DE2923735A1 - METHOD OF MEASURING ANTIGEN RECEPTORS - Google Patents

Description

ALEXANDER R. HERZFELD ₆ Frankfurt amgo

LAWYER 5 ZEPPELINALLEE 71 AT THE LANDGERICHT FRANKFURT AM MAIN * -? TELEPHONE 0611/779125 ^) Q O Q * 7 ^ R

Applicant: Corning Glass Works
Corning, NT, USA

Methods of measuring antigen receptors

The invention relates to a method for measuring antigen receptors on cell membranes.

A crucial process "in an immune reaction is the Antigen binding to antigen-specific, cellular receptors of the lymphocytes. Specificity is a key feature the immune response. The reaction to the action of an antigen "consists of a series of cell reactions. As a result of the diversity of the observed antigen-cell interactions a generally binding discussion is cumbersome and difficult. The following therefore uses the example of the interactions between antigen and lymphocytes derived from the thymus (T lymphocytes or T cells). However, the findings and teachings according to the invention have general validity for antigen-cell interactions.

909850/0916

£ 923735

The immune response is regulated by T cells. Through interaction Genetically competent T cells reactive with the antigen are activated by T cells and antigen. These activated T cells then go through a predetermined sequence of events and cause the immune response against the antigen.

For reasons that are not yet fully understood, the antigen causes an increase in the number of T cells reactive with the antigen. the Quantitative measurability of the antigen receptors on the T cells thus opens up the possibility of diseases before onset more obvious or clinical symptoms, and also monitor the progress of treatment. This can be of particular importance in chemotherapy.

The measurement methods customary up to now are time-consuming and cumbersome. They can be divided into three groups:

1.) Visual evidence

The antigen receptors are determined visually after the cells have been treated with a receptor-specific antigen. The antigens can be radioactively labeled and analyzed using an autoradio * graphic. Another possibility is to create antigen derivatives in the form of solid-phase, complex-forming carriers, such as rosettes on complexes of cell-antigen-red blood cells, or cell-antigen beads, fibers or cells. In both cases the

909850/0916

Identification and enumeration of positive cells a microscopic Investigation. Please refer to the following literature:

L.J. Kennedy et al., J. Immunol., 114, 1670 (1975); G.J. Hammerling and H.O. McDevitt, J. Immunol., 112, 1926 (1974); M. Jondal, Scand. J. Immunol., Supplement 5, 69 (1976); J.B. Natvig and S.S. Froland, Scand. J. Immunol., Supplement 5, 83 (1976); U.O. Butishauser et al., Proc. Nat. Acad. May be. (USA), 69, 1596 (1972); and H. Wekerle et al., Proc. Nat. Acad. May be. (USA), 69 1620 (1972).

2.)! Fluorescence methods

Two detection methods are known for this. One method is using antigens in the form of enzymes the homologous receptor activity is determined by first converting the enzyme with the cells, and the Enzyme-cell complex is incubated with a substrate made fluorescent. Hydrolysis of the substrate gives a soluble, fluorescent molecule that can be determined by microscopic examination. These are obvious only suitable enzymes acting as antigens, see S. Swain, J. Immunol., 119, 1427 (1977).

The second method requires the one that is very difficult to quantify microscopic measurement of the uptake of the fluorescent antigen on the cell surface.

909850/0916

-X-

3.) Determination by equilibrium binding

This third method relies on the binding of a radioactive ligand to cells under equilibrium conditions. • The radioligand-cell complex is separated by centrifugation, washed, resuspended to equal volume and removed sections are counted, see M.J. Benfari et al., J. Immunol., 119, 1427 (1977).

What all these known methods have in common is the difficulty in precisely quantifying the antigen receptors on the cell membranes to eat. The equilibrium binding process has so far given the best results, but it is very time-consuming, conditional considerable amount of work, burdened with sources of error, and requires numerous, time-consuming centrifugation steps.

The invention has a comparatively quick and accurate process for measuring antigen receptors on cell membranes.

The object is achieved by the method of the invention in that the cells are mixed with an aqueous, isotonic solution of an immunoreactive, water-soluble, labeled antigen having a purity of at least 95 % and an immunoreactivity of at least 40 % , the mixture is mixed at a temperature of 4 - 40 ⁰ G is incubated for a period of time sufficient for equilibrium binding, the cells are collected and the amounts of the labeled antigen bound to the cells are measured.

909850/0918

-f '292373S

Further favorable configurations emerge from the description and the demands.

In the explanatory drawings shows:

FIG. 1 shows the cell binding counts of a radioactive as a diagram labeled antigen model, (GAT *) depending on the incubation period;

FIG. 2 shows the percentage GAT * cell binding inhibition as a diagram per / ug GAT (unlabelled or original antigen model), protein or lactic acid dehydrogenase addition;

FIG. 3 shows, as a graph, the percentage of cell-bound cells displaced GAT * pro / Ug GAT or protein addition.

In the following description, "antigen" and "ligand" used synonymously.

As a first step of the method according to the invention, cells are mixed with an aqueous, isotonic solution of an immunoreactive, water-soluble, labeled antigen which is at least 36% pure and at least 40 % immunoreactive. The labeling can be done in different ways. The antigens can be radioactive with a specific activity of at least 50 / uCi // ug, preferably 100-400, more favorable 200-400 / uCi / / Ug, or fluorescent with at least one fluorescent molecule per antigen molecule, or enzymatically with at least one enzyme molecule per 100 antigen molecules, or in

909850/0916

-V-

otherwise marked, provided that the specific Activity of the antigen identified elsewhere is high enough, i.e. roughly equivalent to the values given.

The cells of animal origin must be immune to the immune system, the type of which is mostly irrelevant as far as they are accessible to antigens are. An aqueous suspension of the cells is preferred. A single-cell suspension is very cheap. Very cheap are lymphocytes, especially T lymphocytes or T cells.

Each of the labeled antigens must be substantially equivalent to the original or unlabelled antigen; this means that under unsaturated conditions at least 40 % of the added marker can be bound to an immobilized antibody preparation. This gives a regulated, reproducible model for determining the immunoreactivity. In the case of radioactively labeled antigens, their immunoreactivity is preferably over 70 or even 80 %.

Based on the consideration that there is competition for the antibody cells available for binding between the original, unlabelled antigen and the labeled antigen , the entire bound, labeled antigen can be displaced approximately by an excess of 5 mol. Of the original antigen.

- 7 909880/0916

ORIGINAL INSPECTED

The labeled antigen must be at least 95 % pure. This can be demonstrated by known methods which depend in detail on the particular antigen. In the example of proteinaceous antigens, the required purity is demonstrated, for example, by

1. Gel filtration, with the identified antigen as homogeneous, molecular unit (provided that in the case of radioactive labeling there is no radiolytic Experiences damage).

2. Precipitation with trichloroacetic acid, with precipitation of at least 95 % of the label.

3. Electrophoresis, the marked antigen running as a single band on polyacrylamide gel.

In addition, the invention is not restricted to proteinaceous antigens, even if most of the antigens are proteins, glycoproteins or are polysaccharides.

The labeling of the antigen can be carried out in a known manner. The type of radioactive labeling depends on the antigen, the radioactive atom or isotope, the equipment available, etc. Examples of suitable radioactive isotopes include H ³ , G ¹⁴ , Na ²⁴ , P ⁵² , S ³⁵ , K ⁴² , Ca ⁴⁵ , Fe ⁵⁹ , Co ⁶⁰ ,

125 131
I, I,. Mostly the non-metals are preferred. Particularly

are favorable for the proteinaceous antigens i ¹² -> una

909850/0916

e ■: '

-r-

Usually the radioactive isotope is introduced into the antigen by chemical conversion. However, some antigens can also be synthesized directly from radioactively labeled reaction participants or components. A third possibility consists in the biochemical synthesis of an antigen in the presence of one or more nutrients, the radioactive being Isotope part of the antigen. The former way is likely to be the most practical.

For fluorescent labeling, all known labels are suitable that have at least one fluorescent label for each antigen Supply molecule, e.g. ituorescine isothiocyanate, tetraethylrhodamine, Fluorescamin et al. Production takes place in a known manner, depending on the antigen and the structure of the label. Mostly will the fluorescent molecule is covalently bound to the antigen by standard methods, for example in the case of a proteinaceous amine via amidated ω-carboxy groups of the protein, which are usually supplied by aspartic or glutamic acid, or via the release of free amino groups by lysine. Another possibility is to use lysine or amidated ω-carboxy groups to be incorporated into a synthetic polypeptide, and this via free ω-oxy groups of the protein and free amino groups (e.g. from lysine) of the polypeptide itself to couple to the antigen. Optionally, the coupling of the fluorescent Labeled with the antigen or the polypeptides, or a fluorescently labeled polypeptide with the antigen Carbodiimide activation of the ω-carboxy groups are supported.

909850/0916

A suitable activator is also. Woodward's reagent K, N-ethyl-5-phenylisoxazolium-3 -sulfonate ^t. (See HG Khorana, Ghem. Ind. (London), 1955, 1087J IT, Weliky et al., Immunochemistry, 1, 219 (1964); WF Line et al., Biochim.

Biophys. Acta, 242, 194 (1971) J R.P. Patel et al., Biopolymers, 5, 577 (1967) and J.L. Riggs et al., Am. J. Path., 34, 1081 (1958).

In preparation of the enzymatically labeled substrate, essentially any one is free of covalent attachment to an antigen enzyme capable of greater loss of enzymatic activity. The enzyme molecule: antigen molecule ratio can as a rule be more or less 1: 1-1: 100, depending on the enzyme activity after coupling to the antigen, and the measuring method for determining the labeled bound or attached to the cells Amount of antigen. Examples of suitable enzymes include peroxidase, / 3-galactosidase, alkaline phosphatase and like that.

The enzymatic antigen labeling is carried out in a known manner , depending on the particular enzyme and antigen. In the case of proteinaceous antigens , an enzyme can be coupled, for example, via _ω- oxy groups or amino end groups by way of the oxodiindactivation, or the activation and coupling takes place with the aid of gluteraldehyde and bis-diazobenzidine.

40 ⁰ C for sufficient for equilibrium binding - as a second step of erfindungsgenäöen process, the mixture produced at a temperature in the temperature range 4

809880/0916 - ⁱ⁰ ~

ORIGINAL INSPECTED

Incubated for a period of time. Preferably, the incubation temperature is in the range 4 - 37 ⁰ C, and is particularly favorable an incubation temperature of 4 ° or 37 ⁰ C.

The incubation period is not critical as long as the equilibrium binding occurs. Usually 2 hours are enough, sometimes also more or less.

The third step is to collect the cells from the incubated mixture. All known methods are fundamental suitable, only in the case of radioactive labeling is a filtration, if possible with a fiber optic carrier, indicated. In detail the method used is not critical, only an unspecific collection or leaving behind of the identified antigen is necessary and cell loss can be largely ruled out. The use of an automated, multiple collecting device is particularly favorable (MASH, Microbiological Associates, Bethesda, Maryland, USA), which was also used in the examples below.

Finally, the amount of labeled antigen bound to the collected cells is measured. There are known methods for this and devices suitable.

Since, in the case of radioactively labeled antigens, unspecific uptake of the radioantigen cannot be completely avoided, parallel runs are carried out for control purposes, leaving out the cells. The number of only cell-bound antigens is obtained from the difference between the counts with and without cells. in the

- 11 -

909850/0916

ORIGINAL INSPECTED

-γ.

As a rule, the radioantigen binding by cells exceeds the unspecific uptake around a statistically significant limit value (P S 0.05). When using the MASH device mentioned, The main source of the non-specific uptake is the glass fiber strips necessary for the restraint and isolation of the cells, which intercept the sadioantigen.

Naturally, a bond or restraint must be radioactively labeled Antigen through components other than cells, as well as displacement of cell-bound counts through non-cross-linking antigens or ligands are excluded. Typically it is that under competitive equilibrium conditions amount of radioactive not displaced by a large molar excess of natural (unlabelled) antigen Antigen binding, which is the unspecific cellular binding component represents.

When using a fluorescent or enzymatic marked Antigen, the collected cells are mostly resuspended, or the marked antigen can be replaced by acidic or basic dissociation methods are released. Enzymatic release is also possible, if not disruptive enzymes such as trypsin or papain can be used. As a result of the very high specific activity of some enzymes can also be collected, tightly packed cells in the case of enzymatically labeled antigens can be used immediately.

909850/0916

In the case of fluorescently labeled antigens, the fluorescent label is measured at a certain wavelength (e.g. UV light) and the amount of radiation emitted (mostly in the visible light range) is measured in a known manner.

I ^{1 For} the measurement of enzymatically labeled antigens, the collected cells must be added to a substrate specific for the enzyme label. The substrate is changed by the enzyme, and the decrease in substrate or the increase in the reaction product is usually quantified colorimetrically or fluorometrically. A substrate specific for peroxidase is, for example, O-dianisidine. For the sensitive colorimetric determination, the enzyme: antigen molecular ratio must usually be at least 1: 1 or greater. For the fluorescent measurement, this ratio is regularly at least 1: 100.

An example is described for further explanation without limitation. An arbitrarily immunogenic terpolymer, Poly GIu AIa Tyr, was used as the antigen model, which is described below for the sake of simplification referred to as GAT (molecular weight 45,000).

example Substances and processes

Experimental animals: inbred mice, breeding series (C57BL / 10J) (H-2 ^), hereinafter referred to as ^M B10 ^H , from Jackson Laboratories, Bar Harbor, Maine, USA, all largely of the same age.

909850/0916

Antigens: Crystalline protein albumin (MW 43,000) and milk dehydrogenase, LDH (E.C. Wr. 1.1.1.27, MGew. 135,000, obtained from rabbit muscles, "both" obtained from Sigma Chemical Company, St. Louis, Missouri, USA.

Poly Glu ^6O Ala ⁵ ° Tyr ¹⁰ , hereinafter referred to as “GAT” (MGew. 45,000) was prepared according to the Katchalski-SeIa, Advanc. Protein Chem. 13, 244 (1958) obtained from the aN-carboxyanhydrides of the L-amino acids. An aqueous solution (pH 7.5) of the polymer was dialyzed with several changes of distilled water for 6 days at 4 ⁰ C for the removal of HBr residues and then stored, the polymer solution and the protein at -20 ⁰ C.

Preparation of radioactively labeled GAT

According to the Hunter and Greenwood chloramine-T method, in Nature, 194, 495 (1962) and Greenwood, Biochem. J., 89, 114 ^ _ (1963) GAT was ^{radioactively labeled with I 12} ^ with high specific activity. This radioactively labeled GAT, hereinafter referred to as GAT *, was purified by gel permeation chromatography with a 61 × 1 cm Sephadex G-50 column. 1 ml sections were removed. The process consisted of 0.05M barbital buffer, pH 8.6, containing 0.1% by weight per volume of bovine serum protein, hereinafter referred to as BSA. All protein-bound radioactive materials were collected in one or two sections and stored at -20 ° C. The various GAT * preparations had specific activities of 220 260 / uCi // Ug and could be filled to at least 96% with trichloroacetic acid.

909850/0916

-γ-

Solid phase GAT analysis

Provision of immobilized antibodies

1 g dry weight of the arylamine derivative of glass-regulated pore size, an average particle size of 1 μm in diameter and an average pore size of 550 2 in diameter were diazotized in 10 ml of 2N HCl with an addition of 0.25 g of NaNOp at ⁰ ° C .; for the method used, see WB Jakoby and M. Wilchek, Affinity Techniques, Enzyme Purification Part B, Vol, XXXIV, Methods of Enzymology, pp. 64-6 and 71-2, (1974). After 20 minutes, the diazotized arylamine glass was ^{washed thoroughly with distilled water at 0} ° C. and, after the last wash, suspended in 5 ml of 0.1M phosphate buffer (pH 8.4). Overnight, two sections of the slurry were reacted at 40 °, each with whole sheep anti-GAT and normal sheep serum, respectively. The slurries were then washed thoroughly with the phosphate buffer and the remaining diazonium ^{groups were quenched overnight at 4 °} C. with 1 % BSA (bovine serum protein) in phosphate buffer.

GAT "* analysis

All solid-phase GAT analyzes were carried out in 1M phosphate buffer pH7, containing 1 % BSA and 0.2% polyoxyethylene sorbitan monooleate.

Sections of 20 µl GAT (50 gp) were diluted with buffer increasing amounts of the immobilized antibody and the

- 15 909850/0916

immobilized normal serum was added and the final volume was equalized with buffer. The specificity was proven by the competitive displacement of GAT * ^{1 by GAT.} In each of them, the reaction participants were incubated at 37 ° C. for 1 hour with vortex action at 15 minute intervals. Individual sections were then centrifuged for 15 min. At 4,000 × g, the glass was washed with buffer and centrifuged again. The glass particles were counted with a scintillation spectrometer. The antigen binding was counted after correction for unspecific binding (binding of immobilized normal serum) as a percentage of the 50 pg GAT bound by the immobilized antibody.

The immunoreactivity of GAT was checked immediately after purification and periodically thereafter to ensure that the radioactively labeled antigen retained its original antigenic properties. In a typical experiment, 97 % of the counts bound to 400 / ug of immobilized antibody were displaced by 5 / ug of GAT under competitive binding conditions.

Preparation of single-cell suspensions

The thymus glands of adult mice of the same age and sex (Variety B10) were surgically removed under aseptic conditions and freed from the parathymic lymph nodes. Immediately after removal, these were placed in Hank's balanced saline solution (HBSS), and then one with the pestle sterile disposable syringe (iccm) pressed through a 60 mesh sieve and

- 16 909850/0916

the single cells (thymocytes) are collected in HBSS. This single-cell suspension was pipetted into a sterile 10 ml centrifuge tube and the cell debris was allowed to settle for 2 minutes. The supernatant suspension was poured into a new centrifuge tube (10 ml) and the cells were sedimented at 500 × g. The erythrocytes were lysed by hypotonic NH.Cl treatment. The cells were counted twice with a hemocytometer. The viability of the cell preparations was 0.04% Trypanblaufärbstoffausschluß with 95 - 99% determined.

Cytotoxicity testing

Antisera: The estimate of the T cell content of the unicellular Suspension was made with AKR anti-C5H (anti-thy 1,1) sera and rabbit anti-mouse thymocytt.-nsereri (RAi-II sera) dv '-' unguided. To the BAMT sera preparation was veined and intravenous to adult New Zealand rabbits weighing 3-4 kg injected with 1 χ 10 'viable DBA / 2J thymocytes. The first blood sample was taken 3 weeks after the injection.

• j

Several intravenous re-injections with 1 χ 10 thymocytes were made then carried out, each after 7 days, followed by a blood sample. Heat activated sections of the RAMT sera were adsorbed and generated kidney, liver and bone marrow cells by incubation with DBA / 2J for 30 min at 37 ° C and thereafter Sera clarification by centrifugation (100Oxg) T-cell specificity. The serum to packed cell ratio was 10: 1.

- 17 -

909850/0916

EPMI-1640 (G-rand Island Biological Co., G-rand Island, NY, USA) and heat-inactivated calf embryo serum (referred to here as PCS) were used for dilution. The reactions were carried out in microtiter plates with a U-shaped bottom. Total 1 10 ^ χ viable lymphocytes in 50 / al diluent with 50 / ul diluted heat-inactivated antisera set air, 30 min. At 37 ⁰ C in 5% on carbon dioxide content incubated. In each experiment, 50 μl of guinea pig complement, diluted 1:10, was added and the mixture was additionally ^{incubated at 37 °} C. for 1 hour. The cells were then equilibrated at 4 ° C and the percentage of cytotoxicity determined by 0.04% trypan blue dye deflection. The comparative experiments consisted of (a) cells incubated with normal rabbit serum plus complement or normal serum plus complement (b) cells plus complement (c) cells plus antibody (d) cells plus diluent. The cytotoxicity of the comparative experiments was 2-11%.

Immunofluorescence

The direct immunofluorescence of mouse ethymocytes with fluorescein-labeled rabbit anti-mouse globulin was carried out according to MG-. Mage et al., J. Immunol. Methods, 15, 47 (1977). All conversions were carried out in RPMI-I64O plus 10% PCS. The cells were examined for immunofluorescence with a hemocytometer. For indirect immunofluorescence, thymocytes were reacted with the corresponding antithymocyte serum at 37 ^° C. for 30 minutes. The sensitized cells were washed once and titrated with fluorescent polyvalent antiglobulin of the appropriate specificity according to Mage et al., Op. Cit., And reacted.

- 18 909850/0916

Competitive GAT * analysis

The tests for competitive antigen binding were carried out in microtitration plates with a U-shaped bottom. RPMI-164O (pH 7.4) with 10 % FCS served as the diluent. The FCS sections were individually examined for anti-GAT activity by solid phase antigen binding. 10 µl of GAT ⁺ (about 0.5 ng, about 130,000 CPM) was added to each microtiter plate, then 50 µl of GAT or diluent was added, and the mixture was ^{equilibrated with GAT at 37 °} C. in air containing 5% CO _2. To determine the specificity of the GAT * binding reactions, GAT was replaced by protein albumin or LDH in comparative experiments. 1 × 10 ^ viable T cells, in some cases B10 erythrocytes in 100 μl suspension, were rapidly added to each plate and incubated for a further 2 hours under the same conditions (plateau binding ratios). The contents were individually collected with glass fiber filter paper and 0.15M NaCl washing solution, and each was thoroughly washed 25 times the amount of the washing solution. Glass fiber disks representative of the contents of the titration plates were punched out of the filter paper. Their remaining radioactivity was measured with a scintillation spectrometer. The comparative tests with GAT ⁺ or GAT plus unlabelled inhibitors (brought to the appropriate volume with diluent) were also tested in order to determine the macromolecular binding (GAT * binding) to the glass microfibers of the filter paper. From these

5 #

Data, the GAT bound specifically to 1 × 10 T cells (equal to the cell-bound GAT * less GAT * captured unspecifically by glass fiber strips) was calculated. This Hintergrundzählwerte l _a gen about 8-10 times under the uninhibited (ie

909850/0 916 -19-

GAT-free) binding values. To determine the optimal bonding conditions the reaction of GAT 'with thymocytes was observed for various periods of time at 4 ° and 37 ° C.

Modified displacement attempts were made to determine whether a cell surface receptor was responsible for the observed binding. 100 / ul containing 1 χ 10 ^

/ ul contains n with 50 / different amount or protein pulsed and thoroughly tritu-

B10 thymocytes were treated with 10 / ul containing 0.5 ng GAT * for 2 hrs. At 37 ° C, then with 50 / ul with and without verriert GAT, incubated another 2 hr. At 37 ⁰ C and collected.

Results

In the oytotoxicity tests, the thymocyte suspensions were consistently 95 % thy 1.1 positive with anti-thy 1.1 plus complement and 95 % positive with RAMT complement. Cytotoxicity to comparable bone marrow ropes (Τ cells negative) ranged from 5 to 7 % in both antisera. Indirect immunofluorescence tests with RAMT plus J ¹ ITC conjugated goat anti-rabbit globulin showed 98-100 % fluorescence-positive thymoeytes. Direct immunofluorescence tests with FITG-conjugated polyvalent rabbit anti-mouse globulin showed no direct immunofluorescence, ie no immunoglobulin-positive cells were detectable. The combined data on cytotoxicity and immunofluorescence demonstrate that the thymic derived cell population was free from B-cell contamination and predominantly off

909850/0 916

- 20 -

thymic T cells.

Binding kinetics of GAT * with thymocytes

Preliminary studies have shown that when 1 χ 1Ck viable B10 normal thymocytes are incubated with approximately 0.5 ng GAT * for 1 hour at 37 C, the radioantigen is bound to the cell. However, the optimal bond tests show the influence of time and temperature on the bond. Figure 1 illustrates the GAT cell binding values with 95% confidence for incubation times of 0.5, 1, 2, 3 and 4 hrs. At 4 ° C (4-4) and 37 ⁰ C (oo), for GAT * and 1 χ 10 ⁵ thymocytes. At 37 ° C the binding reaction proceeded rapidly and reached its maximum after 2 hours. At 4 C the binding was slower and reached a lower maximum height. The bond size at 37 ° C is approximately four times the bond at 4 ° C. Therefore all binding tests were carried out at 37 ° C for 2 hours.

Repeatability and Immunological Specificity

The results of the various bonding attempts under equilibrium sb edinguj
summarized.

Weight conditions at 37 ° C for 2 hours are given in Table I.

909850/0916

acpm: C! .E. ^a » T A BELLE I Replica ^d 3833 + 272 χ 10 ' 'Thymocytes 12th 3411 + 224 b % bound ⁰ 9 3901 + 205 2.46 12th GAT * binding by 1 3343 ± 115 2.50 12th attempt 2.80 A. 2.79 B. C. D.

Footnotes: ^a ACEM - cell-bound GAT * count values - GAT * unspecific through fiberglass strips

S.E. = Standard - withheld deviation

^c % bound = (4CPM / total GAT share * counts per implementation) χ 100.

Number of replica samples per attempt

From this table it can be seen that 1 × 10 ^ B10 thymocytes bind repeatably 2.46-2.8%, on average 2.64 % of the total GAT * additive. The standard deviation in these four experiments ranged from 7.1-3.4% ΔΰΉΊ and had the acceptable average of 5.6 %.

If the observed reaction of GAT * and thymocytes is immunochemically specific, then the excess GAT (or other cross-binding antigen) inhibit or block the uptake of GAT *,

- 22 -

909850/0916

if both GAT and G-AT are present in the incubation ^{mixture, i.e. competitive inhibition takes place.} Table II summarizes the data observed in the 4 experiments.

TABLE II

GAT * binding by 1 χ 314 - 3 E. N / D ^a 21st 10 ^ thymocytes bound in the presence of 25 / Ug GAT attempt 44 414 ί 46 % , 21 Replicas A. 359 i ■ not 0, 11 B. a / D = , 30 - C. , 30 12th D. certainly. 6th Footnotes:

The table repeatedly shows competitive inhibition of GAT *,

if a mix of GAT

and GAT was reacted with the thymocytes in molar excess. This inhibition was essentially complete, a decrease from an average of 2.64 % to 0.27 % of the total GAT * added.

To further prove that the radioantigen binding observed represents a specific antigen-receptor interaction, B10 erythrocytes (cells without immune function and a priori receptor-negative) were converted with GAT *. Table III summarizes the results and shows, in contrast to 1 × 10 ^ thymocytes, the inability of 1 × 10 ⁵ B10 erythrocytes to bind GAT *.

- 23 -

909850/0916

TABLE III

Binding by 1 χ 10 ^ cells Cell type ^ CPM-S. E. Replicas

Thymocytes 3593-253 9
Erythrocytes 0 7

Cells without a suitable receptor “do not bind GAT, provided that the "observed GAT * binding response is a true antigen-receptor response and not the result of an original interaction between GAT * and structural membrane components is. Because the B10 erythrocytes show no GAT ^ binding occurs the binding of GAT * to the erythrocytes via a molecular unit, i.e. a receptor that the erythrocytes lack.

The attempts at competitive inhibition by replacing GAT with protein or LDH are summarized in FIG shown. The diagram shows the importance of increasing amounts of GAT (X-X), protein (A-4) and LDH (0-0) for the inhibition of GAT * ' Binding with B10 thymocytes. Neither LDH nor protein, both of which were immunologically non-reactive antigens, could GAT * from the thymocytes displace, while the GAT * displacement by GAT in the investigated Range was concentration dependent. The observed reactive binding of GAT * and the thymocytes is thus for GAT (and GAT *) immunologically specific.

909850/0916

-r-

21 ? 323735

G-AT receptor sites

The interaction of GAT * with its cellular receptor causes at least partial cut-off (see, for example, EK Dunham et al., J. Exp. Med., 136, 403 (1972, as well as Kennedy et al., Op. Cit.), But leaves part of the GAT receptor complex on the cell surface GAT *, which is externally complexed with its plasma membrane receptor, is thus at least partially displaced (exchanged) by GAT. The cell values relating to the cells should, however, be internalized in the macrophages and thus cannot be displaced if the cellular binding observed is based on active phagocytosis by thymic macrophages. The results of the displacement experiments are summarized in Figure 3. From this it can be seen that after treatment with the medium only, more than 70 % of the total cell-bound counts were displaced with 200 / ug GAT (0-0) the GAT concentration in the conversion mixture (the linear semi-logorithmic regression correlation coefficient efficient is 0.98). The diagram also shows that unlabelled, immunochemically non-reactive protein (4-4) does not displace cell-bound GAT *. Examination of the contents of the titration plate with the phase contrast microscope immediately before removal shows a cell viability of 98-100%

U-shaped uniform cell distribution on the / plate bottom without spreading. As a result, the majority of those found in the radioantigen binding experiment
bond to consider.

Binding attempt determined GAT * count values as a surface

- 25 -

909850/0916

Various modifications are possible. So can two or

125 several characteristics of different energy spectra, e.g. I,

131
I for the simultaneous detection of different receptors

can be used on the same or different cells.

909850/0916

'it'

Blank page

Claims (10)

- / ■ 292373 Claims ~ \) Method for measuring antigen receptors on cell membranes, characterized in that the cells are mixed with an aqueous, isotonic solution of an immunoreactive, water-soluble, labeled antigen of a purity of at least 95 % and an immunoreactivity of at least 40 % , the mixture at a temperature incubated 40 ⁰ C for a sufficient period of time to Grleichgewichtsbindung, the cells were collected, and the amounts of the antigen bound to the labeled cells is measured - by the fourth 2. The method according to claim 1, characterized in that the antigens are fluorescent with at least one fluorescent molecule per antigen molecule, or enzymatically with at least one enzyme molecule for 100 antigen molecules, or radioactively with a specific activity of at least 50 / uOi / / ug are. 3. The method according to claim 2, characterized in that the antigens are radioactively characterized, the cells after incubation collected by a filtration method, and the radioactivity of the cells thus collected is counted. 4. The method according to claim 3, characterized in that the specific antigen activity is 100-400 / uCi /, _U g, preferably 200-400 / uCi // ug. 909850/0916 ORIGINAL INSPECTED 5. The method according to claims 3 or 4, characterized in that that a fiberglass support is used for the filtration. 6. The method according to claims 1 or 2, characterized in that the cells are in aqueous suspension. 7. The method according to claim 6, characterized in that a unicellular cell suspension is used. 8. The method according to any one of claims 1-7, characterized in that that the cells are lymphocytes, T lymphocytes and the like are. 9. The method according to any one of claims 1-8, characterized in that the immune reactivity of the antigens is more than 70 % and preferably more than 80 % . 10. The method according to any one of claims 1-9, characterized in that that the antigen is a protein, a glycoprotein, or a polysaccharide. 9098S0 / 0918