AU2660500A - Method for detecting antibodies or antigens and for determining blood groups - Google Patents

Abstract

The invention relates to a method for detecting antibodies or antigens in a test fluid as well as for determining blood groups by reaction with a defined specific binding partner. The antigen or antibody or specific binding partner are either present in free form in the test fluid or bonded to a support. In case of a positive antigen-antibody reaction an agglutinate of antigens or antibodies, the corresponding binding partners and the support materials is formed, which can be optically detected. A microreaction vessel having a cross-section which narrows from the top downwards contains a viscous substance, notably a gel. A test fluid is placed into the vessel. The specific binding partner is added to either the test fluid or the gel, or a liquid containing said binding partner is added to the vessel after the test fluid has been placed in it. The sedimentation image is then evaluated optically. A flat agglutinate of antigens or antibodies, binding partners and support materials indicates a positive antigen-antibody reaction, a deposit of antigens or antibodies and binding partners and/or support materials in the lower, narrow area of the vessel indicates a negative reaction. The above method can be carried out in a fully automated manner, for example by using a pipetting robot.

Description

-2 Method for detecting of antibodies or antigens as well as the determination of a blood group Technical field: Invention relates to a method for detecting of antibodies and/or antigens in a test liquid as well as for the determination of a blood group by reaction with a predetermined specific binding partner, wherein the antigen or, respectively, the antibody, or the specific binding partner in the test liquid is bound or unbound to a carrier and wherein an agglutinate of antigens or, respectively, antibodies to be detected, which agglutinate is formed of the corresponding specific binding partners and the carriers in case of a positive antigen antibody reaction, which agglutinate can be detected optically as a sedimentation picture, according to the preamble of claim 1. The invention relates further to the use of tipped floor micro-titration plates for performing a test for detecting antibodies or antigens by identifying an agglutination reaction as well as for determination of a blood group. State-of-the-art: Blood group materials of the human being, but also a plurality of other substances formed in an organism, are identified and detected by specific antibody antigen reactions. This reaction is detectable by cross-linking, agglutination of antigens (Ag), and antibodies (Ak), and defined carrier substances, which carry antigens or antibodies, to a complex macroscopically capturable, the agglutinate.

-3 Examples for this are the erythrocyte side blood group determination as well as the serum counter test. In the case of the erythrocyte blood group determination blood group antigens bound to the membrane of the donor erythrocyte are detected by reaction with anti-serums, wherein the anti-serums contain antibodies as specific binding partners. The antibodies are known (search and find antibodies), which are disposed in a test liquid, and, for example a test antibody suspension, and wherein the test liquid contains the unknown erythrocytes. The agglutination is caused under favorable reaction conditions by the reaction of antigen and antibody. The system ABO and RH and in addition a plurality of antigens can thereby be determined. The erythrocytes are known in the serum counter test, wherein the erythrocytes exhibit known antigens (search and find antigens). The test liquid, for example serum, comprises the unknown endogenic antibodies (so-called iso-agglutinins), which are detected by reaction with the test erythrocytes. Also the blood group, however only ABO, can thereby be determined. The detection of the regular antibodies, most of the incomplete IgG type, is performed indirectly through the anti-IgG. According to a first step test erythrocytes incubate with a known Ag-pattern with test serum. The antibodies are bound to the antigen (Ag). According to a second step free antibodies are washed out with a washing centrifuge in three washing steps. According to a third step the antihuman globulin (anti-IgG/Coombs serum) is added, whereby a bridge formation occurs and thus a visible agglutination occurs, however -4 only by way of a centrifugation. The agglutination can be made visible in various ways. For example a conventionally known method for blood group determination employs round floor micro-titration plates, wherein the recesses of the plate have pipetted in various anti-sera containing specific antibodies as specific binding partners for the antigens on the membrane of the erythrocytes and wherein the anti-sera are contacted with a test liquid, wherein the tested liquid contains the erythrocytes to be tested in a thinned concentration. The plate is centrifuged after an incubation time and in the following the plate is carefully shaken. The erythrocytes have agglutinated to a lump, which collects in the middle of the recess based on the centrifugation step and wherein the lump if possible is not destroyed by the shaking. A positive reaction therefore is detected optically in the middle of a recess as a point shaped or button shaped agglomeration of erythrocytes. On the other hand, no connection exists between the erythrocytes in case of a negative antigen antibody reaction, that is in case of a non performed agglutination. The erythrocytes collected in the recess center after the centrifugation step correspondingly are distributed by the shaking over a large part of the U-shaped recess floor. A negative reaction is thus optically detectable by a flat extended erythrocyte blot spread over the floor of the recess. However this method can be automated only with substantial expenditures. On the one hand the method is technically very much demanding to realize a centrifugation step and a shaking step. On the other hand the reliability of the detection of a positive reaction -5 depends on the strength of the antibody antigen bond: in case of weak bonds the agglutinated erythrocyte lump can be separated again by external interferences, in particular by shaking up, and thus erroneously a negative reaction can be diagnosed. For this reason also the automatic distinction between positive reaction and negative reaction, for example by an automatic photomatic evaluation, is not possible without further efforts because of the possibly missing clear sedimentation picture but the distinction has to be performed by an experienced operating person or at least has to be checked. Finally the reaction pictures are not stable, because the carrier erythrocytes with or without agglutination always collect at the lowest possible point of the reaction vessel. Thus no reliable checking of the reaction result is possible. No visible reaction and thus detection for the anti body screening is given based on the incomplete antibodies. Similarly, the cross match test, the blood compatibility test, is not furnished. c2 part 1: A further method employs specially prepared micro-titration plates for the detection of antibody antigen reactions, wherein in each case a step shaped structure is milled into the V- shaped floor of the individual recesses of the micro-titration plates. The width of the individual steps is in the order of magnitude several micrometers. Anti-sera or, respectively, test erythrocyte solutions are brought to reaction with erythrocytes or, respectively, blood serum samples in a conventional way in the recesses of the plate for the performing of a blood group determination and are evaluated after an incubation at temperatures of about 30 degrees centigrade. If no antibody antigen -6 reaction does takes place in a recess, then test erythrocytes, which are in this case the carriers of the antigens, collect in the middle of the V-shaped recesses and are detectable as point shaped or button shaped sediment. In case of a positive reaction, however, the erythrocytes cross-link and form a flat agglutinated structure which is held by the step shaped structure. The same reaction in a V-floor micro-titration plate without step structure would lead to an erythrocyte lump in the middle of the recess and would therefore not be distinguishable from a negative reaction. In this case however, a positive reaction is optically detectable a more flat carpet of agglutinated erythrocytes extending over the floor of the recess based on the step shaped structure. This method is relatively easy automatizable as a classic agglutination method, wherein no additional mechanical engagements such as centrifuging, are required. In addition to, the evaluation based on clear sedimentation pictures is possible automatized photometrically or with a video camera, wherein the sedimentation pictures are relatively stable with respect to mechanical interferences. However, the high expenditure requirements for production of the respective micro-titration plates by milling in the staircase structure is problematically. For this reason the prices of the micro-titration plates are so high that a single use cannot economically be borne. In contrast expensive cleaning and sterilizing steps are required for multiple path operation in order not to obtain falsified results. Furthermore the lifetime of the micro-titration plates is limited to about 5 to 80 rounds of employment based on experience,. Furthermore the capturing of weakly positive reactions problematical, -7 whereby a refusal probability of about five percent is observed. This is particularly observable in case of the serum counter test, since the individual anti-body (iso- agglutinin) titer strongly varies from human being to human being. Here also no antibody screening and no blood compatibility test is possible. Methods are known for detecting antigens or antibodies from the European patent document EP 0305337B1 (=US 5338689) and United States patent 5512432, which methods render the positive or, respectively negative antigen antibody reaction optically visible in a reaction vessel. A predetermined amount of a test liquid of carrier bound complexes is added in a transparent micro-reaction vessel, wherein the micro-reaction vessel contains a dispersion or suspension of inert particles in the shape of so-called beads, for example agarose, and the known specific binding partners for the antigens or, respectively antibodies to be detected, wherein the test liquid contains the antigens or, respectively antibodies to be detected bound to a carrier. In the following the mixture is subjected to a centrifugation step for sedimentation; the making visible of a reaction is only possible through such a centrifugation step. A sedimentation to the lowest point of the vessel is not achieved by standard gravitation. During the formation of the antigen antibody complex, the antigen antibody complex adheres on the inert particle in a strongly positive case and is present within the inert particle in a weakly positive case; in case of an absence of an antigen antibody complex, that is, in the negative case, the sediment is disposed below the inert particles. The method is performed in a single elongated narrow micro-reaction -8 vessel, which comprises three sections, that is, an upper section, a middle section and a lower section, wherein the upper section and the lower section are shaped cylindrically and wherein the upper section exhibits a larger diameter as compared to the lower section. The middle section is conically shaped and connects the two neighboring sections each other, a plurality of such vessels can be disposed in the shape of a card and can be employed as a handy unit. The vessel is centrifuged and then the location is optically determined, where the carriers are collecting. If the carriers collect above the inert particle layer, then this points to a strongly positive antigen antibody reaction: the carriers cross linked to an agglutinate are prevented by the particles to penetrate to the floor of the vessel according to the centrifugal forces. In case of a negative antigen antibody reaction, however, the non-cross linked carriers penetrate in contrast to the floor of the vessel, which means that a reaction cannot be recognized without a centrifugation step. In the following the agglutination reaction can be identified by observing the reaction vessel from the side and by capturing optically the position of the carriers. This method can only be automatized in a cumbersome way because a centrifugation step is necessary. Furthermore the capturing of the weak antigen antibody reactions is problematical, wherein a nonuniform, cloudy distribution of agglutinate is observed over the filling high level of the particles in case of a weak antigen antibody reaction. These reactions therefore cannot be reliably automatically captured.

-9 Technical purpose: It is an object of the present invention to furnish a method for detecting of antibodies or antigens as well as for determination of blood groups under avoidance of the disadvantages of the state of the art, which methods can be simply performed and is easily automatizable and which leads to highly reliable test results. Disclosure of the invention as well as of the advantages of the invention: The object is accomplished by a method for detecting of antibodies and/or antigens in a test liquid or, respectively, a test mixture as well as for blood group determination by reaction with a pre-given specific binding partner, wherein the antigen or, respectively, the antibody or the specific binding partner are unbound or bound to a carrier in the test liquid and wherein an agglutinate of the antigens or, respectively, antibodies to be detected, of the corresponding specific binding partners and the carriers is formed in case of a positive antigen antibody reaction, wherein the agglutinate is optically detectable as a sedimentation picture, and wherein the solution comprises that a micro-reaction vessel having a narrowing cross section from the top toward the bottom is employed, wherein the micro-reaction vessel contains a viscous substance; a predetermined volume of the test liquid is added onto the viscous substance to the vessel, wherein the specific binding partner is added either to the test liquid or to the viscous substance, or a further liquid which contains the specific binding partner is added prior to or after the -10 adding of the test to the vessel; the forming or formed sediment/agglutinate is evaluated in a top planar view from above or from below onto the micro-reaction vessel; wherein a flat extending agglutinate (lawn formation) of antigens or, respectively, antibodies, binding partners and carriers points to a positive antigen antibody reaction and wherein a spatially minor extended deposit (button formation) of carriers points to a negative reaction with an absence of an antigen/antibody complex. An advantageous embodiment of the method is characterized in that the reaction is performed in a reaction vessel open at the top and closed at the bottom, wherein the reaction vessel exhibits a narrowing down cross-section at least in the region close to its floor, such that the vessel furnished at least in part with an inclined wall in its region close to the floor, wherein the wall joins with the floor or, respectively, forms the floor; wherein the viscous homogeneous substance, in particular a heated jelly or a heated gel is cast in and allowed to cool down at least in this region with inclined wall of the vessel close to the floor, such that the substance covers or, respectively, fills out the region of the vessel with inclined wall including the floor as a homogeneous layer after the cooling down and thereby forms a cross linking or, respectively a grid structure; wherein the predetermined volume of the test liquid or of the test mixture is placed into the vessel onto the homogeneous layer of the viscous substance; wherein the micro-reaction vessel is at least integrated for such a long time until the visible sediment/agglutinate is formed in the visible boundary layer between the inclined wall of the micro-reaction vessel and the homogeneous layer out of the -11 viscous substance based solely on the force of gravity, wherein a flat shaped agglutinate layer on the inclined wall of the vessel in particular points to a positive antigen antibody reaction, wherein in contrast a forming, spatially small extended deposit in particular in the region of the lowest point of the floor or, respectively, of the inclined wall of the vessel points to a negative reaction with an absence of an antigen antibody complex. According to a further advantageous embodiment of the method, the analysis, if an antibody antigen reaction has taken place in a micro reaction vessel is performed optically by way of the sedimentation picture by an operating person or automatically with a photometer or by way of a video camera or by a charge coupled device (CCD) camera. The analysis if an antibody antigen reaction has taken place in a micro-reaction vessel is also performed photometrically by measuring and recording the absorption of the mixture contained in the recess along a predetermined line and the thereby determined local absorption profile is evaluated. Furthermore the profile length is determined for a fixed width of the profile according to the method and a positive reaction or, respectively, a negative reaction is then diagnosed when length of the profile is found to be located below or, respectively, above a predetermined value. The viscous substance can be a gel which is heated up to the melting temperature and which is filled in into the micro- reaction vessel in a liquid state for forming of a homogeneous layer, wherein the viscous substance is cooled down up to the gel point or up to the neighborhood of the gel point and a homogeneous layer is formed -12 thereby, wherein the homogeneous layer forms a fine cross linking or, respectively, grid structure upon cooling. The gel -preferably of the type XII low viscosity for beading NRA 7299 -exhibits at least a gel strength of 0.01 grams per square centimeter equal to about 0.02 mg per 100 milliliters gel concentration. The preferred concentration is disposed between 0.1 mg per hundred milliliters and 6666 milligrams per hundred ml gel concentration. The region 10 mg per hundred milliliters to 110 mg per hundred millimeters gel concentration is in particular preferred. A particularly preferred region is disposed within the last recited region of between 40 mg per hundred milliliters to 80 mg per hundred milliliters gel concentration. The viscous substance is preferably a gel, advantageously an organic gel, in particular agarose and/or gelatin. The viscous substance can also be composed of cross linked polymers, wherein each viscous substance comprised of cross linked polymers is suitable. When the invention method serves for performing o- a serum countered test, which includes iso-agglutinin, or for detecting of irregular blood group antibodies (antibodies search and find test) or for an erythrocyte side blood group determination with known antibodies, then the test liquid contains blood serum with unknown antibodies or unknown erythrocytes. A liquid is added to the test liquid or to the viscous substance, wherein the liquid contains the test erythrocytes or test antibodies of a known blood group. In a special case, for example for a serum counter test, the carriers are -13 occupied with the specific binding partner antigens of a known blood group, which detect the antibody (iso-agglutinin) to be detected. Again specific binding partner antigens, which are located on the erythrocytes, are concerned in case of blood group antibodies (antibody search and find test). The specific binding partner antigens then show the presence of the irregular antibody. In case of an erythrocyte side blood group determination the carrier antigen is not known, but the known search and find antibody is disposed in the test liquid or in the viscous substance. Protein such as protein A and/or G, and/or antigens, and/or antibodies (anti-IgG (Coombs serum), anti-C3d, anti-IgN) can be added to one of the liquid or, respectively, to the mixture or to the viscous substance at the point in time of the investigation, which performs an influence; on the reaction. The carriers within the test liquid are erythrocytes, leucocytes, blood platelets, latex particles or agarose particles, glass particles or plastic particles. If the required materials of content are present in the liquid, then only a native gel is required. Therefore the method according to the present invention for the erythrocyte side blood group determination, for a determination of irregular antibodies, and for a serum counter test can be employed in an advantageous way. In principle the problem exists here, that each donor has an individual antibody titer. An individual thinning of the donor serum would have to be made in order to obtain a reliable reaction with conventional methods, which in particular impedes the automatization of the method.

-14 The cross-sectional area of the vessel at the filling height level of the viscous substance amounts to at least the fourfold of the cross sectional area of the vessel in the region below the viscous substance. Tipped floor micro- titration plates or round floor microtitration plates are employed, wherein the micro-reaction vessel is the recess of a tipped floor microtitration plate or of a round floor microtitration plate. According to the method incubation takes place after the step 1. 2, preferably at temperatures of at least five degrees centigrade, preferably between 10 and 37 degrees centigrade, in particular at room temperature, and for at least 15 minutes, preferably for at least 50 minutes. One of the liquids or the viscous substance can contain albumin in a concentration of at least 0.01 milligrams per ml, preferably between 0.2 mg per ml and 1 mg per ml. The invention method is associated with the big advantage that it can be easily automatized as a sedimentation method subjected only to gravity forces. This is based on the fact that the gel or the jelly heated above the melting point (that is at least to the melting point temperature) and cast into the micro-reaction vessel forms an irregular micro- cross linking or, respectively, micro-grid structure upon cooling way, wherein the micro-grid structure can be seen under the electron microscope and micro-grid structure operates quasi as a sieve or a brake for the antibodies or antigens in the test liquid, for -15 the binding partner (detecting antigens or, respectively, detecting antibodies) and/or for the carriers (for example erythrocytes, leucocytes, blood platelets, latex particles or agarose particles), that is through which the reaction partner can only diffuse through under braking. In case of a positive antibody antigen reaction with agglutination of the carriers there occurs a formation of an agglutination layer, agglutination lawn, or agglutination carpet in the boundary layer between the vessel wall and.the layer out of a viscous substance, wherein the extent of the agglutination layer, the agglutination lawn or the agglutination carpet in length and width is substantially favored relative to the extension in the height direction, that is relative to a thickening. In other words: one of the decisive core points of the invention comprises the recognition of the inventor that the viscous substance is to be applied as a homogeneous, preferably thin layer or in the form of the meniscus on to the inclined wall of the micro-reaction vessel. Therefore a sequence of viscous substances, such as for example gel or jelly then form a kind of finest, fibrous cross linked or grid like structure in an irregular way, wherein the liquid is disposed, upon cooling to the fixed point (gel point). The antigens or antibodies contained in the test mixture or, respectively, in the test liquid form complexes with the specific binding partners also disposed in the test mixture or, respectively in the test liquid or inside of the viscous layer, which complexes migrate through the homogeneous layer made out of viscous substance based solely on gravity forces and thereby react with the binding partners (the detecting antigens or, respectively, the detecting antibodies) within the layer, wherein the -16 specific binding partners are either disposed within the test mixture or, respectively, the test liquid for within the viscous substance or within a further liquid. This viscous material acts as a delaying brake onto the complex reaction partners sinking based on the forces of gravity. An agglutinate (sedimentation) formed as a connected flat shaped layer deposits in the boundary layer on the inclined wall of the vessel in case of a positive reaction. An agglutinate formed flat extending on the inclined wall called lawn formation and containing antigens or, respectively antibodies, specific binding partners (detecting antibodies or, respectively, detecting antigens) and carriers (for example erythrocytes) to be detected points to a positive antigen antibody reaction. In case of a negative reaction there results no agglutinate, but there is formed a spatially far less extended deposit (of carriers) button formation called -which less extended deposit forms usually in the region of the lowest point of the inclined wall at the floor; this points to a negative reaction. Therefore it is sufficient according to the present invention that the viscous substance in the form of a homogeneous layer, for example a gel layer, is present only in the region of this inclined wall, wherein however, this viscous layer can be selected also thicker, in the region of several millimeters, or can also form a meniscus. Thus the layer of the viscous substance has as the purpose to make visible and to hold an agglutination of the antigen and/or antibody carriers, that is the cross linked antigen and/or antibody carrier, in the form of a flat shaped layer ("carpet" or, respectively, "lawn") in the region of the V-shaped recess or of the bombed recess, wherein the agglutination occurred based on an antigen antibody reaction. In -17 contrast non-cross linked carriers migrate according to the pure gravity force along the inclined wall and collect preferably (because the vessel narrows down) at the lowest part of the vessel floor under formation of a spatially less extended, in an ideal case point shaped, optically capturable structure "button". In addition weaker reactions can be detected with the method according to the present invention. For this purpose the viscous substance is to be formed with a correspondingly high viscosity and correspondingly long sedimentation times/incubation times are to be used until the rendering visible of the reaction. If a lawn layer is formed within the boundary layer (which would correspond to a positive reaction (agglutinate)), however the negative reaction is not yet present as a button, but instead is present more or less as a flat formed distribution at the floor, then this reaction can be forced through in this boundary layer by an increased gravity force, for example by centrifugation, such that not reacted erythrocytes collect quicker in the lowest layer. According to the invention various buffers and/or also the specific binding partners (proteins, as protein A and/or G and/or antigens and/or antibodies, detecting antigens or detecting antibodies, in particular monoclonal antibodies, and/or the carriers) can be incorporated into the gel, wherein the specific binding partners are favoring the antibody or, respectively, antigen reaction. Here both buffers as well as binding partners can already be added during the heating stage of the gel under the precondition that the buffers and -18 the binding partners are stable relatively to heat. Furthermore the method is associated with the advantage that conventional microtitration throw away plates with V-shaped recess or bomb shaped recess can be employed as micro- reaction vessels, which microtitration throw away plates are cost favorable and can be easily handled with a pipetting robot. The micro-titration throw away plates have to be prepared for the performing of the method only with the viscous substance, for example the gel or the jelly, under formation of a homogeneous layer within the V-shaped recess or the bombed recess of the microtitration throw away plates. The evaluation occurs either with the open eye or in particular fully automatically with a video camera or a charge coupled device CCD camera or photcmetrically by a top planar view or a bottom planar view onto the vessel. The photomatic evaluation can be performed under a small technical and economic expenditure as compared to the recordation of the sedimentation pictures with a camera and their evaluation and the photomatic evaluation thus is suitable for a simpler and more cost favorable automatization of the method. The viscous substance can be a gel, wherein the gel is heated up to or above its melting point and in the following is filled into the vessels for forming of a homogeneous layer. Materials can be added to the viscous substance, which materials influence the agglutination reaction, such as for example also the specific binding partner of the antibody to be detected or of the antigen to be detected, that is the specific detecting antigen or the specific detecting antibody.

-19 The viscosity of the viscous substance is to be selected such that an antigen antibody reaction leads to a visible flat shaped agglutination of the carriers in the boundary layer. This can in particular depend on the strength of the antigen antibody binding or on the re-pulsing action of the carriers. In case of a weak antigen antibody bond in general are higher viscosity is required as compared to a strong bond. However, the method is associated with the advantage that the method is in principal suitable for all reaction strengths. Overall the reaction conditions, that is the viscosity of the viscous substance, the concentration of the antigen or, respectively the antibodies and the specific binding partner, surrounding temperature and the like have to be selected such that in case of a positive antigen antibody reaction there is present an aglutination of the carriers in the boundary layer. The respective regions depend in particular on the reaction to be investigated. Furthermore it can be advantageous for this to incubate the sample after the combining of the reagents at a suitable temperature for some time in order for forming the sedimentation picture. The addition of Coombs serum, which contains anti-IgG, which leads to a visible agglutination, is particularly helpful in case of incomplete antibodies, which otherwise would show no reaction. However, Coombs serum does not use the re-pulsing forces. The liquid or, respectively, the liquids should be added free of bubbles to the viscous substance, which can be accomplished in case of a -20 fully automatically performed method by a pipetting device with the suitable mechanical control, for example a pipetting robot. According to advantageous embodiment of the invention gel is an organic gel, in particular agarose and/or gelatin. The gel are associated with the advantage that they can be very easily composed, in particular in connection with throwaway microtitration plates. The gel can be furthermore stored in a good way, they are stable and o. ver short time periods relatively insensitive to changes in the ambient temperature. In particular excellent results obtained with agarose of a gel concentration of about 40 milligrams per hundred milliliters up to 18 milligrams per ml during the performing of serum counter tests and antibody (Ak) search and find tests for blood group determination. Microtitration plates such prepared with and opposed can be stored under cooling for several months. Also the finished prepared samples can be stored under cooling for several weeks, wherein the reaction pictures remain stable. The reaction results therefore can be drawn over a longer time period after performing of the test for control purposes. The carriers, to which the antigen or, respectively, antibodies or the specific binding partners to be detected our bows, are preferably erythrocytes, leucocytes, blood platelets or latex particles. The latex particles can be visibly dyed or marked by fluorescence for simpler optical evaluation. The concentration of the carriers in the reaction liquid is to be selected such that the agglutinate can be detected reliably with the selected optical detection method.

-21 In accordance with the present invention the sedimentation picture is evaluated optically by top planar view from above or below onto the reaction vessel. In case the measurement takes place from below that in the micro-reaction vessel has the transparent. The micro reaction vessel is to serve the also transparent if the evaluation takes place from above in order to increase the contrast of the carriers relative to the background. Based on the different vessel cross sections in the upper region of the viscous substance and in the lower region of the viscous substance or, respectively, in the region of the vessel floor there is generated a flat shaped "carpet" or, respectively "lawn" of cross linked carriers in case of an agglutination in the boundary layer. The antigen antibody reaction can thus be very simply detected optically by measuring of the spacial extension of the collection of the carriers. The sedimentation picture can visually be analyzed by an operating person or automatically on a photomatical way, on a fluorometric way or video controlled. Since the carrier collection essentially is measured from above or from below, there is given a particularly good automatizability of the method. In particular microtitration plates with a plurality of micro reaction vessels which are disposed in a plane, can be evaluated in a planar view from the top or from the bottom onto the plate in a single processing step. The clarity of the visible or, respectively, detectable reaction results depends among others from the selected form of the vessel. Therefore the vessel, at least in the region of the layer of the viscous substance, is formed conically narrowing and is furnished with an inclined running or inclined circulating or, respectively, conical wall, for -22 example a V -recess of a tipped floor microtitration plate or a bombed, rounded recess of a round floor microtitration plate, which in fact exhibits an inclined wall in its lower region, wherein the inclination can be in section an inclined plane or also a curved line. Good results are generally obtained in case the cross-sectional face of the vessel in the region of the filling height level of the viscous substance amounts to at least the fourfold of the cross sectional face of the vessel in the region below the viscous substance. The analysis is performed preferably photometrically relative to the situation if an antibody antigen reaction has taken place in a micro reaction vessel by measuring and recording the absorption of the mixture contained in the recess along a predetermined line and evaluating the thereby determined local absorption profile. This evaluation method is good suitable for the evaluation of a plurality of samples, for example a microtitration plate, in a single processing step and thus in an automatized form of the method. By measuring only a single absorption profile, the data wall you and to be stored and evaluated is decreased relative for example a video evaluation. Advantageously, the profile length is determined for a fixed to profile width. A positive reaction or, respectively, a negative reaction is then diagnosed, when the profile length is disposed below or, respectively, above a predetermined value. This evaluation method has proven to be particularly reliable. The micro-reaction vessel can also in particular in the region of the inclined wall of the micro-reaction vessel can be furnished with an additional coding such that a coding is disposed in the boundary -23 layer. This coding can comprise or, respectively contain in particular directed antibodies so-called catch antibodies as for example with anti-IgG or protein A and/or G with high bond affinity for immuno globulins or anti-IgM, complement, anti-C3d and so on. Such an additional coding is particular then of advantage, it reactions have to be detected, wherein the antibodies are specifically small as is the case for example by incomplete IgG-antibodies or in case of weak antibodies. A coding is not required in case of strong antibodies of the IgG-type as well as in case of complete antibodies, such as for example of the 1gM-type. Such an additional coding is in addition of advantage where free antibodies have to be captured away. Similarly combinations are conceivable of an additional coding of the inclined wall and of specific contents materials in the viscous substance; in principle a practically arbitrary coding of vessel wall and addition to the viscous substance are possible. Alternatively, the picture of the reaction vessels can be recorded by way of a video camera or a charge coupled device CCD camera for the like. Positive reactions can be distinguished from negative reactions by picture analysis. A further possibility for capturing the reaction pictures is the fluorescence marking of the carriers and the detection of the spacial emission of fluorescent light. Advantageously one of the liquids or the viscous substance contains albumine in a concentration of at least 0.01 mg per milliliter, preferably between about 0.2 mg per milliliter and 1 mg per milliliter -24 or another supplement. This serves for stabilizing the erythrocytes and full reducing the repulsive forces of the erythrocytes among each other, thereby a better cross linking is achieved. The diffusion speed of the erythrocytes through the viscous medium is reduced furthermore by addition of albumine and thus the sensitivity is increased. Similarly liss or bromealin or poly ethylene glycol or similar materials can be added, which have similar properties for advancing antigen antibody reaction. Incubation takes preferably place at about room temperature in case of a blood group determination. Thus incubators can be dispensed with which produce a constant increased ambient temperature. The automatization of the method is the thus further alleviated. The drawing shows in the figures 1A through 1E schematically illustrated reaction vessels in a longitudinal section, which are recesses in tipped floor microtitration plates. Figure 1 shows the schematically reaction vessels 1 in longitudinal section, which for example are furnished as recesses in tipped floor microtitration plates. The reaction vessels 1 are shaped cylindrically in an upper region and narrow conical in a lower region. The reaction vessels 1 contain up to a certain filling height level of viscous substance 2, in particular at gel. The surface of the substance 2 is curved and form a meniscus S caused by the casting in process, and for example swinging of the vessel after the filling in of the liquid gel and adhesion at the vessel wall. The gel layer showed at least be of -25 such thickness that the wall of the vessel is covered in the narrowing down region. On the other hand the thickness of the gel layer determines also the time until making visible of a reaction. Therefore a curved gel layer can be advantageous under certain circumstances, wherein the gel layer is as thin as possible at all locations. The size relationships illustrated are only schematically. A certain volume of the test liquid 3 is applied the viscous substance according to the process step 1.2, wherein the test liquid 3 contains carriers antigens and antibodies. The carriers, which are optically detected for identifying an antigen antibody reaction, are illustrated strongly exaggerated as points. Figure 1A showed the situation shortly after adding of the test liquid 3. The carriers 4 start to sediment in the upper region of the gel layer 2 as shown in figure lB. The carriers 4' have sunk down already in the middle region in the gel layer 2 as shown in figure 1C. Figure 1D shows two things: on the one hand a positive reaction and on the other hand where the carriers 4" are formed to a flat shaped agglutinate on the V-shaped floor and are thus stable. In case of a negative reaction, that is a not agglutinating reaction, the carriers 4' have reached the V-shaped floor and migrate (sediment) on the floor in the boundary layer to the lowest possible position of the micro-reaction vessel. A negative reaction of the carriers 4"' is illustrated in figure 1E, wherein possibly the formation of the reaction can be supported by centrifuging. Following to the gravity force/centrifugal force, the not agglutinated carriers collect -26 at the lowest point of the vessel to a spacially narrowly delimited structure 4"' wherein the station narrowly delimited structure 4' can be easily optically identified and can be distinguished relative to the flat agglutinates according to figure 1D. Of course the illustrations are only schematically, because the sedimentation process runs continuously and nested into the other. Embodiments are described in the following: Micro-reaction vessels are prepared with the viscous substance, which is agarose in this case for preparing the performance of the Invention method, that is the micro- reaction vessels are coated. For this purpose one proceeds advantageously in the following ways: 53 milligrams agarose, for example agarose number A 7299 type XII of the company Sigma, are completely dissolved in 100 ml isoftonic phosphate buffered rock salt solution (for exampleiSuwasol, company Ortho, pH value about 7.45 OrPBS, pH 7.4) at a temperature of about 95 degrees centigrade under still ring. The degenerated gel exhibits a gel concentration of about 53 milligrams per hundred milliliters under these conditions. About 20 micro-liters of the gel solution is pipetted in each cavity of a tipped floor microtitration plate at temperatures from about SO to 60 degrees centigrade. The plate is then uniformly shaken and is allowed to gel in a refrigerator at temperatures from about four degrees centigrade eight degrees centigrade. The gel forms a layer which covers the interior wall of the vessel. The plate prepared in this manner is not perishable for time period of at least two to three months when covered and cooled.

-27 1 mg to 2.5 grams of the recited agarose can be dissolved in hundred milliliters liquid in order to vary the desired gel concentration. A gel produced from 61.5 milligrams agarose per hundred ml PBS (phosphate buffered saline) with the pH 7.4 has proven to be good in practical situations. Initially in each case a defined volume, for example 25 microliters of an isoftonic rock salt solution in a ratio of 1:3.3 of a thinned EDTA plasma, citrate plasma or serum of a donor is pipetted into four mixing vessels for performing a method for blood group determination, in this case the serum counter test. The thinning down ratio is adapted to the photometrical valuation based on the proper coloration of the se-um; in case of a visual evaluation or a video evaluation a lesser thinning down can be employed are to an undiluted serum or, respectively, plasma. A certain amount, for example 25 microliters of one percent to 4 percent by volume of test erythrocytes of the groups Al, A2, B and 0, which are in this case the carriers of the specific binding partners antigens Al, A2, B and 0 -are distributed onto the four mixing vessels and are mixed. In the following this test solution is pipetted into the micro-reaction vessels, which are in this case the cavities of the microtitration plate. The thinning down medium of the test erythrocytes can contain preferably beef albumin in a concentration of her from about 2 milligrams to 150 milligrams and in this case 100 milligrams relative to 100 milliliters of physiological rock salt solution NaCl. Alternatively, the patient serum can be added undiluted to the thinned down erythrocyte solution, whereby a processing step can be dispensed with.

-28 After about an hour incubation time at room temperature the result can be evaluated photometrically, by measuring the absorption of radiation in the red region or, respectively, in the infrared region as a function of the location of the profile. A refusal rate of less than 1 percent was determined in a fully automated method under these reaction conditions. A still better identification succeeds upon visual evaluation. The method can be automated with high put through values because commercially available microtitration plates usually exhibit 96 cavities. A Coombs test (antibody search test/anti-globulin test) can be constructed without washing based on this foundation by adding in excess anti-C3d and anti-lgG to the agarose mixture (for the production of the gel layer at the vessel wall) The mixture of search and find cell (specific antigen and carrier erythrocyte) and the donor serum are pipetted onto the gel. The irregular antibodies take position at the search and find cell, wherein it incubation at the temperature of about 37 degrees centigrade occurs. The charged search and find cells take anti-IgG during the following sedimentation and sediment through the gel or, respectively the viscous layer braked on the inclined wall of the plastic layer, where the search and find cells is then _and thus in the boundary layer form a lawn. The method can also be employed for the detection of IgG molecules by adding protein A -a surface protein of the bacteria cell wall of many strains of staphylococcus aureous -- or protein G -- a protein of the bacteria cell wall of streptococcus --. Protein A and protein G are -29 very stable against high temperatures and bind many immuno globulins; protein G has a high bond affinity especially relative to immuno globulins of the class G (IgG). Anti-IgM can be added for capturing of irregular 1gM anti-bodies. The reaction occurs in the boundary layer between the plastic wall, possibly also the plastic floor, of the micro-Y reaction vessel and the viscous material according to the present Invention method, wherein two principals can be performed. According to the first principal the method is performed without washing -and in particular without centrifuging .The result can be read out after approximately one hour. According to the second principal a centrifugation step _again without washing -can be added, wherein the centrifugation step accelerates the making visible of the formation of the negative reaction. A lawn formation in each case indicates here a positive reaction, and a button formation indicates a negative reaction. The addition of Coombs serum, which contains anti-lgG, which leads to a visible agglutination, is particularly helpful in case of incomplete antibodies, which otherwise would show no reaction. However, Coombs serum does not reduce the repulsing forces. The liquid or, respectively, the liquids should be added free of bubbles to the viscous substance, which can be acoomplished in case of a fully automatically performed method by a pipetting device with the suitable mechanical control, for example a pipetting robot. According to an advantageous embodiment of the invention the gel is an organic gel, in particular agarose and/or gelatin. These gels are -30 associated with the advantage that they can be very easily composed, in particular in connection with throwaway microtitration plates. The gels can be furthermore stored in a good way, they are stable and over short time periods relatively insensitive to changes in the ambient temperature. In particular excellent results have been obtained with agarose of a gel concentration of about 40 milligrams per hundred milliliters up to 18 milligrams per ml during the performing of serum counter tests and antibody (Ak) search and find tests for blood group determination. Microtitration plates such prepared with agarose can be stored under cooling for several months. Also the finished prepared samples can be stored under cooling for several weeks, wherein the reaction pictures remain stable. The reaction results therefore can be drawn over a longer time period after performing of the test for control purposes. The carriers, to which the antigen or, respectively, antibodies or the specific binding partners to be detected are bound, are preferably erythrocytes, leucocytes, blood platelets or latex particles. The latex particles can be visibly dyed or marked by fluorescence for a simpler optical evaluation. The concentration of the carriers in the reaction liquid is to be selected such that the agglutinate can be detected reliably with the selected optical detection method. In accordance with the present invention the sedimentation picture is evaluated optically by a top planar view from above or below onto the reaction vessel. In case the measurement takes place from below then the micro-reaction vessel has to be transparent. The micro reaction vessel is preferably also transparent if the evaluation takes place from above in order to increase the contrast of the carriers -31 relative to the background. Based on the different vessel cross sections in the upper region of the viscous substance and in the lower region of the viscous substance or, respectively, in the region of the vessel floor there is generated a flat shaped "carpet" or, respectively "lawn" of cross linked carriers in case of an agglutination in the boundary layer. The antigen antibody reaction can thus be very simply detected optically by measuring of the spacial extension of the collection of the carriers. The sedimentation picture can visually be analyzed by an operating person or automatically in a photometrical way, or in a fluorometric way or can be video controlled Since the carrier collection essentially is measured from above or from below, there is given a particularly good automatizability of the method. In particular microtitration plates with a plurality of micro-reaction vessels which are disposed in a plane, can be evaluated in a planar view from the top or from the bottom onto the plate in a single processing step. The clarity of the visible or, respectively, detectable reaction result depends among others on the selected form of the vessel. Therefore the vessel, at least in the region of the layer of the viscous substance, is formed conically narrowing and is furnished with an inclined running or inclined circulating or, respectively, conical wall, for example a V-recess of a tipped floor microtitration plate or a bombed, rounded recess of a round floor microtitration plate, which in fact exhibits an inclined wall in its lower region, wherein the inclination can be when seen in section an inclined plane or also a curved line. Good results are generally obtained in case the cross sectional face of the vessel in thc region of the filling height level of -32 the viscous substance amounts to at least the fourfold of the cross sectional face of the vessel in the region below the viscous substance. The analysis is performed preferably photometrically relative to the situation if an antibody antigen reaction has taken place in a micro reaction vessel by measuring and recording the absorption of the mixture contained in the recess along a predetermined line and evaluating the thereby determined local absorption profile. This evaluation method is good suitable for the evaluation of a plurality of samples, for example a microtitration plate, in a single processing step and thus in an automatized form of the method. By measuring only a single absorption profile, the data volume to be stored and evaluated is decreased relative for example to a video evaluation. Advantageously, the profile length is determined for a fixed to profile width. A positive reaction or, respectively, a negative reaction is then diagnosed, when the profile length is disposed below or, respectively, above a predetermined value. This evaluation method has proven to be particularly reliable. The micro-reaction vessel can also -- in particular in the region of the inclined wall of the micro-reaction vessel -- be furnished with an additional coding such that a coding is disposed in the boundary layer. This coding can comprise or, respectively contain anti-globulin - antibodies directed against immunoglobulins -- , so-called "catch antibodies" as for example with anti-IgG or protein A and/or G with high bond affinity for immuno-globulins or anti-IgM, complement, anti C3d and so on. Such an additional coding is particular then of -33 advantage, if reactions have to be detected, wherein the antibodies are specifically small as is the case for example with incomplete IgG antibodies or in case of weak antibodies. A coding is not required in case of strong antibodies of the IgG-type as well as in case of complete antibodies, such as, for example, of the 1 gM-type. Such an additional coding is also of advantage where free antibodies have to be captured away. Similarly combinations are conceivable of an additional coding of the inclined wall and of specific contents materials in the viscous substance; in principle a practically arbitrary coding of vessel wall and addition to the viscous substance are possible. Alternatively, the picture of the reaction vessels can be recorded by way of a video camera or a charge coupled device CCD camera or the like. Positive reactions can be distinguished from negative reactions by picture analysis. A further possibility for capturing the reaction pictures is the fluorescence marking of the carriers and the detection of the spacial emission of fluorescent light. Advantageously one of ithe liquids or the viscous substance contains albumine in a concentration of at least 0.01 mg per milliliter, preferably between about 0.2 mg per milliliter and 1 mg per milliliter or another supplement. This serves for stabilizing the erythrocytes and full reducing the repulsive forces of the erythrocytes among each other, thereby a better cross- linking is achieved. The diffusion speed of the erythrocytes through the viscous medium is reduced furthermore by addition of albumine and thus the sensitivity is increased.

-34 Similarly liss or bromealin or poly ethylene glycol or similar materials can be added, which have similar properties for advancing the antigen antibody reaction. Incubation takes preferably place at about room temperature in case of a blood group determination. Thus incubators can be dispensed with which produce a constant increased ambient temperature. The automatization of the method is thus further alleviated. The drawing shows in the figures 1A through 1E schematically illustrated reaction vessels in a longitudinal section, which are recesses in tipped floor microtitration plates. Figure 1 shows schematically reaction vessels 1 in longitudinal section, which for example are furnished as recesses in tipped floor microtitration plates. 'The reaction vessels 1 are shaped cylindrically in an upper region and narrow conically in a lower region. The reaction vessels 1 contain up to a certain filling height level of viscous substance 2, in particular a gel. The surface of the substance 2 is curved and forms a meniscus 5 caused by the casting in process, and for example swinging of the vessel after the filling in of the liquid gel and adhesion at the vessel wall. The gel layer should at least be of such thickness that the wall of the vessel is covered in the narrowing down region. On the other hand the time until the making visible of a reaction plays a part in the thickness of the gel layer. The illustrated size ratios are only schematically. A certain volume of the test fluid 3 is applied onto the viscous -35 substance in the process step 1.2, which contains carriers, antigens and antibodies. The carriers, which are optically detected for identifying an antigen antibody reaction, are illustrated strongly exaggerated as points. Figure 1A showed the situation shortly after adding of the test liquid 3. The carriers 4 start to sediment in the upper region of the gel layer 2 as shown in figure lB. The carriers 4i have sunk down already in the middle region in the gel layer 2 as shown in figure 1C. Figure 1D shows two things: on the one hand a positive reaction and on the other hand where the carriers 47 are formed to a flat shaped agglutinate on the V-shaped floor and are thus stable. In case of a negative reaction, that is a not agglutinating reaction, the carriers 4' have reached the V-shaped floor and migrate (sediment) on the floor in the boundary layer to the lowest possible position of the micro-reaction vessel. A negative reaction of the carriers 4"' is illustrated in figure 1E, wherein possibly the formation of the reaction can be supported by centrifuging. Following to the gravity force/centrifugal force, the not agglutinated carriers collect at the lowest point of the vessel to a spacially narrowly delimited structure 4"', wherein the spacially narrowly delimited structure 4' can be easily optically identified and can be distinguished relative to the flat agglutinates according to figure 1D. Of course the illustrations are only schematically, because the sedimentation process runs continuously and nested into each other.

-36 Embodiments are described in the following: Micro-reaction vessels are prepared with the viscous substance, which is agarose in this case for preparing the performance of the invention method, that is the micro-reaction vessels are coated. For this purpose one proceeds advantageously in the following ways: 53 milligrams agarose, for example agarose number A 7299 type X11 of the company Sigma, are completely dissolved in 100 ml isotonic phosphate buffered rock salt solution (for example "Suwasol", company Ortho, pH value about 7.45 or PBS, pH 7.4) at a temperature of about 95 degrees centigrade under stirring. The generated gel exhibits a gel concentration of about 53 milligrams per hundred milliliters under these conditions. About 20 micro-liters of the gel solution is pipetted into each cavity of a tipped floor microtitration plate at temperatures from about 50 to 60 degrees centigrade. The plate is then uniformly shaken and is allowed to gel in a refrigerator at temperatures from about four degrees centigrade eight degrees centigrade. The gel forms a layer which covers the interior wall of the vessel. The plate prepared in this manner is not perishable for a time period of at least two to three months when covered and cooled. 1 mg to 2.5 grams of the recited agarose can be dissolved in hundred milliliters liquid in order to vary the desired gel concentration. A gel produced from 61.5 milligrams agarose per hundred ml PBS (phosphate buffered saline) with the pH 7.4 has. proven to be good in practical situations.

-37 Initially in each case a defined volume, for example 25 microliters of an isotonic rock salt solution in a ratio of 1:3.3 of a thinned EDTA plasma, citrate plasma or serum of a donor is pipetted into four mixing vessels for performing a method for blood group determination, in this case the serum counter test. The thinning down ratio is adapted to the photometrical evaluation based on the proper coloration of the serum; in case of a visual evaluation or a video evaluation a lesser thinning down can be employed up to an undiluted serum or, respectively, plasma. A certain amount, fcr example 25 microliters of one percent to 4 percent by volume of test erythrocytes of the groups Al, A2, B and 0, which are in this case the carriers of the specific binding partners -antigens Al, A2, B and 0 _are distributed onto the four mixing vessels and are mixed. In the following this test solution is pipetted into the micro-reaction vessels, which are in this case the cavities of the microtitration plate. The thinning down medium of the test erythrocytes can contain preferably beef albumin in a concentration of from about 2 milligrams to 150 milligrams and in this case 100 milligrams relative to 100 milliliters of physiological rock salt solution NaC. Alternatively, the patient serum can be added undiluted to the thinned down erythrocyte solution, whereby a processing step can be dispensed with. After about an hour of incubation time at room temperature the result can be evaluated photometrically, by measuring the absorption of radiation in the red region or, respectively, in the infrared region as a function of the location of the profile.

-38 A refusal rate of less than 1 percent was determined in a fully automated method under these reaction conditions. A still better identification succeeds upon visual evaluation. The method can be automated with high put through values because commercially available microtitration plates usually exhibit 96 cavities. A Coombs test (antibody search test/anti-globulin test) can be constructed without washing, based on this foundation, by adding in excess anti-C3d and anti-IgG to the agarosemixture (for the production of the gel layer at the vessel wall) The mixture of search and find cell (specific antigen and carrier erythrocyte) and the donor serum are pipetted onto the gel. The irregular antibodies take position at the search and find cell, wherein an incubation at the temperature of about 37 degrees centigrade occurs. The chargecI search and find cells take anti-IgG during the following sedimentation and sediment through the gel or, respec t ively the viscous layer braked on the inclined wall of the plastic layer, where the search and find cells then and thus in the boundary layer -form a lawn. The method can also be employed for the detection of IgG molecules by adding protein A -- a surface protein of the bacteria cell wall of many strains of staphylococcus aureous -- or protein G -- a protein of the bacteria cell wall of streptococcus -- Protein A and protein G are very stable against high temperatures and bind many immuno globulins; protein G has a high bond affinity especially relative to immuno globulins of the class G (IgG). Anti-IgM can be added for capturing of irregular 1gM anti-bodies.

-39 The reaction occurs in the boundary layer between the plastic wall, possibly also the plastic floor, of the micro- reaction vessel and the viscous material according to the present invention method, wherein two principals can be performed. According to the first principal the method is performed without washing -- and in particular without centrifuging --. The result can be read out after approximately one hour. According to the second principal a centrifugation step -- again without washing -- can be added, wherein the centrifugation step accelerates the making visible of the formation of the negative reaction. A lawn formation in each case indicates here a positive reaction, and a button formation indicates a negative reaction. An additional coding of the inclined wall of the micro-reaction vessel can be performed in both cases, wherein the coding is to be performed prior to the coating of the vessel with the viscous material. The substances employed for the coding are here applied in a solid way (covalent or in physical bond) onto the wall, only in the following the viscous substance is filled into the vessel. In particular codings with protein A or G or mixture of protein A and G, for example with the mixing ratio 1:1, or with anti-globulins, antibodies directed against immuno globulins such as for example anti-IgG are to be considered for the purpose of performing a Coombs test, wherein the employed microtitration plates exhibit a high binding force to proteins. In case of coding of the inclined to vessel wall with anti- IgG the procedure is preferably as follows: 1 ml anti-IgG Rabbit is mixed with 19 milliliters carbonate bicarbonate buffer pH 9.6 which corresponds -40 to a thinning of 1:20. In each case 50 microliters of the mixture are pipetted into a cavity of a tipped floor microtitration plate, for example and ELISA plate (company Greiner, Germany) and left standing overnight at room temperature. In the following three times washing is performed with 150 microliters PBS-buffer, pH 7.1, for example with PBS -buffer; anti-IgG adheres solidly at the wall. Twenty-five microliters gel are pipetted into each cavity preferably at about 52 degrees centigrade for the following coating of the vessel with the viscous substance, for example gel of the 61.5 milligrams agarose (type XIII, low viscosity, Co. Sigma) per hundred ml PBS buffer pH 7.4. Such a treated tipped floor or round floor microtitration plate can be stored at the temperature of 4 to 8 degrees centigrade. An additiona! coding of the micro-reaction vessels is performed in connection with a further embodiment prior to the coating with viscous substance, for example with the protein A and/or protein G. For this purpose a mixture of one miiligram protein A and/or protein C per hundred ml PBS buffer, pH 7.1 is produced. In each case 50 microliters of this mixture with a concentration of 0.01 milligrams per ml per cavity of a tipped floor microtitration plate, for example ELISA plates of the company Greiner, Germany are pipetted in and are incubated at room temperature overnight. In the following three times washing is performed with the PBS buffer. In the following the cavities- as precedingly described at the additional coding with anti IgG- with gel or another viscous material- coated. Also a modified Liss buffer or buffer with proteins can be employed instead of the PBS for the dissolution of the gel.

-41 Antibodies or protein as well as a further coding substances can be adhesively attached the wall of the micro-reaction vessel for coding of the vessel wall, then a coating with the viscous material is performed. These two microtitration plates for the investigation of irregular antibodies are composed as follows in accordance with the further embodiment. An about 0.6 vols. pct. search and find cells suspension from commercially available search and find cells of about 3 vols. pct. as follows: a mixture of one part 3 vols. pct. search and find cell suspension 4 2 parts modified Liss solution + 2 parts modified bromealin. The serum to be investigated is thinned down: 1 part serum + 1 part Liss solution. Now twenty-five microliters of the thinned serum plus twenty-five microliters of the about 0.6 vols. pct. use search and find cell suspension are mixed and are pipetted onto the gel or, respectively, the viscous substance. In the following one hour long at 37 degrees centigrade incubation takes place and then the result is read and evaluated. Commercial applicability: the method according to the invention is particularly suitable for an anti-body search and find test, for a serum counter test, for blood group determination on the erythrocyte side (ABO and RH formula), as well as a cross match test. Each antigen antibody reaction, which is determinable over particles, can be performed with the method. The method is in particular commercially applicable in hospitals, in medical laboratories or in installations for blood and plasma recovery. The particular usefulness of the method -42 is present in the fully automatic performability of the method. List of reference numerals: 1 micro-reaction vessel 2 viscous substance or gel layer 3 test liquid or liquid test mixture 4, 4 ', 4", 4'" carrier 5 meniscus

Claims (21)

1. 1. A micro-reaction vessel is employed with a narrowing cross section from top to bottom, which micro-reaction vessel contains a viscous substance; 1. 2. A predetermined volume of the test liquid is added onto the viscous substance the vessel, wherein the specific binding partner is added either to the test liquid or to the viscous substance, or a further liquid, which contains the specific binding partner, is added prior to or after adding of the test liquid to the vessel; 1. 3. The forming or formed sediment/agglutinate is evaluated in a planar view from the top or from below onto the micro-reaction vessel, 1. 4. Wherein a flat shaped agglutinate (lawn formation) of antigen or, respectively, antibodies, binding partners and carriers points to a -44 positive antigen antibody reaction and a spatially slightly extended deposit (button formation) of carriers points to a negative reaction with the absence of an antigen /antibody complex.

2. Method according to claim 1 characterized in that 2.1. The reaction is performed in a micro-reaction vessel open on the top and closed at the bottom,, wherein the micro-reaction vessel exhibits at least in its region close to the floor a narrowing down cross-section, such that the vessel has at least partly inclined wall at least in the region close to the floor, which inclined wal! transitions into the floor or, respectively forms the floor, 2.2. The viscous homogeneous substance, in particular a heated jelly or a heated gel cast and allowed to cool down at least in this floor close region with inclined wall of the vessel, such that the substance covers or, respectively, fills after the cooling down the region of the vessel with inclined wall including the floor as a homogeneous layer and thereby forms a cross linking or, respectively, a grid structure; 2.3. The predetermined volume of the test liquid is placed into the vessel onto the homogeneous layer of the viscous substance; 2. 4. The micro-reaction vessel is incubated for at least such a long time until the visible sediment/agglutinate forms out of the viscous substance alone based on gravity force in the boundary layer between the inclined wall of the micro-reaction vessel and the homogeneous layer out of the viscous substance, wherein in -45 particular a flat formed agglutinate layer on the inclined wall of the vessel points to a positive antigen anti-body reaction and wherein a spatially slightly extended deposit formed in particular in the region of the lowest point of the floor or, respectively, inclined wall of the vessel points to a negative reaction without antigen/antibody complex.

3. Method according to claim 1 or 2, characterized in that the analysis, in an anti-body antigen reaction has taken place in a micro-reaction vessel is performed optically by an operator or automatically by way of a photometer or by way of a video camera or a charge coupled device CCD-camera or by way of a fluorometer.

4. Method according to one of claim 1,2, or 3, characterized in that the analysis, in an anti-body antigen reaction has taken place in the micro-reaction vessel, is performed photometrically by measuring and recording the absorption of the mixture contained in the recess along a predetermined line and by evaluating the thus determined local absorption profile.

5. Method according to claim 3 or 4, characterized in that the profile length is determined for fixed profile width and a positive reaction or, respectively a negative reaction is diagnosed in case the profile length disposed below or, respectively, above a predetermined value.

6. Method according to claim 1 or 2 characterized in that the viscous substance is a gel, which was heated up to the melting -46 temperature and was filled into the micro-reaction vessel in a liquid state for forming of a homogeneous layer, wherein the gel cools down up to the gel point or, respectively into the neighborhood of the gel point.

7. Method according to claim 6 characterized in that gel exhibits a gel concentration of at least 0.02 mg per hundred milliliters, preferably in the region of the 0.1 mg per hundred milliliters to 6.66 grams per hundred milliliters (6666 mg) preferably between 10 mg per hundred milliliters and under 10 mg per hundred milliliters, particularly preferred between 40 mg per hundred milliliters and 80 mg per hundred mifliliters.

8. Method according to one of the preceding claims characterized in that the gel is an organic gel, in particular either agarose and/or gelatin.9. Method according to claim 6 or 8 characterized in that the viscous substance is formed of cross linked polymers.

10. Method according to one of the preceding claims characterized in that the carriers within the test liquid are erythrocytes, leucocytes, blood platelets, latex particles or agarose particles, glass particles or plastic particles.

11. Method according to one of the preceding claims characterized in that the cross-section of face of the vessel in the region of the filling -47 height level of the viscous substance amounts to at least a fourfold of the cross-section of face of the vessel in the region below the viscous substance.

12. Method according to one of the preceding claims characterized in that tipped floor or round floor microtitration plates are employed and that the micro- reaction vessel is a recess of a tipped floor or round floor microtitration plate.

13. Method according to claim 1 characterized in that incubation is performed according to the step 1.2, preferably at temperatures from at least five degrees centigrade, preferably between 10 and 37 degrees centigrade, in particular room temperature and for at least 15 minutes, preferably at least 50 minutes.

14. Method for performing of a serum counter test for detecting of blood group antibodies (irregular antibodies) according to one of the preceding claims characterized in that the test liquid contains blood serum, which is to be investigated relative to the presence of certain blood group antibodies, and wherein a liquid is added to the test liquid, wherein the liquid contains test erythrocytes of a known blood group, which are connected as carriers with specific binding partners for the antibodies or, respectively, iso-agglutinins to be detected.

15. Method for performing of blood group determination according to one of the preceding claims characterized in that the test liquid, which is to be investigated relative to the presence of certain -48 antigens contained erythrocytes with unknown antigen and wherein a liquid is added to the test liquid, which liquid contains known search and find antibodies.

16. Method according to claim 14, characterized in that albumine in the concentration of at least 0.01 mg per ml and preferably between 0.2 mg per ml and 1 mg per ml is added to one of the liquids or, respectively, the mixture or. the viscous substance.

17. Method according to claim 14 or 15 characterized in that proteins as protein A and/or protein G and/or antigens and/or antibodies are added to one of the liquids or, respectively, the mixture or to the viscous substance at the point in time of the investigation.

18. Method according to one of the preceding claims characterized in that the region of the inclined wall of the micro-reaction vessel is coded with capture materials, namely proteins, as protein A and/or G, and/or antigens and/or antibodies and only thereupon the coating of the micro-reaction vessel with the viscous substance is performed.

19. Method according to one of the preceding claims characterized in that various buffers and/or specific binding partners (detecting antigen or detecting anti-body, in particular monoclonal antibodies) and/or carriers are contained in the gel, which are added in a liquid heating state of the gel under the precondition of heat stability. -49

20. Use of tipped floor or round floor microtitration plates with a plurality of recesses, wherein a viscous substance is contained in the form of a homogeneous layer at least in the region of the V-shaped or bombed recess under formation of a boundary layer between the inclined wall of the micro-reaction vessel and the homogeneous layer out of the viscous substance for performing a test for detecting of antibodies or antigens by detecting and agglutination reaction or for blood group determination.

21. Use of tipped floor or round floor microtitration plates with a plurality of recesses wherein capturing materials, namely protein, as protein A and/or protein G and/or antigens and/or antibodies are applied ir the plurality of recesses at least in the region of the V shaped or bombed recesses as a coding.

22. Use of tipped floor or round floor microtitration plates with a plurality of recesses. wherein capturing materials, namely protein, as protein A and/or protein G and/or antigens and/or antibodies are applied as a coding at least in the region of the V-shaped or bombed recesses and wherein a viscous substance in the form of a homogeneous layer is contained in the recesses of the mitral reaction vessels at least in the region of the V-shaped or bombed recesses under formation of a boundary layer between the inclined wall of the micro-reaction vessel and the homogeneous layer out of the viscous substance for performing a test for detecting of antibodies or antigens or for blood group determination by detection of an agglutination reaction.