CH631550A5 - Process for the determination of cell types or of cell compatability on a solid support material - Google Patents

Description

The aim of the present invention was to develop an easy-to-carry out method for determining cell types or cell compatibility on a solid support material, which method should be able to be carried out both as an agglutination test and as a cell lysis test. This test should also be able to be carried out with human blood cells and should not have the previously described disadvantages of previously known methods for determining the compatibility of blood cells.

Surprisingly, it has been shown that the desired goals can be achieved with a novel method for determining cell types or cell compatibility on a solid support material, by irreversibly binding a first monolayer to cells on the support material, these cells containing antigens or with antigens were provided, and this first layer is brought into contact with a solution which contains antibodies and then a suspension of second cells which carry antigens is applied, and the extent of the immunoadhesion of the second cells caused by antibodies to the first cell layer is determined.

The present invention therefore relates to a method for determining cell types or cell compatibility on a solid support material, which is characterized in that

(a) irreversibly binds a first monolayer of cells to the carrier material, the cells containing or having been provided with antigens,

(b) bringing this first layer (a) into contact with a solution containing antibodies, antibodies being bound by immunoabsorption to such an extent that the antibodies of this solution are reactive with the antigens of the cells of the first layer.

(c) subsequently applying a suspension of second cells, these second cells carrying antigens, and

(d) the extent of immunoadhesion of the second cells

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determined by antibodies bound to the first cell layer, a fibrinogen solution and a subsequent treatment. a polylysine solution under such conditions

In this context, the term "will be mistaken for the fact that the chemically reactive groups form on the surface-bondable binding of a first monolayer to cells to the surface of the carrier material.

solid carrier material »the binding of reactive cells to it s As already mentioned, preferred cell types are understood to mean this carrier material by molecular forces. This should be tested according to the method according to the invention — this binding can take place through the formation of covalent cells, or whose cell compatibility is to be determined, blood mixing bonds between the cell surface and reactive cells, in particular red blood cells, that is, Ery groups of substrates , or the binding can be caused by throcytes. In this preferred embodiment of the invention, weaker molecular forces are achieved, for example in accordance with the method according to the invention, those in step (a) are irreversible by van der Waals forces, electrode forces or by bound cells erythrocytes which contain antigens or hydrogen bonds. The irreversibly to the solid support on which antigens are bound, and the cells bound in the process by subsequent work steps (c) are likewise erythrocytes not detached from the support. which carry antigens, and in step (d)

With the aid of the method according to the invention, not only is the degree of immunoadhesion of the second erythrocytes determined, the sensitivity of the test method is increased, but it is also possible due to the antibody of the erythrocytes of the first layer, and the result of the immune reactions is also easily caused.

Measure using simple devices. With this method, the first layer of ery-

When carrying out the method according to the invention, subject throcytes to lysis before the second erythrocyte can be applied to the first cell layer and the second cell layer from cells 2 <> cytos.

of a first test person, and who in step (b)

used solution be a serum, which is from a second test according to a further embodiment of the invention

person was removed. According to the method, agglutins can be applied in a serum

When carrying out the method according to the invention, by making a monolayer of such in step (a), the second cell layer can be formed in method step (c), producing 25 cells, in which or on which antigens are cells by testing one of them Cell population located in a cell with respect to the cells to be tested; in step (b), the essentially just sufficient number is applied so that a solution is formed which forms a monolayer which is too small for the presence of the agglutins, the extent of the immunoadhesions being; in step (c) a second suspension of such cell of this monolayer uses a function of the percentage of oils known to be in or on tested cells that carry specific antigens. 30 they have antigens for the agglutins on their

According to a further preferred embodiment of the test is now; and by in step (d) the extent of the method according to the invention, the solution which contains the anti-immunological adhesion of these second cells to the first cell body is a mixture which layer determines.

(1) a solution of an antibody which is immunoadactivated. Another preferred embodiment of the present sorption with an antigen carried out by the cells of layer (a) 35 is carried out in step (a) , and binds beautiful erythrocytes to the solid support material, in which

(2) contains a second solution or suspension that is on antiches or on which antigens are located; in step (b), by inhibiting the antibodies in the solution (1) with the antibody-containing solution, this is checked by immuno-testing. absorption binds to the first monolayer of the erythrocytes;

The method according to the invention can be carried out according to a further preferred embodiment in step (c) and a suspension of second erythrocytes in such a way that uses, in or on which there are antigens, the antigens of the first cell layer (a) are antigens, that of and that these second erythrocytes are supported on the solid immunoglobulins or complementary components as a second monolayer above the first layer, which is bound to the surface of the cell population and allows the erythrocytes to settle and after formation, and that the antibodies are in the Solution which is used in the process 45 second monolayer of protamine sulfate to the suspension in a step (b), anti-immunoglobulin or an anti-sufficient amount, so that an immunoadsorption complement to the antigenic determinants by the immune of the second erythrocytes the antibodies carried on the noglobulins, or complementary components - first monolayer are bound, and that it is washed to remove second erythrocytes that are not bound, which are to be tested on the first cell monolayer, and that the cells of the second layer are antigenic - 50 then the extent of the immunoadhesion of the second erythrocytes, which have an immunological adhesion to the antioxidants, which are due to the antibodies to the first layer of erythro-bodies, which were used in step (b). zytes are bound.

In this last-described embodiment of the invention, as already explained, the method according to the invention is

In accordance with the method according to the invention, the immunoglobulins or for determining cell types or the cell compatibility complementary components on the cells of the cell layer (a) 55 can be particularly advantageous if they are bound with the aid of this method after this cell layer (a) has already been determined. The Erfin has been built. On the other hand, however, it is also possible that the cells that are bound in step (a) are now explained in more detail using tests for determining the blood group.

ne or complementary components to be tested for. According to the method according to the invention, a test may or should not be carried out, or it is suspected that they carry it. 60 blood types can be determined in three steps:

According to a further preferred embodiment of the er 1. A monolayer of erythrocytes (for example the type according to the method of the invention becomes the monolayer of cells A) is irreversibly bound to a solid support; (a) are formed by placing a suspension of these cells on the 2nd layer on the cell layer as an immunoadsorbent.

applies solid support material to be coated, with groups such as anti-A antibodies being placed and specific adsorber-reactive surface of the support material being present;

able to bind the cells to the support, 3. the antibody-coated cells can now be tested, a preferred support material consisting of a polystyrene, either for their susceptibility to immune lysis on which the reactive groups are treated with by complement (solid phase lysis) or on their ability

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a second monolayer of cells bearing a corresponding one; 2. the cell layer is in contact with a mixture

Wear antigen to bind (solid phase agglutination). brought that (a) from a solution more reactive, to the immunoad-

The test results can be evaluated in a simple manner. Sorption of antibodies enabled with the aforementioned antigen, e.g. through the microscopic examination. Of loading and (b) of a second solution or suspension, which is of particular importance, however, that in the s according to the invention contains unknown antigen, the unknown antigen method, the test results specifically for the evaluation with the help of the competitive binding with the in contain the mixture

of densitometry using a standard and antibodies. Then 3.

conventional devices. For solid-phase hemagglutination, the degree of immunoadsorption of the antibodies in the solution, those (a) prepared in the manner described above can be measured on the cell monolayer (a).

Test plates according to either the static or the flow 10 2. The detection of antigen by competitive inhibition

Density determination to be examined. With this method one can first of all make 1. a monolayer

Work wavelengths at which the hemoglobin of the test- a cell suspension with known antigen absorbs the light through irreversible erythrocytes; e.g. is suitable blue binding made on a solid support. On this cell

Light with a wavelength of 415 nm. The flow-through layer is 2. given a solution which contains antibodies, the determination of which only requires the use of known 15 to react with the antigen of the cell layer and to immunoad-

Laboratory equipment and allows quantitative responses to lead to sorption. Then 3. a mixture is added

de questions: ben who a) a suspension with the cells and b) a solution

1. Has a second monolayer been formed; or contains suspension of antigen that competes with the

2. how many cells are contained in the second monolayer; immunoadsorbed antibodies of solution 2) react

3. What is the distribution of the second monolayer? 20 can. The degree of the second monolayer formation is now measured. The uniformity of the second of the second cell suspension is measured here as a “distribution”.

Called layer. A uniform second monolayer shows 3. Detection of antibodies in a cell population indicates that 100% of the cells in the overlaid cell suspension In this test 1. a cell population to be tested will carry the necessary antigen in order to establish a binding to the first cell Carrier bound in such a way that an irreversible happens. An uneven second monolayer-bound first cell monolayer is formed on the carrier. The formation, on the other hand, means that some «negative» cells in the cell monolayer are 2. covered with a solution that contained an applied suspension. Antigen with a known specific ability to immuno-In the alternative method described above contains noadsorption with antibodies. Finally, 3. the level of the immunoadsorption that arises for the detection of immunological factors is measured.

The presence of antibodies or antigen can be detected in the presence of antibodies which have been bound from a solution by a cell population by means of immunoadsorption, conveniently by applying a second suspension. In this test, 1. is applied to a solid Carrier an antibody

sion of test cells can be checked. If the first monolayer has given perus-containing cell suspension which reacts on this support with the antigen (or antibody) from the solution, it forms irreversibly bound monolayer. The resulting layer can be formed by overlaying the special cell layer 2. with a mixture. This mi

cifical antigen (or antibody) of known specificity as a mixture contains a) a solution with which the antigen-testing agent acts, the cells of the second suspension and b) a second, antibody-containing cell suspension or solution.

binds, creating a second monolayer. solution, these antibodies being able to competitively

The method according to the invention can also be used to inhibit immunoadsorption of the antigen. Subsequently, the competitive reaction between solutions and 40 will be 3. the level of immunoadsorption of the antigen in question

Suspensions of materials suspected of being common in solution (a) measured on the monolayer.

me to check antigenic properties. For each of these methods, the degree of immunoadversible cell monolayer bound to the template, such as presorption, can be established by one or more of the methods generally described above. This cell layer will be determined using the methods described.

stratifies with a mixture of two solutions, the 45 of which, despite the longstanding difficulties, contain the Kli-first antigen or antibody of known specificity and which operate in blood bank serology, and despite the ability to immunoadsorb to the bound cell monolayer Methods to improve and by appropriate, and the second is the test solution or suspension. To simplify the devices and automation, there is only competitive binding of the known antibodies or antigens, little known work in this area. For many years, factors that were contained in the second suspension or solution 50 by the so-called “Eldon” cards for the blood group are reflected in a known reduction in the immunization level. These have been described e.g. in noadsorption to the irreversibly bound cell layer. No. 2,770,572. A test card is correspondingly described in this patent specification. This method can also be used for the typing of human blood. The tests are being carried out to map the degree of formation of a second cell monolayer on their surface in different areas. Here, a cell suspension which is capable of forming a second monolayer mixed with antibody factors mixed with antibody factors is mixed with a second Lö-conglutinin or conglutinin substitute for the typing of solution or suspension to be examined . The human blood. In the relevant blood typing tests with competitive binding between these two immunological Eldon cards, the blood sample to be typed is shown in drops of partners in the mixing of the second monolayer on the different serum fields on the card. 60 given. After a sufficient reaction time, the memory of these basic procedures can be read different specific results on the basis of the agglutinations, le methods are possible: However, these tests are based on the detection of anti-A, anti-B 1. The detection of antigen by the competitive immunoad- and anti-Rh (Rh0 or D) and even these have sorption significant error sources in their interpretation.

As a first step, this test method includes the preparation of another diagnostic test. US Pat. No. 3,666,421 is another diagnostic test

Position of a cell monolayer that is irreversibly described on a solid, in which the serological reagents as drops

Carrier is tied. This monolayer is transferred from a cell slide to a test slide and dried as a stain.

suspension formed which is known to become an antigen net. This dried material can subsequently be like

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The solved and for the reaction for the identification of blood-purposes of the typing of blood or tissues as well as for groups with the help of agglutination or of others through the compatibility tests has not yet been used. Agglutination Detectable Antibody Reaction Systems The following will be used with respect to the invention. However, the test slides have the currently used tests for the determination of erythrocytes disadvantages, which are also explained in the case of ordinary agglutination.

siger phase occur. The test shows only that the pre-polystyrene test tube is

presence or absence of agglutination, coated with fibrinogen (concentration of fibrinogen

and it requires the practiced assessment by the technical assi solution (0.5 mg / ml), which irreversibly adheres to the polystyrene stentine, in order to determine whether the agglutination binds with a special. After a wash, the fibrinogen layer is treated with a fish antigen-antibody reaction or by unspecific io a polylysine solution (concentration 0.1 mg / ml).

Cell aggregates has emerged. It is also difficult or after a repeated wash, a suspension of even becomes impossible to determine whether free, non-agglutinated cells are either normal or protease-treated erythro-

len in addition to the clumped cells of the specific agglutinate cyte (RBC) added. These RBCs are irreversible (for exist. Test purposes) as a monolayer to those with polylysine and fibrin

In US Pat. No. 3,770,380, a device for the coated polystyrene surface is bound. The bin

tion of the immune adherence response. There must be a density of 2 X IO6 RBC per square centimeter

At this point it should be emphasized that the immune adherence response speaks to the diameter of the RBC of 7 micrometers.

which is described in this patent, differs from the immuno-monolayer of the bound RBC and is stable and serves noadsorption phenomenon, which is used in the method according to the invention as an immunoadsorbent for binding antibodies. Immune adherence is based on20 from any solution (or serum) if that contains an unspecific clumping of particles or cells specific for the antigenic determinants due to the presence of complement antibodies. At the Immunadhä- cell membranes are the bound RBC.

As a result, the complement creates the bond. Immune Adhesion The method of binding blood cells as a monolayer reference is characterized by non-specific reactions between a solid support and is not limited to the above-described complement and the particles or cells to which the 25 methods are limited. The literature on coupling it binds. In contrast, the immunoadsorption of proteins on polymers (cf.Cuatrecases and Anfinsen, loc. Cit.) By a specific bond between the antigenic detectors shows that, as an alternative, preparations of minerals from a cell membrane and the polymers contained in the serum (foils or plates of, for example Polyurethane or polyAntibodies, which is why imstyrene) can be used, the surface of which, in contrast to immunoadherence, reak-munoadsorption refers to an active compound or group, such as glutardialdehyde, bromine-specific binding between antigens and antibodies. eyan, amino or carboxyl groups, which allow covalent binding of the blood cells to the support in the device described in US Pat. No. 3,770,380. It is itself a flat, cell-like structure that is understandable that the binding substances must be immunologically active in coating their soil with bacterial or viral material against any antigens or antibodies that are detected. The coating is bound by means of a transparent, dried protein intermediate layer to the base of the cell-like substrates, or substrates for use in the invented structure. The immunoadherence reactions between the methods according to the invention can be all substances if (i) they see the cell which has been prepared with bacteria or viruses in such a way that a cell monolayer is located in the front and the antibody-bearing erythrocytes and the hend described irreversibly bind to it, and complement in the test liquid is determined by the degree of 40 (ii) if they are suitable for the Bäk- in view of the detection method used adherence of the specifically prepared erythrocytes. Having evaluated the most convenient detection method for coating containing virgin or viral material. light transmittance based - like the microscopic. The method described in U.S. Patent No. 3,770,380 has no counting and flow density determination - is found to be of practical clinical value because the immunoadherence preferred carriers that are translucent. If the measurement itself is rarely used and because the immunoadherence of the radioactivity is used, the use of light-specific and therefore not cheap for the detection of specific permeable material is of course not necessary.

fish antigen-antibody reactions. For the purposes of the invention, the substrate or layer of blood cells embedded in a solid support, simply the inside surface of a test tube, may have been used for scientific purposes when the flow density measurement is used to evaluate the test -that nothing should be taken into account with blood group determinations and compatibility- 50 test results have to do with flat surfaces. These layers are not best suited by kova-. Lente or other molecular forces can be bound for larger investigation approaches. Goodman (Na- strips of cell-binding material can also be used, ture, vol. 193 (1962), p. 350) prepared columns from with mold to produce the first cell layer. As a result, the anti-aldehyde-treated erythrocytes, which were recognized in a polyurethane resin body and examined for their ability to be embedded, can neither immunolysis in the presence of complement to throcyte antibodies based on their binding intensity for the fractionation of human anti-Ery-55 support or a second monolayer of cells of an erythrocyte. Edelman et al., Proc. Nat. Acad. Sei., Vol. 68 of unknown type. Quantitative results (1971), p. 2153, obtained fractionated blood cells by taking their one time with the help of flow density determination, whereby ability to specifically bind lectins, antibodies a loss of color lysis, the increase in color formation or antigens , which previously indicated partially hydrolyzed nylon fibers 60 of a second monolayer of erythrocytes. For this were tied up, exploited. the latter purpose is advantageous if the first monolayer, for example of antigens, antibodies or enzymes, is removed from one another as part of the punctiform principle of binding prosthetic groups, with preparation by hypotonic lysis, so that it no longer subtracts as a background solid matrix, is a known method; see. Cuatrecases and must be. Hypotonic lysis only removes no touch, Ann. Rev. Biochem., Vol. 40 (1971), p. 259. 65 significant amounts of the antibody bound to the membrane are used in the radioimmunoassay in the solid phase; see. Brit. by. The formation of the second monolayer can be done with mere Med. Bull., Vol. 30 (1974), pp. 1-103. The irreversible binding of the eye or the optical density determination by means of blood or other cells on a solid base for hemoglobin content can be recognized. When using the

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Flow density determination can e.g. at 415 nm are currently being carried out quantitatively in the liquid phase, with operative answers to questions such as “a second layer could be set up with the same success in the solid phase”, “how many cells (in relation to a clear one) This also includes the use of additives, the maximum) contains the second monolayer »,« how is the distribution known to improve agglutination, eg second monolayer ». 5 symmetrical and asymmetrical hydrophilic colloids, protea

In order to answer the last question, the cell suspensions, the ion concentration, polyelectrolytes, the tonicity and sion should be given to the buffer systems on the antibody-loaded monolayer to control the pH. These factors are going to have a concentration sufficient to have a second e.g. by Berkman et al., Transfusion, Vol. 11 (1971), p. 317, monolayer. The second monolayer is then described.

see to remove unbound cells. According to this io The invention can also be applied to other problems - washing is the distribution of the bound portions of the second, in which immuno-specific cellular reactions ten monolayers occur as a function of the percentage of positive. One such problem is the antiglobulin test for the ven »cells which are bound and the« negative »cells, determination of cells and their loading with IgG, IgM, IgA which have not been adsorbed. The resulting holes or IgD and IgE immunoglobulins and with C3, C4 and other islands can be detected by means of the microscopic flow density as complement components or bound activation mood. Their frequency and intensity will produce; see. Rosenfield et al., Vox-Sang., Vol. 26 (1974), S. expressed as the rate of the screened surface. 289-333.

The method according to the invention of binding the anti- Another possible application is testing the solid body on a monolayer of erythrocytes by immunoad phase with the quantitative evaluation of a passive or e-sorption has various important advantages. Sera with a 20 reverse passive hemagglutination. Passive hemaggluti antibody concentrations that are too low for the conventional national tests can be used for direct analysis of the antibody tests in the liquid phase, can be concentrated by binding to concentration, but also for the indirect analysis of soluble cell monolayers. For tests in the liquid antigen concentrations due to the competitive binding on the sigen phase, it is usually not possible to use undiluted sera based on common antigens; see. different serum proteins can interfere. This 25 Nusbacher et al., J. Immunol., Vol. 108 (1972), p. 893. Reverse disturbance does not play a role in tests in the solid phase, since the passive tests measure the soluble antigen directly; see. Cook, antibodies are selectively adsorbed and disruptive proteins Immunol., Vol. 8 (1965), p. 74 and Juji and Yokochi, Japan, J. are removed by washing. Many serums are inappropriate - exptl. Med., Vol. 39 (1969), p. 615. In addition, however, tests for the liquid phase cannot be carried out, since they contain antibodies with an undesired specificity which also do not remove bacteria, protozoa, fungi and culture cells from cell lines Tissues or tumors on their antigenic determinants. According to the invention, by appropriate selections on their surfaces, after the cells according to the invention have been formed for the formation of the first monolayer, the antibodies which are to be tested can only be adsorbed in the solid phase like the blood cells on their sensitivity. possibility of antibody-mediated agglutination or lysis

The present invention is suitable both for determination and analysis. The tests according to the invention in fixed pha-mation of cell types as well as cell compatibility. For the 35 se can also be used to solve problems with molecular cell typing e.g. the first monolayer with the bound antibody concentrations, the K value of the antibody-internal antibodies from cells and antibodies of a known type and the degree of K value heterogeneity.

Type manufactured. The determination of the cell type of a patient. The method according to the invention using one or a donor is then carried out by assessing whether these cell-cell monolayers in the solid phase are particularly suitable for those who have formed a second monolayer. The donor cells, the first and other cells, are used for compatibility and blood compatibility determination and for compatibility tests with blood test tests.

To form monolayer. The examples illustrate the invention.

These can then react with the possible antibodies of the patient serum, which are bound by immunoadsorption, example 1. This is demonstrated either by the addition of 45 immunolysis by human anti-A

of complement, which results in lysis, or through a white polystyrene test tube with an inner diameter of tere addition of the same donor cell and the determination of 10 mm and a height of 75 mm are on the inner antibody capacity to form a second monolayer (under surface 2 Minutes with 0.2 ml of a fibrinogen solution (one of the prerequisites that a first binding took place). Concentration of 0.5 mg / ml coated, washed and

The choice of the determination 50 suitable according to the invention for 2 minutes with 0.2 ml of a solution of poly-D-lysine methods makes no difficulties. Erythrocytes have a hydrobromide of molecular weight 140,000 of a concentration-their own label treated by the pigmented protein hemation of 0.1 mg / ml. After a further wash globin that has its maximum at a wavelength of 415 nm will be 0.2 ml of a 2 to 5 percent (v / v) suspension of absorption. The presence or absence of type Ar cells in 0.9 percent saline for 2 minutes of erythrocytes can thus be easily and specifically added in the 55. This creates a monolayer to be detected as described above by adherence. Others of erythrocytes with a flat surface and a density of labeling methods can also be used of 2 X 106 / cm2. These erythrocytes also dissolve after. Such methods are radioactive labeling, e.g. repeated washing no longer from their pad. Also with 51Cr or 125J, biochemical processes, e.g. a selected - the addition of strong human anti-A is not capable of intracellular enzyme, the fluorescence technique, e.g. Use-60 to solve more. If, after the addition of anti-A, 0.2 ml of a molecular probe for identification of dead plement is added, the hemoglobin of the adherent or living cells. Duck erythrocytes are released for all of these procedures. The level of immunolysis becomes talking devices and they can be automated. They are now either measured with the help of the remaining hemoglobin - equally usable for the serology of blood banks, or it is released by the release of radioactivity for medical blood tests, the typing of tissues and 65 form of 51Cr, which was used in order to mark the compatibility of the monolayer before blood transfusions and dying cells. With a standard transplant. th dose of complement can reduce the lytic potential of anti

It is known that a whole range of methods can be quantified. Conversely, a standard

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The dose of anti-A defines the lysing complement and can be measured as a whole by titration. The classic way of complement action using guinea pig erythrocytes (cf. L. Pillemer et al., Science, Vol. 120 (1954), p. 279) or the other way of properdin action using EDTA Complexation of calcium ions, but not magnesium ions in human serum.

None of these attempts to study immune A-anti-A lysis can be carried out so sensitively and reproducibly in a test in the liquid phase. IgM, IgG and IgA anti-A are all very effective agglutinins for type Aj cells and agglutination interferes with immunolysis. In solid phase experiments according to the invention, the lytic potential of anti-A was not only measurable, but 50 times more perceptible than in tests in the liquid phase. The diagnostic possibilities are therefore considerably improved with the method of the invention.

Example 2

Determination of blood groups

The problem of typing human erythrocytes and detecting human anti-erythrocyte antibodies prior to transfusion to determine compatibility is considerable. Indeed, the only means of detecting some human blood groups through direct agglutination are very expensive and require complex equipment; see. Berkman et al., Transfusion, Vol. 11 (1971), p. 317. According to the invention, it was possible to adapt the complicated methods in the liquid phase to a test system in the solid phase, and with this technique a specific typing for Rh, Kell , Kidd, Duffy, Xga, Lewis, Lutheran and MNSs with a sensitivity that far exceeds that in the liquid phase.

For blood group determination, a monolayer of erythrocytes is produced using the same method as that described for immunolysis in Example 1. Only this monolayer was first exposed to a known specific antibody (0.2 ml) and then hypotonic with aqua dest. lysed. At this point, a second dose of erythrocytes (0.1 ml) in 0.2 percent suspension (v / v) was placed in the test tube. The suspension is allowed to settle so that a light second monolayer is formed. It is now possible to increase the specific binding of the antibodies to this second monolayer in different ways. E.g. the low ion method according to Berkman et al., cited above, was successfully used. First the supernatant liquid is replaced by a solution (pH 6.0) containing 5% mannitol and 0.0025% protamine sulfate. Secondly, the monolayer is washed after 5 minutes at room temperature with a 0.0014 M phosphate-buffered saline solution (0.85%) (pH 7.3). In this way, non-antibody-bound, known negative cells are removed. Other methods of enhancing cell antibody binding were even more advantageous. Some of these could not be successfully used for Berkman's liquid phase instrumental method.

The specific antibody binding to the second cell monolayer could be improved by successively replacing the supernatant liquid with a buffer (pH 7.0) containing 2.5% PVP. Then it is replaced by the same buffer with a pH of 6.0 and a content of 0.01% protamine sulfate. Finally, the tests are washed with a 0.2 M phosphate buffer (pH 7.3). It is clear that the possibilities for the detection of agglutination in the solid phase are very numerous because this method avoids many problems associated with the tests in the liquid phase.

The solid phase tests have been shown to be extremely sensitive to the detection of Rh antibodies of the IgG type. Approximately the same sensitivity for one antibody molecule per red blood cell was achieved as was recently described for detection in the auto-analyzer (cf. Rosenfield et al., Cited above), namely 10 antibody molecules per cell with 50 percent hemagglutination. With the method in the solid phase, however, only 1/1000 erythrocytes as used in the auto-analyzer are required and the sensitivity is 10000 times stronger, which detects approximately 1 pg of antibody protein / ml. This surpasses the sensitivity of all tests described so far, including those that use the survival rate of the cells “in vivo”.

Tests with anti-Rh and protease-pretreated erythrocytes were also successfully carried out. These tests were set up in microtiter polystyrene plates with flat bottoms. The flat bottoms made it possible to examine the specific adherence of the erythrocytes microscopically. Bound cells were only present if they were Rh positive. “Holes” were observed in artificial mixtures of Rh-positive and Rh-negative cells, which were caused by the presence of non-adherent cells in these areas. They corresponded to the percentage of Rh-negative cells contained in the artificial mixture. This result shows that, according to the invention, the proportion of unbound cells in a blood sample can be determined quantitatively. This is the crucial problem in the detection of the survival rate of transfused cells according to the Ashby method (Arch. Int. Med., Vol. 35 (1925), p. 516) and in the characterization of human chimeras; see. Race and Sanger, "Blood Groups in Man", Davis, 1968, pp. 475-490.

Example 3

Direct antiglobulin tests

These tests were performed like the blood grouping tests, except that the washed erythrocytes were from a patient suspected of having acquired hemolytic anemia. These were used to make the first and second cell monolayers. The first monolayer (bound by polylysine fibrinogen) was evaluated with a single xenogeneic anti-human globulin serum and seven (serum) specificities. These sera were individually specific for IgG, IgM, IgA, IgD, IgE, C3 and C4. The antibody coated cell monolayer was then hypotonic lysed, washed and loaded with a cell suspension a second time to form the second monolayer. The binding of this second monolayer was strengthened by adding 0.9 percent saline solution containing 1 percent polyvinylpyrrolidone (PVP, average molecular weight 300,000 IK value 90). Finally, the second monolayer was washed with pure 0.9% saline. This procedure is similar to the method described by Hsu et al., Vox-Sang., Vol. 26 (1974), p. 305. However, the results obtained in the solid phase were clearly superior to those of Hsu, which came from tests in the liquid phase. A patient with an active acquired hemolytic anemia, whose adhesive proteins could not be typed in PVP because of a pronounced spontaneous agglutination in the liquid phase, was clearly positive for IgG, IgM, IgA, IgE, C3 and C4 using the method of the invention.

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Claims (12)

631550 2nd PATENT CLAIMS 1. A method for determining cell types or cell compatibility on a solid support material, characterized in that (a) irreversibly binds a first monolayer of cells to the carrier material, the cells containing or having been provided with antigens, (b) contacting this first layer (a) with a solution which contains antibodies, antibodies being bound by immunoabsorption to such an extent that the antibodies of this solution are reactive with the antigens of the cells of the first layer, (c) subsequently applying a suspension of second cells, these second cells carrying antigens, and (d) the extent of the immunoadhesion of the second cells is determined by antibodies bound to the first cell layer. 2. The method according to claim 1, characterized in that the first cell layer and the second cell layer are cells of a first test person, and the solution is a serum that was taken from a second test person. 3. The method according to claim 1, characterized in that in step (c) the second cell layer is formed by applying cells of a cell population to be tested in an essentially just sufficient number so that a monolayer is formed, the extent of the immunoadhesion of this Monolayer is a function of the percentage of cells tested that carry specific antigens. 4. The method according to claim 1, characterized in that the solution containing the antibodies is a mixture which (1) a solution of an antibody that reacts by immunoadsorption with an antigen carried by the cells of layer (a), and (2) contains a second solution or suspension which is tested for antigens by inhibiting the antibodies in solution (1). 5. The method according to claim 1, characterized in that the antigens of the first cell layer (a) are antigens which are carried by immunoglobulins or complementary components which are bound to the surface of the cell population, and that the antibodies in the solution which used in process step (b) is anti-immunoglobulin or an anti-complement to the antigenic determinants carried by the immunoglobulins, or complement components to be tested on the first cell monolayer, and that the cells of the second layer carry antigenic determinants which have immuno-adhesion to the antibodies used in step (b). 6. The method according to claim 5, characterized in that the immunoglobulins or complement components are bound to the cells of the cell layer (a) after this cell layer (a) has already been built up. 7. The method according to claim 5, characterized in that the cells which are bound in step (a) carry immunoglobulins or complementary components to be tested for, or one suspects that they carry them. 8. The method according to claim 1, characterized in that the monolayer of the cells (a) is formed by applying a suspension of these cells to the solid support material to be coated, with reactive groups on the surface of the support material that are capable of the cells are to be bound to the support, a preferred support material consisting of a polystyrene on which the reactive groups are formed by treatment with a fibrinogen solution and a subsequent treatment with a polylysine solution under conditions such that the chemically Form reactive groups on the surface of the carrier material. 9. The method according to any one of claims 1 to 8, characterized in that the irreversibly in step (a) 5 bound cells are erythrocytes, which contain antigens or on which antigens are bound, and that the second cells used in process step (c) are also erythrocytes which carry antigens, and in step (d) the extent of the immunoadhesion of the second erythrocytes is belo true, which is caused by the antibody of the erythrocytes of the first layer. 10. The method according to claim 9, characterized in that the first layer of the erythrocytes is subjected to lysis before the second erythrocytes are applied. 11. The method according to claim 1, characterized in that agglutins are determined in a serum by producing in step (a) a monolayer from cells in which or on which there are antigens with respect to the cells to be tested; in step (b) use the solution 20 to be tested for the presence of the agglutins; in step _ (c) a second suspension of cells is used which are known to contain or contain antigens for the agglutins, the presence of which is being tested; and that in step (d) the extent of the immunoadhesion 25 of these second cells is determined on the first cell layer. 12. The method according to claim 1, characterized in that in step (a) washed erythrocytes bind to the solid support material in which or on which antigens are located; in step (b) with the antibody-containing solution, it binds to the first monolayer of the erythrocytes by immunoabsorption; and in step (c) a suspension of second erythrocytes in which or on which antigens are located, and that these second erythrocytes are allowed to settle on the solid support material as a second monolayer above the first layer of the erythrocytes, and after the formation of this second monolayer adds protamine sulfate to the suspension in an amount sufficient to immunoadsorb the second erythrocytes on the antibodies bound to the first monolayer and to wash for second erythrocytes that are not bound are to be removed and then the extent of the immunoadhesion of the second erythrocytes, which are bound by the antibodies to the first layer of erythrocytes, is determined. 45 Many medical procedures require cell compatibility to be determined and, for example, the compatibility of blood cells between donor and patient must be determined prior to a blood transfusion or transplant. The tolerance or compatibility of blood cells is determined by the lack of an immunological reaction between antibodies contained in the blood serum and an anti-55 gene which is contained in the blood cells of the donor. For example, if a patient's erythrocytes are of type A, i.e. the erythrocytes have the antigen "A", their serum contains anti-B antibodies, i.e. his antibodies would react with "B" cells. Such incompatibility can have serious consequences due to the resulting intravascular hemolysis. There are two different types of tests for typing and demonstrating the compatibility of blood cells: 1. Tests that determine whether the addition of a 65 specific antibody causes agglutination of the blood cells, and 2. Tests that demonstrate that a specific antibody together with complement causes the lysis of the tested blood cells. The first type of these tests, agglutination, is based on the clumping of the blood cells. If the cells e.g. Containing type A antigen, they are agglutinated by anti-A antibodies in the absence of complement. The A-antigen and the anti-A antibodies react specifically with one another by an immunological reaction, the antibodies forming bridges between the adjacent cells. This leads to a clumped mass of blood cells that are connected to each other by the added antibodies. The second type of the aforementioned tests, the cell lysis, is based on the detection of the destruction of the cell membranes and the resulting cell death and the release of the cell contents. Cellisis is the result of a reaction between antibodies bound to the cell membrane and a group of potentially destructive normal serum proteins called "complement". Both methods described above are used for the typing and compatibility studies of the cellular blood elements, erythrocytes, granulocytes, lymphocytes and thrombocytes. Although they are often only used qualitatively, they can also be used quantitatively and then serve to detect antigen, antibodies and serum complement. The test methods that are currently routinely used in clinics for blood group determination and compatibility testing of blood cells, the agglutination test and the cellisis test are both carried out in the liquid phase. Antibody-containing serum with or without complement is mixed with a blood cell suspension, the blood cell type being selected according to the question. Usually constant volumes are used. Assessing the results of agglutination tests requires the technical assistant's ability to distinguish between specific agglutinations that result from the molecular bridging between specific antigen and antibodies and non-specific cell aggregates caused by different forces that cause a certain degree of clumping. The technical assistant must also be able to recognize free, non-agglutinated cells that may be present in addition to clumped or agglutinated cells. This requires either experienced, well-trained personnel or precise particle measurement and counting using expensive instruments. In addition, the degree of specific agglutination is either only to be recorded semi-quantitatively or it requires expensive and complicated detection methods. Although various devices for the typing of erythrocytes have been developed with the aid of agglutination, this type of equipment is always expensive and complicated to use. For example, a device that is recommended for the typing of erythrocytes is known under the name “Auto-Analyzer”; see. Berkman et al., Transfusion, Vol. 11 No. 6 (1971), p.317. In the auto-analyzer, the blood samples and the antibody-containing serum are agglutinated in tube coils under certain conditions. The tube coils are connected to a «T» connector, the foot of which is directed downwards, which removes the agglutinates that have formed. Agglutination can now be determined either by measuring the optical density in the “T” piece that contains the outflowing liquid that contains the non-agglutinated fraction (Berkman et al., Op. Cit.), Or by collecting the agglutinates the “T” piece on filter paper (cf. Shield et al., Transfusion, Vol. 9 (1969), p. 348). This apparatus is expensive and complicated and is not suitable for simple automation for routine use in blood banks. Another very expensive device is known under the name "Groupamatic"; see. Garretta et al., Vox Sang., 631 550 Vol. 27 (1974), p. 141. Serum and blood cells are brought together for agglutination in the Groupamatic device. The presence of agglutination is demonstrated by passing the suspension through two beams of light. One light beam passes through the center of a reaction cuvette, the other through the periphery. The difference between the transmission of the light rays is taken as a measure of the strength of the agglutination. This device is only worthwhile for very large blood banks. The tests based on immunolysis are not problematic as long as the cell surface antigen-antibody reaction reacts sufficiently with complement, as in the typing of tissues, e.g. HL-A. Unfortunately, this is rarely the case with human erythrocytes. Either the reaction between the cell membrane antigen-antibody complexes and the complement is not sufficient, or the anti-body concentration has to be limited in order to prevent agglutination that is too intensive, as this mechanically interferes with immunolysis. These problems place a heavy burden on blood banks' serological laboratories, since even the routine typing of erythrocytes remains a time-consuming, manual activity that requires more skilled and experienced personnel than is available. In addition, only a few blood banks can afford to perform lymphocytotoxic tests for tissue typing. There is also no direct immunological test for the compatibility of granulocytes before a transfusion and only an indirect test for platelets by the release of 14C-serotonin. Granulocytes and platelets can be identified as HL-A types by selected and suitable tests, but their compatibility cannot be guaranteed.