¬ eral photoconductors (9) , wherein the connections of the photoconductors to the cover plate or the cassette corre- spond to the sample placed on the multiple-sample-carrier with regard to location and number, in that the photons emitted by the samples (5) are led by the photoconductors (9, 10) to a detector system (12) which converts the sig¬ nal of each sample individually into signals which can be detected by a computer, these converted signals being di¬ rected to a computer evaluation system (14) which evalu¬ ates the signals emitted by the samples (5) and detected by the detector unit (12) on the basis of their intensity and in relation to the emission time.
2. A process according to claim 1 wherein the ac¬ tivators are mitogens or antigens
3. A process according to claim 1 or 2 wherein the T lymphocytes are in whole blood.
4. A process according to claim 1 or 2 wherein the T lymphocytes are in a mononuclear cell suspension.
5. A Process according to claim 1 or 2 wherein the T lymphocytes are in a supernatant from a lymphocyte culture
*
6. A device for carrying out the process accord- ing to one of the claims 1 to 5, consisting of a receiv¬ ing unit (1) for chemiluminescence emissions, a detector unit (12) and a data evaluation unit (14) , characterized in that the receiving unit (1) for the chemiluminescence emissions comprises a cover (6) for covering multiple- sample-carriers (3) or a cassette for holding multiple- sample-carriers wherein a multiplicity of photoconductors (9) proceed from this receiver, and lead to the detector unit (12), wherein the number of photoconductors (9) cor¬ responds to the foreseen number of samples (5) to be mea- sured and the inlets (7) of the photoconductors (9) are disposed as near as possible to the foreseen position of the corresponding luminescing sample (5), wherein the de¬ tector unit (12) is capable of intensifying the signals for each photoconductor (9) individually and of convert- ing them into electrical signals which can be processed by a computer system and the detector unit (12) is directly connected to the evaluation computer (14).
7. A device according to claim 6 characterized in that the detector unit (12) contains a position resolu- tion photomultiplier or a position resolution microchan- nel plate.
8. A device according to claim 6 or 7, character¬ ized in that it is equipped for simultaneous measurement of 50 to 200 samples (5).
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