CA2495728C

CA2495728C - Method for the detection of antibodies and/or antigens in a test liquid, particularly for determining the blood group

Info

Publication number: CA2495728C
Application number: CA2495728A
Authority: CA
Inventors: Joerg Spindler
Original assignee: Deutsches Rotes Kreuz Blutspendendienst Baden-Wurt Gemeinnu GmbH
Current assignee: Deutsches Rotes Kreuz Blutspendendienst Baden-Wurt Gemeinnu GmbH
Priority date: 2002-08-23
Filing date: 2003-08-13
Publication date: 2012-08-21
Anticipated expiration: 2023-08-13
Also published as: CA2495728A1; EP1532454A2; AU2003260413A1; WO2004019038A3; DE10239568A1; WO2004019038A2; AU2003260413A8

Abstract

The invention relates to a method for detecting antibodies or antigens in a test liquid by means of a reaction with a given specific bonding partner. According to the inventive method, the antigens are tied to a carrier, and the resulting sample mixture is subjected to an incubation step and then centrifuged by means of a particle-buffer mixture. An agglutinate comprising the antigen that is to be detected, the carrier, and antibodies is formed in case of a positive antigen-antibody reaction. A predefined quantity of the incubated sample mixture is added to a predefined quantity of a buffer-particle mixture which is disposed inside a detection receptacle. The buffer-particle mixture is provided with an embedding buffer consisting of an aqueous solution of dextran and/or polyethylene glycol, glycine, and/or trisodium citrate.

Description

Method for the detection of antibodies and/or antigens in a test liquid, particularly for determining the blood group The invention relates to a method for the detection of antibodies and/or antigens, particularly for blood group determination, antibody search test, serum check test and infection serology, in a test liquid by reaction with a given specific binding partner, the antigens or the antibodies or the specific binding partners in the test liquid being unbound and/or bound to a carrier, and the sample mixture prepared in this manner being subjected to an incubation step, and, in the case of a positive antigen-antibody reaction, an agglutinate being formed from antigens or antibodies to be detected or the specific binding partners and the carriers, which agglutinate is optically detectable as a sedimentation image.

Human blood groups, but also a large number of other substances formed in an organism, are detected by specific antigen-antibody reactions This reaction is detectable through crosslinking, i.e. agglutination, of antigens (Ag), antibodies (Ab) and optionally certain carrier substances which carry antigens or antibodies to form a macroscopic optically detectable complex, the agglutinate. This may be a three-dimensional agglutinate or so-called monolayer, which is frequently observable when the surfaces of the test plates are coated (coded) with antibodies.

Examples are the erythrocyte blood group determination and the serum check test. In the first-mentioned one, wO 2004/019038 2 PCT/EP2003/008995 blood group antigens bound to the membrane of the donor erythrocytes are detected by reaction with antisera which contain antibodies as specific binding partners.
The antibodies which are present in a test liquid are known (search antibodies), for example test antibody suspension and the unknown erythrocytes with unknown antigens are added. The agglutination is caused by reaction of antigen and antibody. Consequently, the system ABO and RH and also a large number of antigens can be determined.

In the serum check test, the erythrocytes which have known antigens (search antigens) are known. The test liquid, for example serum, contains the unknown endogenous antibodies (so-called isoagglutinins), which are detected by reaction with the test erythrocytes.
This also makes it possible to determine the blood group, but only ABO (Biotest, Lexikon der Immunologie [Biotest, Lexicon of Immunology], 2nd Edition, Medical Service Munich, pages 36, 37).

This technique works with unmodified microtitre plates having a round bottom, a series of different antisera, in particular anti-A, anti-B, anti-D and rhesus control serum, for determining the blood group in the ABO
system, being pipetted into the wells of the plate, and the erythrocytes to be tested being added in dilute form. After an incubation time of at least 20 minutes, the plate is centrifuged and carefully shaken. The total processing time is about 40 minutes. A positive antigen-antibody reaction is visible as agglutinate, and a negative reaction is evident as an erythrocyte suspension which, when left to stand, settles out to give a knob on the bottom of the test plate. The serum check test works in a similar manner.

The method can be automated only by a complicated procedure and is often not unambiguous. The reliability of the detection of a positive reaction depends on the strength of the antibody-antigen bond: in the case of weak bonds, the agglutinated erythrocytes can be separated again by external disturbances, in particular by shaking, and thus incorrectly diagnosed as a negative reaction. For this reason, owing to the possible lack of a clear reaction picture, the automatic distinction between positive and negative reaction, for example by means of automatic photometric evaluation, is virtually impossible and must be carried out by an experienced operator or at least visually checked and is therefore very time-consuming.

EP 0 363 510 A discloses the so-called solid phase technique for finding irregular antibodies, which is based on coded microtitre plates without gel. This requires multiple complicated washing and centrifuging steps. The total processing time, incubation for 20 min, centrifuging and washing steps, is about 40 min. The positive results can be seen as monolayer (lawn), and the negative results as a knob on the bottom of the plate. As a result of these steps and the long duration, the flexibility of the processing of the samples and hence automation is poor. Moreover, the ABO-Rh and serum check test can be worked through by this technique only to a limited extent and with considerable effort.

EP 0 305 337 Al has disclosed, for the detection of antigens or antibodies, a method for the optical visualization of complexes of carrier-bound antigens with antibodies in the reaction vessels, which is referred to as the ID gel technique. This uses six parallel columns filled with gel beads, in the form of a card. In the upper region above the column, a relatively large container serving for holding the sample is present. Between the lower column filled with gel and the upper container, an air cushion must remain on filling with the sample; an incubation phase which comprises standing for 15 min at 37 C is effected; the subsequent centrifuging time of the card is 10 minutes at 85 x g. The result is read by viewing the column laterally. In the case of a positive reaction, the agglutinates are present on the gel of the column or are distributed in the form of flocks in the gel of the column. In the case of a negative reaction, the erythrocytes are present in compact form at the bottom of the column. A reduction of the incubation time is not possible because the samples are not permitted to be mechanically agitated, because otherwise the sample would come into contact directly with the gel during the incubation phase and a false negative reaction would thus be indicated. Furthermore, the g number cannot be increased and hence the centrifuging time cannot be decreased, because otherwise a false negative reaction would likewise be indicated. Moreover, separate cards are necessary for the serum check test and ABO-RH system just as separate columns of a card are required for distinguishing between, for example, IgG and IgM. Owing to the still long incubation and centrifuging time and the card form and separate columns for the individual determinations, this technique is not suitable for full automation.
Furthermore, the sending of the cards to the customers is problematic since, if the columns are turned upside down, the gel can run out of said columns. It should also be mentioned that pipetting of the gel subsequently into the column is problematic owing to the high viscosity and the adhesion thereof.

EP 0 849 595 B1, which is based on EP 0 305 337 Al, states that (quote) "commercially available standard particles having a diameter of 3-7.5 m settle out only incompletely into the gel matrix, whereas larger, 11.9 m, or smaller synthetic particles < 1 m could penetrate only slightly into the gel. An increase in the parameter of centrifuging time from 10 min to 50 min or centrifuging speed from 1030 to 1300 rpm resulted in better but still incomplete sedimentation.
Even if it had been possible to achieve optimum sedimentation by these two measures, a change/increase in the centrifuging time and centrifuging speed is in fact very disadvantageous for the following reasons:
1. The method of EP 0849595 B1 is intended to permit a method which takes a particularly short time in comparison with comparable methods. In the case of a possible centrifuging time of 50 min, the desired test duration of 20 minutes, including incubation, would thereby triple. 2. As is known to the person skilled in the art, the sensitivity in the gel centrifuging method decreases with increasing centrifuging speed" (end of quote). For detection of the reaction, centrifuging for 10 minutes at 85 g is used.

An advantage of the invention consists in providing a novel method for the ABO and RH determination, for the serum check test and for the antibody search test, which method meets the modern requirements of the market, namely to provide flexible and rapid automatic processing of the samples via a fully automatic system with emergency input for patients and donors. For this purpose, the processing time has to be as short as possible, which means that the incubation time may be not more than 5 to 7 minutes, as far as possible separately in a plate, and the centrifuging time may be 2 to 4 minutes, likewise separately in a reaction plate, which must also meet the requirements that they can be individually loaded. It is also advantageous if the test apparatus can be easily loaded with the individual components in a simple manner, for example shortly before the investigation.

Moreover, no washing steps are required. In order to be able to give preference to emergency patients, it is necessary, in the case of emergency input, additionally to ensure that the incubating patient sera or plasmas which have no priority can incubate for a relatively long time, up to about 40 minutes, without adversely affecting the results, so that an emergency patient can be quickly handled in between; the normal routine is then resumed. It is also necessary for the processing of the samples to be capable of being carried out with, as far as possible, the same base reagents, and the easy shippability of the reagents must also be ensured.
it is also advantageous that the detection method can be easily adapted to the specific circumstances.

While avoiding the disadvantages of the prior art, the method according to the invention therefore provides a method for the detection of antibodies and/or antigens, such as for blood group determination, antibody search test, serum check test and infection serology, with high sensitivity, which can be carried out in a simple manner and can be effected with a high degree of automation. The method leads, in particular in a very short reaction time, to very reliable test results of the samples, and as far as possible also to automatic processing of the samples by means of an automatic system. Emergency inputs of investigation samples of emergency patients are also possible without there being any impairment of the other samples as a result.
In the method according to the invention, a predetermined amount of the incubated sample mixture is added to a predetermined amount of a mixture of a buffer, in particular an embedding buffer, with particles, which is referred to below as buffer-particle mixture, abbreviated to PP mixture. This PP
mixture is present within a detection vessel, the embedding buffer having the property of minimizing the distance between the individual particles within the PP
mixture. Moreover, the embedding buffer ensures that the PP mixture has a low viscosity, which is essential for problem-free pipetting. The detection vessel is then subjected to centrifuging, which leads to visualization of the reaction of the sample mixture in the detection vessel, the PP mixture promoting the positive reaction.

In an advantageous embodiment of the method, the embedding buffer consists of an aqueous solution of dextran [ (C6H10O5) n ] and/or polyethylene glycol [HO (C2H4O) nH] , laboratory water (bidistilled water) advantageously being used for the preparation of all aqueous solutions. In a further advantageous embodiment of the method, glycine (H2NCH2COOH) and/or trisodium citrate (C6H5O7Na3) are added to the aqueous solution of dextran [ (C6H100O5) n] and/or polyethylene glycol [HO(C2H4O),,H] .

The present invention further provides a method for the detection of antibodies, antigens, or both, in a test liquid by reaction with a given specific binding partner, the antigens being bound to a carrier, and a sample mixture prepared in this manner being subjected to an incubation step and then being centrifuged through a particle-buffer mixture, in the case of a positive antigen-antibody reaction an agglutinate of antigen or antibody to be detected and carrier being formed, which agglutinate is optically detectable as a sedimentation image, and that a predetermined amount of incubated sample mixture is added to a predetermined amouno of a buffer-particle mixture which is present within a detection vessel, the buffer-particle mixture comprising an embedding buffer, characterized in that the embedding buffer consists of an aqueous solution of:
(a) (i) 0.1 to 500 g/l of dextran, (ii) polyethylene glycol, (iii) glycine, and (iv) trisodium citrate ;
(b) (i) 0.1 to 500 g/l of dextran, (ii) glycine, and (iii) trisodium citrate; or (c) (i) polyethylene glycol, (ii) glycine, and (iii) trisodium citrate.

8a In a preferred embodiment of the method, a high molecular weight dextran is used for the embedding buffer. The dextran has a molecular weight approximately of 100 to 40,000,000 g/mol, preferably of 2,000,000 (2-10 ") g/mol, and is present in the embedding buffer in a range from 0.1 g/l to 500 g/l, preferably 5 g/1 to 40 g/l, more preferably of 18 g/1 to 22 g/l and in particular of 20 g/l.

In a further embodiment of the method, the polyethylene glycol has a molecular weight approximately of 50 to 40,000 g/mol, preferably 3500-4500 g/mol, and is present in the embedding buffer in a range from 1 g/l to 100 g/l, preferably from 10 g/l to 70 g/l, more preferably from 35 g/1 to 45 g/l and in particular 40 g/l.

In a further embodiment of the method, the glycocoll (glycine) is present in the embedding buffer in a range from 1 g/l to 100 g/l, preferably 10 g/1 to 25 g/l, more preferably 16 g/l to 20 g/1 and in particular 18.0168 g/l.

In a further embodiment of the method, the trisodium citrate (C6H507Na3) is present in the embedding buffer in a range from 0.01 g/l to 10 g/l, preferably from 1 g/1 to 5 g/l, more preferably from 2 g/l to 3 g/l and in particular 2.581 g/l. In the case of the trisodium citrate, the dihydrate can be used. The amounts stated above relate to the anhydrous form. If hydrated forms are used, the amounts increase correspondingly.

In a further embodiment of the method, the embedding buffer may be present in a pH range from 4 to 9, preferably from 6.9 to 7.1, in all variants.

Sodium azide (NaN3) is preferably added for stabilizing the PP mixture and is present in a range from 0.01 g/litre to 10 g/l, preferably from 0.5 g/l to 2 g/l, in particular 1 g/1 (corresponding to 0.1%). An antibacterial and/or antiviral agent, such as sodium azide (NaN3), or antibiotics are preferably added to the embedding buffer for stabilization. Consequently, the embedding buffer is preservable and stable for a long time, for example 1 year. The buffer can be stored at room temperature, but it should preferably be stored at refrigerator temperatures for long storage times.

In a particularly preferred embodiment, an embedding buffer is prepared as follows:
First, 0.24 M (18.0168 g/1) glycine and 0.01 M
(2.581 g/1) trisodium citrate (C6H507Na3) are made up to 1 litre with bidistilled H20. A pH between 6.9 and 7.1 results. 20 g of dextran (2.0%) having a molecular weight of 2 x 106 are then added to this buffer solution and dissolved by stirring. Furthermore, 40 g/l (4%) of polyethylene glycol having a molecular weight of about 4000 (PEG
4000) and 1 g of sodium azide (0.1 %) are dissolved. A pH of 7.1 is then established.

In a preferred embodiment of the method, beads are used as particles 5 within the buffer-particle mixture. Preferably used beads are acrylic, gelatin, polyacrylamides, solid nets, silica, FicollTM, PercollTM, phthalate esters, agarose or dextran gel. The particles, preferably the beads, have a diameter of 1 pm to 300 pm, preferably a dry bead diameter of 1 Jim to 300 pm. In a preferred embodiment, the use of the dextran gel 10 SEPHADEXTM G-50 superfine, in particular from 20 Nm to 50 {gym, has proved to be advantageous for the preparation of the PP mixture.
SEPHADEXTM beads from 20 /am to 300 Nm are also suitable and can be used. It has been found that beads which can effect fractionation of the dextran are most suitable.

A SEPHADEXTM G-50, namely SEPHADEXT"" G-50-50 superfine, has a fraction range of dextran between 500 and 10,000 MWt (molecular weight) and globular proteins of 1500 to 30,000 MWt. For swelling of the beads, 9-11 ml of liquid per g of beads are required in the case of SEPHADEXTM G-50-50 superfine. The less liquid required for swelling of the beads, the greater is the required basic centrifuging. For example, with the use of SEPHADEXTM G-25-50 superfine with globular protein 1000-5000 MWt, dextrans 100 to 5000 MWt and 4-6 ml/g of beads for swelling of the beads, a higher gravitational force is required in the case of a constant centrifuging time, for example 2 minutes, the bead size being unchanged, namely being 20 .pm to 50 pm. A

decisive factor is thus the swelling index of the beads, and the fraction range of the proteins and dextrans (stated in molecular weight MWt). If the swelling index and the fraction range are chosen to be larger, for example dextran gel SEPHADEXTM G-100-50 superfine is chosen, a lower gravitational force is required for a desired constant centrifuging time. This means that, with decreasing fraction range of the dextran or of the globular proteins, the g number of the centrifuging is increased with constant centrifuging time, and vice versa.

If, therefore, a particle-buffer mixture is to be prepared from the embedding buffer, particularly preferably 0.250 g of SephadexTM G-50 (G-50-50) and 2.5 ml of embedding buffer are mixed with one another.
Thereafter, the SephadexTM gel is allowed to swell for about 1 hour at room temperature, and 6.5 pi of 22% bovine serum albumin solution are added. This PP mixture is then allowed to stand overnight and then stored in a refrigerator at 2-8 C.

The embedding buffer has the advantage that, with the use of dextran, the colloidal solution of the dextran reduces the interstices between the particles after the preparation of the buffer-particle mixture. This fact is evidently due to the chains of the dextran. It is thus possible to operate with a much higher gravitational force (g) in the centrifuging step. In particular, the centrifuging step can be carried out at an acceleration of about 80 x g to 20,000 x g for a centrifuging time of 0.1 minute to < 10 minutes, preferably 1000 x g to 4000 x g for a preferred centrifuging time of 1 minute to 5 minutes, in particular 1900 to 2600 x g for 2 minutes, or particularly preferably at about 1900 g for 4 minutes.

Owing to the fact that the PP mixture promotes and stabilizes the visualization of the positive reaction, the centrifuging time and the gravitational force depend substantially only on the time after which, and the gravitational force, g number, at which, a negative reaction is unambiguously detectable. This means that in this case the g number can be increased in order further to reduce the centrifuging time. This is not possible in the prior art because, for example in the ID gel technique, a narrow window of g number, 85 x g, and centrifuging time, 10 min, is essential.

A further advantage of the method is that the PP
mixture decisively promotes the positive reaction or the visualization of the monolayer. This is because, under high pressure with the use of coded vessels, the erythrocytes loaded with IgG are pressed and held against the wall coded with anti-IgG. Owing to the PP
mixture or the embedding buffer, the spatial distances between the reactants or particles involved is very small, necessitating the high g number, but with the result that the centrifuging time is also reduced. Even at a high g number of 2500 and a centrifuging time of > 10 minutes, the positive reaction of the monolayer remains stable. This means, owing to the PP mixture or the embedding buffer, the results remain practically unchanged over a large gravitational force range.
Furthermore, the embedding buffer had the advantage of promoting and stabilizing the binding between anti-antibody and antibody-erythrocyte complex, namely the binding between Fab and Fc parts of the antibodies.
Thus, a pronounced lawn is positively formed: plastic wall/anti-antibody/antibody-erythrocyte complex. The PP
mixture furthermore has the advantage that this monolayer remains stable and is held. This means that there is increased sensitivity for the detection of antibodies.

Without wishing to be bound to a theory, the surprisingly advantageous properties might be attributable to the unexpected advantageous mechanical properties provided by the PP mixture. It would be possible for the interstices between individual beads to be reduced by the addition of the PP mixture and for a surprisingly good cleaning effect on passage of the erythrocytes through the gel to be thus achieved, which is why, for example, no additives, such as anti-IgM
antibodies are required for IgM antibodies. This means that the agglutination complex cannot be forced through the gel structure even by relatively high gravitational forces. The weaker agglutinates are possibly forced through up to the walls of the detection vessel by the gravitational forces acting, but, owing to the spatial size and the high g number, they migrate more slowly than individual erythrocytes.

These unexpected advantages have practical effects in carrying out the method. The monolayer is rapidly formed and is relatively stable, which permits a short centrifuging time at a high g number.

On the other hand, the viscosity of the PP mixture according to the invention is, however, so low that the mixture can easily be filled without bubbles into the detection vessels and distributed uniformly.

Surprisingly, it was also found that good results can be obtained with the use of coded detection vessels. The antibodies bound to the detection vessels are not curved or displaced by the gravitational force during centrifuging when the PP mixture according to the invention is used. By adding small amounts of bovine serum albumin or TweenTM
20, this effect can even be enhanced.

The term "coded" vessels or walls is understood as meaning that antibodies, or compounds which bind antibodies, such as, for example, protein A, are bound to the preferably used microtitre plates having a pointed bottom. If, for example, antibodies which are directed against the Fc part of antibodies are bound to the coded vessels, the antibodies bound to the wall react with the Fc part of antibodies which are present in the solution. Consequently, the antibodies present in the detection solution are bound to the walls of the detection vessels. When the antibodies present in the detection solution have bound to erythrocytes, this ensures that the erythrocytes are bound to the vessel wall by means of the antibodies and the antiantibodies.

For example, the standard method which leads the market, the ID gel technique, as a reference method in the detection of irregular antibodies which are known, of 95 high-titre patient sera has, in comparison with the method according to the invention, sensitivity of 87.2% relative to 98% in the case of the invention. In the case of the 95 high-titre patient sera, the following antibodies were relevant: anti-D 11, -E 9, -c 2, -Cw 2, -D+C 4, -D+E 1, -C+Cw 1, -E+CD7 2, -C+E 4, -c+E+C"' 1, -D+Fya+Cl, -Kell+E+CW 1, -S+Cw 3, -E+Jka 2, -Jka 3, -Kell 18, -Lua 1, -Lua+Leb 2, -Lua+C 1, -Fya 4, -Fyb 2, -Leb 1, -M 5, AIHA 4, Fya+E 1, -E+S-M+C`"-Jkb 1, -Jka+Kell 2, -D+Kell 1, -S+Kel l+C+Fya 1, Lub 1, Lea 3.
The number behind the designation of the sera indicates the number of the respective sera.

The specificity of 104 negative patients corresponded in the ID gel technique to 97% and in the subject of the invention to 93%. This is plausible because, the more sensitive the test, the lower is its specificity.
Of course, it is possible by fine adjustment to establish the balance between sensitivity and specificity optimally and to the desired extent, for example by fine adjustment of the dextran by changing the concentration or by metered addition of albumin, preferably from bovine serum, or by addition of the standard buffer "Liss".
Below, a test result of a reference method of the prior art is shown in comparison with the invention:

Antibody Reference Invention Invention specificity method 7 minutes 19-minutes ID technique 25 min Titre Titre Titre Fya 256 256 512 LUa 256 512 512 Kell 32 256 128 JKa 256 2048 2048 Kell 2048 8192 8192 c 1024 2048 8192 Column 1 shows the antibody specificities. The antibody specificities stated in column 1 represent conventional abbreviations for certain antibodies. The titres of column 2 give the values of a comparative method. The titres of column 3 are based on an incubation time of 5 min and a centrifuging time of 2 min (7 minutes altogether) at 2054 g; the PP mixture is finely adjusted by the addition of albumin. The titres of column 4 are based on an incubation time of 15 min and a centrifuging time of 4 min (19 minutes altogether) at 2054 g; the PP mixture was present in the basic formulation according to the invention with dextran and without albumin.
For the interpretation of the test results, a higher titre value means that the test method is more sensitive because the detection was still possible at a higher dilution. This can be explained by way of example for the antibody specificities c and C. In the reference method according to the prior art, a detection was still possible at a titre of 1:1024. With an incubation time of 5 minutes and centrifuging for 2 minutes (column 3), a positive reaction was still detectable in the case of a titre of 1:2048, i.e. the method according to the invention is twice as sensitive as the reference method. With an incubation of minutes (column 4), detection was still possible at 10 a titre of 1:8192, i.e. the method according to the invention is 8 times as sensitive as the reference method.

In order to confirm the sensitivity, in a further 15 example another 58 patient plasmas with known clinically significant antibodies were investigated with a very low titre. These were the following antibodies: anti -Lua+C"` 1, -C 2, -E 7, -D 3, -P12, -C'"3 , -S 3, -Fya+C 1, -M+Jka 1, -Lua 12, -C+Kell 1, -M 5, -Lea 1, -Kell 7, -Jka 2, -Fya 4, -Lea+Ieb 1, -Jkb+M 1 and -E+c 1. The standard method which is a market leader, the ID gel technique, had a sensitivity of 35%. In the method according to the invention, the sensitivity was 48% for 5 minutes incubation and 4 minutes centrifuging time, 48% for 10 minutes incubation and 4 minutes centrifuging time, and 76% for 15 minutes incubation and 4 minutes centrifuging time. This shows that a higher sensitivity can be achieved in the method according to the invention than in the reference method. In the case of 15 minutes incubation time, the antibodies having a very low titre had time to bind and thus exceeded the limit of detection of the novel method. This example shows that the sensitivity is substantially higher than that of the reference method.
In a further example, 101 patients of known blood group were investigated for ABO, Rh and serum check test. The agreement was 100%.

As a result of these tests, it is evident with regard to the invention that, contrary to the prior art in EP 0849595B1, in the invention the sensitivity does not decrease with increasing centrifuging speed or increasing g and hence shorter centrifuging times. Even with a shorter incubation time, the sensitivity is still substantially above that of the reference method.
With the use of centrifuging at 2500 x g for a centrifuging time of 2 min, the results are completely comparable.

Owing to the very small distances between the particles, due to the embedding buffer, no added anti-IgM of any kind is required in the detection of irregular IgM antibodies, so that neither coding of the vessel wall nor coding of the particles or beads and mixture with anti-IgM is required; the three-dimensional structure of Ery/IgM/Ery complex is too large so that, during the centrifuging, the complex either remains on the PP mixture or sticks in the PP
mixture or between PP mixture and wall.

The same applies when no coded vessels are used, but either the particles or beads are coded or the scavenger antibodies are added directly to the PP
mixture.

In a further embodiment of the method, the PP mixture used is one which has such a low viscosity that the PP
mixture is present in liquid or pasty form which, compared with the prior art, permits pipetting of the PP mixture by means of a stepper or pipette into the detection vessel.

In a particularly preferred embodiment, the PP mixture is pipetted into the detection vessel shortly before the investigation. This is a further advantage over the solutions known from the prior art. The standard gels known from the prior art are highly viscous and are therefore difficult to introduce into plates or columns. Moreover, the problem of the formation of air bubbles within the mixture frequently occurs thereby.
However, air bubbles in gels have an interfering effect on the migration of the solution to be detected through the gel. This disadvantage is avoided by the solution according to the invention.
In a particularly preferred variant for detecting antibodies and/or antigens, particularly for blood group determination, antibody search test, serum check test and infection serology, in a test liquid by reaction with a predetermined specific binding partner, the antigens or the antibodies or the specific binding partners in the test liquid being unbound and/or bound to a carrier, and the sample mixture prepared in this manner being subjected to an incubation step, and, in the case of a positive antigen-antibody reaction, an agglutinate being formed from antigens or antibodies to be detected or the specific binding partners and the carriers, which agglutinate is detectable optically as a sedimentation image, said method comprises adding a predetermined amount of the incubated sample mixture to a predetermined amount of a mixture of a buffer, embedding buffer, having particles, buffer-particle mixture, abbreviated to PP mixture, which is present within a detection 5 vessel. The embedding buffer has the property of minimizing the distance between the individual particles within the PP mixture. The detection vessel is then subjected to centrifuging for visualization of the reaction of the sample mixture in the detection vessel, the PP mixture promoting the positive reaction. The embedding buffer is prepared from 10 an aqueous solution of dextran [(C6H1o05)n] and/or polyethylene glycol [HO(C2H40)n"], it being possible to add glycocoil (H2NCH2COOH) (glycine) and/or trisodium citrate (C6H5O7Na3) to the embedding buffer.
The particles added to the embedding buffer are beads which are preferably dextran gel SEPHADEXTM G-50 superfine having a dry 15 diameter of 20 pm to 50 Nm, the centrifuging step then preferably being carried out at an acceleration of 1900 to 2600 x g for a centrifuging time of about 2 minutes for visualization of a reaction.

The sample mixture is first subjected to an incubation step which is preferably carried out in an incubator and takes between one minute 20 and 40 minutes, forced mixing of the test liquid being effected during the incubation. Continuous forced mixing is not part of the prior art in immunohaematology. This forced mixing is preferably achieved by shaking or stirring the test liquid. The forced mixing shortens the incubation time and thus accelerates the reaction time of the antigen-antibody reaction, and furthermore the forced mixing increases the sensitivity of the method. Preferably, an incubation time of 5 to 7 min has proved to be advantageous in the antibody search test, an incubation time of 1 to 5 min has proved to be advantageous in the blood group determination and an incubation time of 2 to 3 min has proved to be advantageous in the serum check test. There is therefore a possible standardization time of 5 min, so that blood group determination, antibody search test and serum check test can be carried out with the same PP mixture and one and the same gravitational number and the same centrifuging time as well as with one and the same vessel, for example microtitre plates.

For carrying out the incubation step and the forced mixing, Liss and/or modified Liss can be added to the sample mixture or the erythrocytes. Here, Liss can be used without modification or after modification with bovine albumin. The following Liss based on laboratory water (bidistilled H20) is preferably used:
Sodium chloride, preferably 1.788 g/1 Disodium hydrogen phosphate Na2HP04, preferably 0.204 g/1 Potassium dihydrogen phosphate KH2PO4, preferably 0.233 g/1 Glycine H2NCH2COOH, preferably 18 g/l Thymerosal CgHgHgO2SNA, preferably 0.20 g/l (preservative) gives Liss with 0.03 mol at a pH of about 6.7.
For example, 4 ml of this Liss buffer plus 0.5 ml of laboratory water plus 0.5 ml of 22% bovine albumin are taken and give modified Liss. The pH is about 7.1.

In a further embodiment of the method, albumin, such as bovine serum albumin, is added to the PP mixture for fine adjustment of the reaction, and, simultaneously or independently thereof, the dextran concentration can be appropriately adapted. In the table above, a fine adjustment of the PP mixture by means of bovine albumin is shown, for example, in column 3.

In a further embodiment of the invention, in the application thereof to infection serology, coloured beads which are coded with protein and have about the density of the erythrocytes are used instead of erythrocytes as given specific binding partners. For this purpose, standard particles - also in contrast to the prior art of EP 0849595B1 - having a diameter of 3 Am to 7.5 m are advantageously used, which particles have been advantageously coloured. This reaction mixture which contains the analytes to be determined is then added to the PP mixture for visualization of the reaction.

In this preferred embodiment, the carriers (coloured beads) are coupled with binding partners, for example antigens. Such antigens are preferably antigens which play a role in blood analysis. If it is, for example, an antigen of the hepatitis C virus (HCV), it is also possible to detect in a test whether the serum to be investigated contains antibodies against this pathogen.
In this case, an antibody which is directed against HCV
will bind to the antigen coupled to the coloured beads and, if the detection vessel is coded with anti-antibodies, the coloured beads remain adhering to the walls of the detection vessel and, after centrifuging, the knob in the tip of the detection vessel, which would indicate a negative reaction, is not formed.
Instead of the HCV antigen discussed above by way of example, other antigens can also be coupled to the beads, for example antigens of other hepatitis viruses, such as hepatitis A or B, or HIV viruses and any other antigens.

In a further embodiment of the method, the sample mixture is reacted in a separate reaction vessel, and the sample mixture is then applied to the PP mixture within the detection vessel, it being possible for the PP mixture to be introduced individually into the detection vessel as required during the processing, and even greater flexibility thus being ensured. Owing to the use of two vessels, namely reaction vessel and detection vessel, greater flexibility of the method is ensured; in addition, the continuous forced agitation of the sample mixture can be carried out. This embodiment is particularly suitable for automatic processing of samples because, while the first plate is being incubated, in an emergency an individual subsequent load can be introduced into this plate, which sample, after the short incubation time of 5 minutes, can then be further processed in the second plate or a strip for the centrifuging step, whereas the longer incubation time applied to the remaining samples of the first plate has no adverse effects on the subsequent results.
Of course, with the use of coded vessels, it is also possible to add the sample mixture directly to the PP
mixture present in the vessel prior to the incubation;

forced agitation of the sample mixture does not take place here. The detection vessels used for carrying out the method and intended for holding the PP mixture are preferably tapered-bottom vessels.

For the antibody search test by means of the PP mixture, it has proved to be advantageous if the inner surfaces of the detection vessels are coded with proteins, in particular with anti-IgG, anti-IgA, anti-IgM, protein A, protein G or anti-C3d and/or with a mixture thereof. The lawn formed in a known manner in the case of a positive reaction is, according to the invention, extremely stable, and the reaction is extremely sensitive. Moreover, it has been found that plates prepared in this manner can be shipped vacuumpacked or packed under inert gas in a very simple manner and can be stored for a long time in a refrigerator.

In a further preferred embodiment of the method, it has proved advantageous if the scavenger proteins, for example anti-IgG, are stabilized. Stabilizing substances, such as Triton TM X-100 or albumin or casein or gelatin or TweenTM 20 or mixtures thereof within a buffer, are used for stabilizing the proteins present on the inner surfaces of the detection vessels. These known buffers comprising said substances, which are used in the ELISA technique for blocking unspecific reactions (false positive or increased background), serve here for the mechanical stabilization of the proteins and hence for the visualization of the positive reaction. One explanation is that the stabilizing substances envelope the scavenging proteins so that the scavenging proteins cannot be mechanically curved away or displaced at the high centrifugal force.
This stabilization can be carried out either immediately after the coding or just before the use of the coded vessels. In this context, it has been found that, if no stabilization of the scavenging proteins takes place, no reasonable positive reaction is detectable on carrying out the method, although such a reaction would have been expected on the basis of the experimental conditions.

In a further preferred embodiment of the method, a separate reaction vessel for the reaction of the sample mixture during the incubation is closed at the bottom with a membrane, reaction vessel and detection vessel in which the PP mixture is present being capable of being inserted into one another or pushed onto one another and, for visualization of the reaction, the reaction vessel being inserted into or pushed onto the detection vessel and the two being centrifuged jointly, the membrane of the reaction vessel being at least partly permeable to the sample mixture and the latter entering the detection vessel during the centrifuging of the two vessels.
This embodiment which uses a membrane which forms the bottom of the reaction vessel is particularly advantageous for manual processing of samples. This is because, in manual processing of the samples, the pipetting step for pipetting the reaction mixture into the detection vessel is omitted.

In a further embodiment of the method, the reaction vessel and the detection vessel are one and the same vessel both for carrying out the incubation and reaction and for carrying out the detection of the reaction, the vessel being divided into two parts and the PP mixture, which is covered by a membrane, being present in the lower part of the vessel and, above said membrane, the reaction space for the reaction of the sample mixture during the incubation being present in the upper part. During the subsequent centrifuging of the vessel, the membrane is at least partly permeable for the sample mixture. This embodiment has the general advantages that the gel or PP mixture used can already have been introduced into the vessel and is firstly securely closed by the membrane. The further advantage is that the incubation step with the forced agitation of the sample mixture can now be carried out using one and the same vessel.

In this embodiment of a sample vessel having a membrane, the vessel may have virtually any desired shape, for example may be a tapered-bottom or a round-bottom or flat-bottom microtitre plate or a column. A
tapered-bottom or round-bottom microtitre plate or strip is evaluated from above or below; a column or flat-bottom plate or strip is evaluated from the side.
The use of a membrane can also be applied to any desired particle tests, for example gel tests. Such a container for carrying out a method for detecting antibodies and/or antigens, such as for the blood group determination, antibody search test, serum check test and infection serology, in a test liquid by reaction with a predetermined specific binding partner, the antigens or the antibodies or the specific binding partners in the test liquid being unbound and/or bound to a carrier, and the sample mixture prepared in this manner being subjected to an incubation step, and, in the case of a positive antigen-antibody reaction, an agglutinate being formed from antigens or antibodies to be detected or the specific binding partners and the carriers, which agglutinate is detectable optically as a sedimentation image, is characterized in that a container which is a round-bottom or tapered-bottom or flat-bottom vessel or a column is separated by a membrane into two chambers, namely into an upper and a lower chamber, the membrane covering the detection layer present in the lower chamber, for example a gel, for the optical visualization of a reaction which has taken place, and the upper chamber representing the reaction space for holding the sample mixture and reaction of the latter, the membrane being at least partly permeable as a result of the centrifuging step, and the sample mixture thus passing from the reaction space into the detection space.

In the general case, the container may also consist of two separate vessels, the first vessel being closed at the bottom by the membrane and serving for holding the sample mixture and carrying out any incubation and the reaction, and the second vessel containing the detection layer, for example a gel, and serving for visualization of the reaction, and the two vessels being capable of being inserted one into the other or pushed one onto the other and both vessels being centrifuged together, the membrane of the reaction vessel being at least partly permeable for the sample mixture during the centrifuging of the two vessels and said mixture thus entering the detection vessel. In both cases, forced agitation of the sample mixture can of course be effected during the incubation.

The membrane may consist of plastic or rubber or of a rubber blend or a latex or a gel.

Examples for carrying out the method are described below. In the drawing, Figure 1 shows a V-bottom vessel, for example in a microtitre plate, having a membrane Figure 2 shows a round-bottom vessel of a microtitre plate having a membrane, Figure 3 shows a V-bottom vessel having a spike present below a membrane for piercing the membrane and Figure 4 shows a V-bottom vessel having a spike arranged above a membrane and movable downwards for piercing the membrane.

The following example starts from coded vessels.
Commercial V-bottom ELISA plates which can be divided into 12- or 8-well strips are preferably used. A coding buffer with specific proteins, such as anti-antibodies, is diluted in the ratio of about 1:800 with PbS buffer and a solution is prepared. About 50 Al of said solution are pipetted into each vessel. The vessel is subjected to incubation overnight at room temperature.
The solution or remaining solution comprising coding buffer and specific proteins is removed. A repeated washing step is not required. 200 l of a PbS buffer which contains about 1% bovine serum albumin solution is then added to each vessel. This last step with bovine serum albumin is also required for the uncoded V-bottom plates for the investigation of the blood groups. This is because coating with proteins is generally required for adhesion. This means that, on filling with the PP mixture, the PP mixture is uniformly distributed over the bottom wall of the microtitre plate and the plate can thus be better filled.

The vessel is incubated for 1 to 2 hours at room temperature, after which, for example, the vessel is washed out by means of a wash buffer which preferably contains Tween 20 and bovine albumin. The vessel has now been completely prepared as a coded vessel and is available for further use as a detection vessel. These can be kept for a very long time after vacuum packing or packing under inert gas. Using a plate coded in this manner, a work sequence for an antibody search test, for example antibody search cells of 1, 2 and 3, can be carried out as follows:

3 times 25 Al of serum or plasma plus 50 Al of search cell suspension 1, 2, 3 of 0.3% 0.1% are introduced into separate reaction vessels, which may be untreated, commercial V-bottom vessels, microtitre plates, the working dilution being effected with modified Liss buffer. The search cell suspension can be prepared from a commercial 3% +/- 1% stock search cell suspension as required. This has the advantage that these search cells are very stable for a long time in their specific buffer, which may be problematic in Liss, and these can be prepared more easily on an industrial scale owing to the higher search cell fractions. For example, 270 l of modified Liss plus 30 l of stock search cell suspension can be added to an extra microtitre plate (or tube in the case of a larger series).

The vessels are incubated at about 37 C and at the same time at a shaking frequency of 700 25 rpm for 5 minutes, and a sample mixture is produced. During the incubation, the buffer-gel mixture is pipetted into the detection vessel by means of a 50 Al stepper;
thereafter, in each case 50 Al of the sample mixture is pipetted onto the PP mixture in the detection vessels.
The detection vessels are subjected to a centrifuging step for about 2 minutes at about 2054 g. The result of the reaction is then optically evaluated either visually or automatically within the detection vessels.
A positive reaction is evident as a monolayer, lawn, and a negative one as a knob. In the case of knob formation, the unaggregated erythrocytes collect at the lowest point of the well in the microtitre plate. Since it is a microtitre plate, the result is read by inspection either from above or below.

The plate coded with anti-IgG exhibits on the one hand IgG antibodies and, owing to the PP mixture, simultaneously also any IgM antibodies present. In the case of a positive reaction, a selective distinction as to whether IgG and/or IgM antibodies are present can be implemented as follows by using a second, uncoded plate with PP mixture and carrying out the test again. If a negative reaction is obtained with the second plate, an IgG antibody is present. If a positive reaction appears, identically to the coded plate, an IgM
antibody is present.

If the reaction in the second plate is weaker compared with that in the first plate, a mixed form of IgG and IgM antibody is present. Likewise, no coding of anti-C3d is required since the detection is so sensitive that the few IgG antibodies dependent on complement which form antigen C3d on the erythrocyte are sufficient for the detection. If, owing to any unspecific reactions, no IgM antibodies are desired, known reducing agents, for example sodium dithionite, may be employed for reduction or decrease of the IgM
antibodies.
For example, once again an untreated microtitre plate or microtitre plate treated with bovine albumin is used as a reaction vessel for the ABO-RH determination and additionally for carrying out the serum check test. The following are pipetted into said plate:

a) 50 l antiA (Biotest Clone, A003 IgM) of a 1:100 solution prepared by means of Liss buffer or with isotonic NaCl 50 Al antiB (Biotest Clone, B005 IgM) of a 1:100 solution prepared by means of Liss buffer or with isotonic NaCl 50 Al antiD (Immucor, Clone, RUM-i IgM) of a 1:30 solution prepared by means of Liss buffer or with isotonic NaCl 50 Al RH mono-Control of a 1:30 solution prepared by means of Liss buffer or with isotonic NaCl, b) in each case 25 Al of a 0.6 0.2% erythrocyte patient suspension which contains Liss buffer or isotonic NaCl are pipetted onto the above-mentioned four loaded vessels (this is necessary for ABO-RH
erythrocyte determination), c) 4 times 50 l of plasma (serum check test) are pipetted into a further four reaction vessels, d) in each case 25 l of a 0.6 0.2% suspension which contains Liss buffer are prepared with Al cells, A2 cells, B cells and 0 cells and pipetted into the reaction vessels.

The iso search cells can be prepared from a commercial 3% +/- 1% stock cell suspension as required. This has the advantage that these iso search cells are stable in their specific buffers, which may be problematic in Liss, and these can be prepared more easily industrially owing to the higher search cell fractions.
For example, 240 gl of modified Liss plus 60 Al of stock cell suspension can be taken in an extra microtitre plate (or tube in the case of a larger series);
e) the sample mixtures are incubated for about 2 to 5 minutes at room temperature from 18 to 25 degrees Celsius and at the same time at a shaking frequency of 700 25 rpm, f) 8 times 25 Al of PP mixture are pipetted by means of a stepper into eight uncoded detection vessels which are untreated or treated with albumin, g) 50 Al each of the sample mixtures are now pipetted into the detection vessels onto the PP mixture, h) the detection vessels are subjected to a centrifuging step for about 2 minutes at about 2054 g and the results of the reaction are evaluated visually or automatically. A negative reaction is evident as a knob; a positive reaction is evident either on or within the PP mixture, but gives the impression of a lawn. Since it is a microtitre plate, the result is read by inspection from either above or below.

A test kit consists, for example, of the following components:
Plate coded with anti-IgG (detection plate), bottle containing PP mixture, blood group plate treated with albumin, bottle containing modified Liss, 3 bottles each containing test cells 1, 2, 3, commercial reaction plate, 4 bottles containing iso cells A1, A2, B, 0 A particular advantage of such a test kit is the easy shipability and its flexibility.

Figures 1 and 2 show one V-bottom vessel (Figure 1) and one round-bottom vessel (Figure 2) each, having a membrane 2 or 4, respectively, which is fastened to the vessel wall above the V-bottom or the round bottom over the total cross-section and separates the vessel into two parts separated from one another. The PP mixture 5 is present below the membrane 2, 4 inside the vessel 1 or 3.

Regarding the membrane 2, 4 used, the following embodiments are possible. A membrane 2, 4 of latex can be made permeable by prior perforation, preferably having a diameter of 6 to 8 m. Since latex is very flexible, these holes are closed in the rest state.

When a vessel which has been filled beforehand with 50 gl of a sample mixture is centrifuged at, for example, 2500 x g, the sample mixture has a weight of about 125 g, which is sufficient to expand the membrane orifices and to allow the sample mixture or the erythrocytes to pass.

Alternatively, a latex membrane 2, 4 has a small membrane thickness and has initial tension such that it tears at a weight of only 10 g during centrifuging of the vessel 1, 3.

Figures 3 and 4 show two further possibilities for implementation. A latex membrane 7 is arranged inside a V-bottom vessel 6 above the V-bottom over the total cross-section of the vessel 6 and in particular is subjected to initial tension. A spike 8 which projects in the direction of the membrane and extends up to the membrane 7 is present below the latex membrane 7 - in addition to the PP mixture 5 - on the sloping wall of the V-bottom.

The spike may also be formed by a sharp edge which is formed on the wall of the V-bottom directly below the membrane.

During the centrifuging, the membrane 7 bulges downwards in the direction of the bottom of the vessel owing to the centrifugal force, and is penetrated by the spike 8 or presses onto the edge and is thus caused to tear.

Figure 4 shows a further V-bottom vessel 9 having a membrane 10 which is stretched above the V-bottom over the total cross-section of the vessel 9. A movable spike 11 which extends down to the membrane 10 is arranged above the membrane 10, on the wall of the vessel 9. If said spike has, for example, a weight of 0.5 g, the resulting spike weight is 1250 g on centrifuging at 2500 x g, so that the membrane is pierced and thus caused to tear.

The method according to the invention is specific and very sensitive. It can be used for the complete blood group determination, the antibody search test and the cross-matching. It is a one-step centrifuging method which requires no washing steps. A complete blood group determination and an antibody search test is possible within 7 minutes, and it is for this reason in particular that the method is suitable for emergency use. Owing to the variable incubation times, the method is suitable for emergency use and is very suitable for semiautomation and full automation. Owing to the identical base reagents, incubation times and centrifuging times, the complete blood group determination with serum check test, antibody search test and cross-matching is possible on a microtitre plate. It is also possible to perform infection serology, such as, for example, hepatitis B, on the same microtitre plate.

Claims

1. A method for the detection of antibodies, antigens, or both, in a test liquid by reaction with a given specific binding partner, the antigens being bound to a carrier, and a sample mixture prepared in this manner being subjected to an incubation step and then being centrifuged through a particle-buffer mixture, in the case of a positive antigen-antibody reaction an agglutinate of antigen or antibody to be detected and carrier being formed, which agglutinate is optically detectable as a sedimentation image, and that a predetermined amount of incubated sample mixture is added to a predetermined amount of a buffer-particle mixture which is present within a detection vessel, the buffer-particle mixture comprising an embedding buffer, characterized in that the embedding buffer consists of an aqueous solution of:
(a) (i) 0.1 to 500 g/l of dextran, (ii) polyethylene glycol, (iii) glycine, and (iv) trisodium citrate ;
(b) (i) 0.1 to 500 g/l of dextran, (ii) glycine, and (iii) trisodium citrate; or (c) (i) polyethylene glycol, (ii) glycine, and (iii) trisodium citrate.

2. The method according to Claim 1, characterized in that the polyethylene glycol, if present, is at a concentration of 1 to 100 g/l; the glycine is at a concentration of 1 to 100 g/l; and the trisodium citrate is at a concentration of 0.01 to 10 g/l.

3. The method according to Claim 1 or 2, characterized in that the dextran has a molecular weight of about 100 to 40,000,000 g/mol, and is present in the embedding buffer in a range from 5 g/l to 40 g/l.

4. The method according to Claim 3, characterized in that the dextran has a molecular weight of 2,000,000 (2.10 6) g/mol.

5. The method according to Claim 3 or 4, characterized in that the dextran is present in the embedding buffer at a concentration of 20 g//l.

6. The method according to any one of Claims 1 to 5, characterized in that the polyethylene glycol has a molecular weight of about 50 to 40,000 g/mol, and is present in the embedding buffer in a range from 10 g/l to 70 g/l.

7. The method according to Claim 6, characterized in that the polyethylene glycol has a molecular weight of 3500-4500 g/mol.

8. The method according to Claim 6 or 7, characterized in that the polyethylene glycol is present in the embedding buffer at a concentration of 40 g/l.

9. The method according to any one of Claims 1 to 8, characterized in that the glycine is present in the embedding buffer in a range from 10 g/l to 25 g/l.

10. The method according to Claim 9, characterized in that the glycine is present in the embedding buffer at a concentration of 18.0168 g/l.

11. The method according to any one of Claims 1 to 10, characterized in that the trisodium citrate (C6H5O7Na3) is present in the embedding buffer in a range from 1 g/l to g/1.

12. The method according to Claim 11, characterized in that the trisodium citrate (C6H5O7Na3) is present in the embedding buffer at a concentration of 2.581 g/l.

13. The method according to any one of Claims 1 to 12, characterized in that the embedding buffer has a pH of 4 to 9.

14. The method according to Claim 13, characterized in that the embedding buffer has a pH of 6.9 to 7.1.

15. The method according to any one of claims 1 to 14, characterized in that beads are used as particles within the buffer-particle mixture.

16. The method according to Claim 15, characterized in that the beads used are acrylic, gelatin, polyacrylamides, solid nets, silica, Ficoll.TM, Percoll.TM., phthalate esters, agarose or dextran gel.

17. The method according to Claim 16, characterized in that beads having a dry diameter of from 1 gm to 300 gm are used for the preparation of the buffer-particle mixture.

18. The method according to Claim 17, characterized in that the beads are SEPHADEX.TM. G-50 superfine beads.

19. The method according to Claim 17 or 18, characterized in that the beads have a dry diameter of from 20 gm to 50 gm.

20. The method according to any one of Claims 1 to 19, characterized in that, for fine adjustment of the reaction, albumin is added to the particle-buffer mixture and the dextran concentration is correspondingly adapted.

21. The method according to Claim 20, wherein said albumin is bovine serum albumin (BSA).

22. The method according to any one of Claims 1 to 21, characterized in that the particle-buffer mixture has such a low viscosity that it is present in liquid or pasty form which permits pipetting of the buffer-particle mixture into the detection vessel by means of a stepper or pipette.

23. The method according to Claim 1, characterized in that the sample mixture is incubated in a separate reaction vessel and then the sample mixture is applied to the particle-buffer mixture within the detection vessel.

24. The method according to Claim 1, characterized in that the incubation step takes from 1 minute to 40 minutes, forced mixing of the test liquid being effected during the incubation.

25. The method according to Claim 24, characterized in that the incubation step takes 5 minutes.

26. The method according to Claim 24 or 25, characterized in that the forced mixing of the test liquid is effected by shaking or stirring.

27. The method according to any one of Claims 1 to 26, characterized in that, for carrying out the incubation step and forced mixing of the test liquid, low ionic strength salt solution (Liss) and/or modified Liss is added to the test liquid.

28. The method according to Claim 1, characterized in that the centrifuging is carried out at an acceleration of from 80 × g to 20,000 × g and for a centrifuging time of from 0.1 minute to < 10 minutes.

29. The method according to claim 28, characterized in that the centrifuging is carried out at an acceleration of from 1000 × g to 4000 × g and for a centrifuging time from 1 minute to 5 minutes.

30. The method according to claim 28 or 29, characterized in that the centrifuging is carried out at an acceleration of from 1900 to 2600 × g for 2 minutes.

31. The method according to Claim 1, characterized in that a separate reaction vessel for the reaction of the sample mixture during the incubation is closed at the bottom with a membrane, the reaction vessel and the detection vessel in which the buffer-particle mixture is present being capable of being inserted into one another or pushed onto one another and, for visualization of the reaction, the reaction vessel being inserted into or pushed onto the detection vessel and the two being centrifuged jointly, the membrane of the reaction vessel being at least partly permeable to the sample mixture and the latter entering the detection vessel during the centrifuging of the two vessels.

32. The method according to Claim 1, characterized in that a reaction vessel and the detection vessel are one and the same vessel both for carrying out the incubation and reaction and for carrying out the detection of the reaction, the vessel being divided into two parts and the buffer-particle mixture, which is covered by a membrane, being present in the lower part of the vessel and, above said membrane, the reaction space for the reaction of the sample mixture during the incubation being present in the upper part, the membrane being at least partly permeable for the sample mixture during the subsequent centrifuging of the vessel.

33. The method according to Claim 31 or 32, characterized in that the membrane consists of plastic or rubber or rubber blend or latex or gel.

34. The method according to any one of claims 1 to 33, characterized in that the detection vessel for holding the particle-buffer mixture are tapered-bottom vessels or columns.

35. The method according to any of claims 1 to 34, characterized in that the inner surfaces of the detection vessel are coded with proteins.

36. The method according to Claim 35, characterized in that the proteins are anti-IgG, anti-IgA, anti-IgM, protein A, protein G or a mixture thereof.

37. The method according to Claim 35 or 36, characterized in that stabilizing substances are used for stabilizing the proteins present on the inner surfaces of the detection vessel.

38. The method according to Claim 37, characterized in that the stabilizing substances are Triton.TM. X-100, albumin, casein, gelatin, Tween.TM. 20 or a mixture thereof.

39. The method according to Claim 1, characterized in that either proteins are mixed into the particle-buffer mixture or beads are coded with proteins.

40. The method according to Claim 39 for use in infection serology, characterized in that the antigens bound to a carrier are coloured beads which are coded with proteins and have about the density of erythrocytes.