WO2004019038A2 - Method for the detection of antibodies and/or antigens in a test liquid, particularly for determining the blood type - Google Patents

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 anti-quenas in a test liquid, in particular when determining blood groups

The invention relates to a method for the detection of antibodies and / or antigens, in particular for blood group determination, antibody search test, serum cross-test and for 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 is unbound and / or bound to a carrier and the sample mixture thus produced is subjected to an incubation step, 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 is optical is 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 can be detected via crosslinking, i.e. Agglutination, of antigens (Ag), antibodies (Ak) and, if appropriate, certain carrier substances which carry antigens or antibodies, to form a macroscopically detectable, optically detectable complex, the agglutinate. This can be a three-dimensional agglutinate or a so-called monolayer, which can often be observed when the surfaces of the test plates are coated (coded) with antibodies.

Examples are the blood group determination on the erythrocyte side and the serum cross-test. In the former, blood group antigens bound to the membrane of the donor erythrocyte are detected by reaction with antisera which contain antibodies as specific binding partners. The antibodies that are in a test liquid are known (search antibodies), for example test antibodies. Flooding and the unknown erythrocytes with unknown antigens are added. Agglutination is caused by the reaction of antigen and antibody. This means that the ABO and RH system and, in addition, a large number of antigens can be determined.

In the case of the serum cross-test, the erythrocytes which have known antigens (search antigens) are known. The test liquid, for example serum, contains the unknown body's own antibodies (so-called isoagglutinins), which are detected by reaction with the test erythrocytes. This can also be used to determine the blood group, but only ABO, Biotest, Lexicon of Immunology, 2nd edition, Medical Service Munich, pages 36, 37.

This technique works with native round bottom microtiter plates, with 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, pipetted into the wells of the plate and with the test erythrocytes is added in a diluted form. After an incubation period of at least 20 minutes, the plate is centrifuged and carefully shaken. The total processing time is approximately 40 minutes. A positive antigen-antibody reaction is visible as agglutinate, a negative reaction is shown as a suspended erythrocyte suspension which, when left standing, settles as a button on the bottom of the test plate. The serum cross-check works in a similar way.

The process can only be automated in a complex manner and often does not provide uniqueness. The reliability of the detection of a positive reaction depends on the strength of the antibody-antigen binding: 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, because of the possibly missing clear reaction picture, the automatic distinction between positive and negative reaction, e.g. by means of automatic photometric evaluation, practically not possible, but must be carried out by an experienced operator or at least checked visually and is therefore very time-consuming.

From EP 0 363 510 A, the so-called solid phase technique for finding irregular antibodies is known, which is based on coded microtiter plates without gel. This requires multiple complex washing and centrifuging steps. The total processing time, incubation 20 minutes, centrifugation, washing steps, is approximately 40 min. The positive results can be seen as a monolayer (turf), the negative results as a button on the slab floor. Due to these steps and the long duration, flexibility in processing the samples and thus automation is poor. In addition, the ABO-Rh and serum cross-sample can only be processed with this technique to a limited extent and with great effort.

From EP 0 305 337 A1 a method for the optical visualization of complexes of carrier-bound antigens with antibodies in reaction vessels is known for the detection of antigens or antibodies, which is called ID-gel technique. This uses six parallel columns filled with gel beads in the form of a card. In the upper area above the column there is a larger container that holds the sample. An air cushion must remain between the lower column filled with gel and the upper container when the sample is filled; there is a rest incubation phase of 15 min at 37 ° C; the subsequent centrifugation time of the card is 10 minutes at 85xg. The result is read by looking at the column from the side. If the reaction is positive, the agglutinates are flaked on the gel of the column or in the gel of the column. In the event of a negative reaction, the erythrocytes are in a compact form at the bottom of the column. The incubation time cannot be shortened because the samples must not be moved mechanically, because otherwise the sample would come into direct contact with the gel during the incubation phase and a false-negative reaction would be indicated. The g-number cannot be increased and the centrifugation time cannot be reduced because otherwise a false negative reaction would also be indicated. Separate cards are also required for the serum cross-test and the ABO-RH system, as are separate columns of a card for differentiating between IgG and IgM, for example. Due to the still long incubation and centrifugation time and the card shape as well as separate columns for the individual determinations, this technique is not well suited for full automation. Sending the cards to the customers is also problematic since the gel can run out of the columns if the columns are upside down. It should also be mentioned that pipetting the gel later is problematic due to the high viscosity and its sticking into the column.

EP 0 849 595 B1, which is based on EP 0 305 337A1, shows that (quote) "commercially available standard particles with a diameter of 3-7.5 μm sediment only incompletely into the gel matrix, while larger ones, 1 1 could microns 9, or smaller synthetic particles <1 micron weak in the gel penetrate. An increase in the parameter ^{'centrifugation,} 10 min to 50 min and Centrifugation speeds from 1030 to 1300 rpm brought better, but still no complete sedimentation. Even if optimal sedimentation could have been achieved by these two measures, a change / increase in the centrifugation time and speed is very disadvantageous for the following reasons: 1. The method of EP 0849595 B1 is intended to be a particularly time-consuming method in comparison to comparable methods allow. With a possible centrifugation time of 50 minutes, the desired test duration of 20 minutes including incubation would triple. 2. As is known to the person skilled in the art, the sensitivity in gel centrifugation processes decreases with increasing centrifugation speed "(end of quotation). A centrifugation at 85 g for 10 minutes is used to detect the reaction.

An advantage of the invention is to provide a new method for the ABO and RH determination, for the serum cross-test and for the antibody test which corresponds to the modern requirements of the market, namely a flexible and fast automatic processing of the samples via a fully automatic machine with emergency input to provide for patients and donors. For this purpose, the processing time must be as short as possible, which means that the incubation time may be a maximum of 5 to 7 minutes separately in a plate and the centrifugation time 2 to 4 minutes separately in a reaction plate, which still have to meet the requirement that these be loaded individually can be. It is also advantageous if the test device can simply be connected to the individual components in a simple manner, e.g. shortly before the examination.

No washing steps are required either. In order to be able to prefer emergency patients, when entering an emergency, it must also be ensured that the incubating patient sera or plasmas, which have no priority, can also incubate for longer periods without negative effects on the results, up to around 40 minutes, so that an emergency patient can quickly in between can be processed; then the normal routine is processed again. It must also be possible to process the samples with the same basic reagents as possible, and it is also ensured that the reagents are easy to ship. It is therefore advantageous that the detection method can be easily adapted to the special circumstances.

The method according to the invention therefore, while avoiding the disadvantages of the prior art, provides a method for the detection of antibodies and / or antigens, such as for blood group determination, antibody screening test, serum cross-test and for infection serology, with high sensitivity, which can be carried out in a simple manner and can be managed with a high degree of automation. The method leads to highly reliable test results of the samples, especially in a very short reaction time; if possible, automatic processing of the samples using an automatic machine. Emergency entries of test samples from emergency patients are also possible, without impairing the other samples.

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, namely an embedding buffer, with particles, which is hereinafter referred to as buffer-particle mixture, abbreviated PP mixture. This PP mixture is located within a detection vessel, the embedding buffer having the property of minimizing the distance between the individual particles within the PP mixture. In addition, the embedding buffer causes the PP mixture to have a lower viscosity, which is essential for problem-free pipetting. The detection vessel is then subjected to centrifugation, which leads to the 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 [(C ₆ H ₁₀ O ₅ ) _n ] and / or polyethylene glycol [HO (C ₂ H ₄ O) _n H], with laboratory water being advantageous for the preparation of all aqueous solutions (Aqua bidest) is used. In a further advantageous embodiment of the method, the aqueous solution is made of dextran [(C ₆ H ₁₀ O ₅ ) _n ] and / or polyethylene glycol [HO (C ₂ H ₄ O) _n H] glycine (H ₂ NCH ₂ COOH) and / or Trisodium citrate (C ₆ H ₅ O ₇ Na ₃ ) added.

In a preferred embodiment of the method, a high molecular weight dextran is used for the embedding buffer. The dextran has a molecular weight of approximately from 100 to 40,000,000 g / mol, preferably from 2,000,000 (2 10 ⁶ ) g / mol, and is in a range between 0.1 g / l to 500 g / l, preferably 5 g / l to 40 g / l, more preferably from 18 g / l to 22 g / l and in particular from 20 g / l, in the embedding buffer.

In a further embodiment of the method, the polyethylene glycol has a molecular weight of approximately 50 to 40,000 g / mol, preferably 3500-4500 g / mol, and is in a range between 1 g / l to 100 g / l, preferably between 10 g / l up to 70 g / l, more preferably from 35 g / l to 45 g / l and in particular 40 g / l in the embedding buffer. In a further embodiment of the method, the glycocoll (glycine) is in a range between 1 g / l to 100 g / l, preferably 10 g / l to 25 g / l, more preferably 16 g / l to 20 g / l and in particular 18.0168 g / l, in the embedding buffer.

In a further embodiment of the process, the trisodium citrate (C ₆ H ₅ O ₇ Na ₃ ) is in a range between 0.01 g / l to 10 g / l, preferably 1 g / l to 5 g / l, more preferably 2 g / l to 3 g / l and in particular 2.581 g / l, in the embedding buffer. The dihydrate can be used with the trisodium citrate. The above quantities refer to the anhydrous form. If hydrated forms are used, the amounts increase accordingly.

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

For preservation, sodium azide (NaN ₃ ) is preferably added to the PP mixture, which in a range between 0.01 g / liter to 10 g / l, preferably between 0.5 g / l to 2 g / l, in particular 1 g / l (corresponds to 0.1%). An antibacterial and / or antiviral agent, such as sodium azide (NaN ₃ ) or antibiotics, is preferably added to the embedding buffer for preservation. As a result, the embedding buffer is long-lasting and stable, for example 1 year. The buffer can be stored at room temperature, preferably it should be stored for longer periods at refrigerator temperatures.

In a particularly preferred embodiment, an embedding buffer is produced as follows:

First, 0.24 M (18.0168 g / l) glycine and 0.01 M (2.581 g / l) trisodium citrate (C ₆ H ₅ O ₇ Na ₃ ) are made up to 1 liter with H ₂ O bidest. This results in a pH between 6.9 and 7.1. 20 g of dextran (2.0%) with a molecular weight of 2 × 10 ^{6 are then} added to this buffer solution and dissolved by stirring. Furthermore, 40 g / l (4%) of polyethylene glycol with 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 set.

In a preferred embodiment of the method, beads are used as particles within the buffer-particle mixture. Acrylic, gelatin, polyacrylamide, solid nets, silica, Ficoll, Percoll, phthalate ester, agarose or dextran gel are preferably used as beads. The particles, preferably the beads, have a diameter between 1 μm and 300 μm, preferably a dry bead diameter between 1 μm and 300 μm. In a preferred embodiment, the use of the dextran gel SEPHADEX G-50 superfine, in particular between 20 μm to 50 μ, has become Production of the PP mixture proved to be advantageous. SEPHADEX beads between 20 μm and 300 μm are also suitable and can be used. It has been shown that beads which can fractionate the dextran are best suited.

A SEPHADEX G-50, namely SEPHADEX G-50-50 superfine, has a fraction range of dextran between 500 and 10,000 MWt (molecular weight), and globular proteins of 1,500 to 30,000 MWt, for swelling the beads at SEPHADEX G-50 -50 superfine 9-1 1 ml / g beads liquid required. The less liquid needed to float the beads, the greater the basic centrifugation required. For example, when using SEPHADEX G-25-50 superfine with globular protein 1,000-5,000 MWt, Dextrans: 100 to 5,000 MWt as well as for suspending the beads 4-6ml / g beads, a higher gravity with constant centrifugation time, for example 2 minutes, the bead size being unchanged, namely 20 μm to 50 μm. The swelling number of the beads, the fraction range of the proteins and dextran (given in molecular weight MWt) are thus decisive. If the swelling number and the fraction range are chosen to be larger, for example dextran gel SEPHADEX G-100-50 superfine is selected, less gravitation is required if the desired centrifugation time remains the same. This means that if the fraction range of the dextran or the globular proteins decreases, the g number of the centrifugation is increased with the same centrifugation time and vice versa.

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

The embedding buffer has the advantage that when dextran is used, the colloidal solution of dextran reduces the spaces between the particles after the production of the buffer-particle mixture, PP mixture. This fact obviously comes from the chains of dextran. This makes it possible to work with a much higher gravity (g) in the centrifuging step. Namely, the centrifugation step with an acceleration of approximately 80xg to 20,000xg during a centrifugation time of 0.1 minutes to <10 minutes, preferably LOOOxg to 4,000xg during a preferred centrifugation time between 1 minute and 5 minutes, in particular 1,900 up to 2,600xg at 2 minutes, or particularly preferably at about 1,900 g for 4 minutes.

Due to the fact that the PP mixture promotes and stabilizes the visualization of the positive reaction, the centrifugation time and the gravitation essentially only depend on the time after which and at which gravitation, g-number, a negative reaction can be clearly demonstrated. This means that in this case the g-number can be increased to further reduce the centrifugation time. This is not possible in the prior art because, for example, in the ID gel technique, a narrow window of g-number, 85xg, and centrifugation time, 10 minutes, is absolutely necessary.

Another advantage of the method is that the PP mixture decisively promotes the positive reaction or the visualization of the monolayer layer. Because the erythrocytes loaded with IgG are pressed under high pressure when using coded vessels against the wall encoded with anti-IgG and held. Due to the PP mixture or the embedding buffer, the spatial distances between the reactants or particles involved are very small, which is why the high g-number is required, which also reduces the centrifugation time. Even with a g-number of 2500 and a centrifugation time of> 10 minutes, the positive reaction of the monolayer remained stable. This means that, due to the PP mixture or the embedding buffer, the results remain practically unchanged over a large gravitational range.

Furthermore, the embedding buffer has 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. This creates a positive lawn: plastic wall / anti-antibody / antibody-erythrocyte complex. The PP mixture also has the advantage that this monolayer layer remains stable and is retained. This means that there is an increased sensitivity for the detection of antibodies.

Without wishing to be bound by theory, the surprising advantageous properties could be due to the unexpected advantageous mechanical properties caused by the PP mixture. It would be possible for the spaces between individual beads to be narrowed by the addition of the PP mixture, and for this to achieve a surprisingly good purification effect when the erythrocytes pass through the gel, which is why, for example, no additives such as anti-IgM for IgM antibodies -Antibody used become. This means that the agglutination complex cannot be pushed through the gel structure even by relatively high gravitational forces. The weaker agglutinates may be pushed through to the walls of the detection vessel by the gravitational forces, but due to the spatial size and the high g-number, they are slower to migrate than individual erythrocytes.

These unexpected advantages have practical implications when performing the procedure. The monolayer layer is formed quickly and is relatively stable, which enables a short centrifugation time with a high g number.

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

Surprisingly, it was also found that good results can be achieved when using coded detection vessels. The antibodies bound to the detection vessels are not bent or displaced by the gravitation during centrifugation by using the PP mixture according to the invention. This effect can be increased even further by adding small amounts of bovine serum albumin or Tween-20.

The term “coded” vessels or walls is understood to mean that antibodies are bound to the preferably used microtiter plates with a pointed bottom, or compounds that bind antibodies, such as, for example, protein A. If, for example, antibodies are bound to the coded vessels that are against the Fc- The antibodies bound to the wall react with the Fc part of antibodies that are in the solution, thereby binding the antibodies in the detection solution to the walls of the detection vessels located antibodies bound to erythrocytes, it is achieved in that the erythrocytes are bound to the vessel wall via the antibody and the anti-antibody.

For example, the market-leading standard method, the ID-gel technique, as a reference method for the detection of irregular antibodies which are known, of 95 high-titre patient sera compared to the method according to the invention has a sensitivity of 87.2% compared to 98% in the invention. With the 95 high-titer The following antibodies were used in patient sera: anti-D 11, -E 9, -c 2, -Cw 2, -D + C 4, -D + E 1, -C + C ^w 1, -E + C ^w 2, - c + E 4, -c + E + C ^w 1, -D + Fy ^a + C 1, -Kell + E + C ^w 1, -S + C ^w 3, -E + Jk ^a 2, -Jk ^a 3 , -Kell 18, -Lu ^a 1, -Lu ^a + Le ^b 2, -Lu ^a + C 1, -Fy ^a 4, -Fy ^b 2, -Le ^b 1, -M 5, AI HA 4, -Fy ^a + E 1, -E + S-M + C ^w + Jk ^b 1, -Jk ^a + Kell 2, -D + Kell 1, -S + Kell + C + Fy ^a 1, Lu ^b 1, Le ^a 3 The number after the name of the sera indicates the number of the respective sera.

The specificity of 104 negative patients corresponded to 97% with the ID-gel technique and 93% with the object of the invention. This is plausible because the more sensitive a test is, the lower its specificity. Of course, it is possible to adjust the balance between sensitivity and specificity optimally or to the desired extent by fine adjustment, such as by fine adjustment of the dextran by changing the concentration or by metered addition of albumin, preferably from bovine serum or by adding the standard buffer “Liss”.

The following is a test result of a reference method of the prior art with respect to the invention:

Column 1 shows the antibody specificities. The antibody specificities specified in column 1 are common abbreviations for certain antibodies. The titers in column 2 indicate the values of a comparison method. The titers of column 3 are based on an incubation time of 5 minutes and a centrifugation time of 2 minutes (7 minutes in total) at 2054 g; the PP mixture is due to the addition of albumin fine-tuned. The titers of column 4 are based on an incubation time of 15 minutes and a centrifugation time of 4 minutes (19 minutes in total) at 2054 g; the PP mixture was in the basic setting according to the invention with dextran without albumin.

For the interpretation of the test results, a higher titer value means that the test procedure is more sensitive because the detection was still possible at a higher dilution. This can be explained by way of example using the antibody specificities c or C. In the reference method according to the prior art, detection was still possible with a titer of 1: 1024. With an incubation period of 5 minutes and centrifugation for 2 minutes (column 3), a positive reaction was still detectable with a titer of 1: 2048, i.e. the method according to the invention is twice as sensitive as the reference method. With an incubation of 15 minutes (column 4), detection was still possible with a titer of 1: 8192, i.e. the method according to the invention is 8 times as sensitive as the reference method.

To ensure sensitivity, another 58 patient plasmas with known clinically significant antibodies with a very low titer were examined in another example. The antibodies were as follows: anti-Lu ^a + C ^w 1, -C 2, -E 7, -D 3, -P- | 2, -C ^w 3, -S 3, -Fy ^a + C 1, -M + Jk ^a 1, -Lu ^a 12, -C + Kell 1, -M 5, -Le ^a 1, -Kell 7, -Jk ^a 2, -Fy ^a 4, -Le ^a + Ie ^b 1, -Jk ^b + M 1 and -E + c 1. The market-leading standard method, the ID gel technique, had a sensitivity of 35%. In the method according to the invention, the sensitivity was 48% at 5 minutes of incubation and 4 minutes of centrifugation, 48% at 10 minutes of incubation, and 15 minutes of incubation and 4 minutes of centrifugation at 76%. This shows that a higher sensitivity can be achieved with the method according to the invention than with the reference method. With an incubation time of 15 minutes, the antibodies, which were very low in titer, had time to bind and thus exceeded the detection limit of the new method. This example shows that the sensitivity is significantly higher than that of the reference method.

In another example, 101 patients with known blood types were examined for ABO, Rh and serum cross-checks. The agreement was 100%.

As a result of these tests, it can be seen with respect to the invention that, contrary to the state of the art in EP 0849595B1, the sensitivity of the invention does not decrease with increasing centrifugation speed or increasing g and thus shortened centrifugation time. Likewise, the sensitivity is still significantly higher than that of the incubation period Reference method. When using a centrifugation of 2,500xg with a centrifugation time of 2 minutes, the results are completely comparable.

Due to the very small spacing of the particles from one another caused by the embedding buffer, no additions of anti-IgM of any kind are necessary for the detection of irregular IgM antibodies, so that neither coding of the vessel wall, nor that of the particles, beads, or as a mixture with Anti-IgM is required; the spatial structure of the Ery / IgM / Ery complex is too large, so that the complex either remains on the PP mixture during centrifugation or gets stuck in the PP mixture or between the PP mixture and the wall.

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

In a further embodiment of the method, a PP mixture is used which has such a low viscosity that the PP mixture is in liquid or pasty form, which, in contrast to the prior art, involves pipetting the PP mixture into the detection vessel by means of a stepper or pipette allowed.

In a particularly preferred embodiment, the PP mixture is pipetted into the detection vessel shortly before the examination. This represents 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 fill into plates or columns. In addition, the problem of the formation of air bubbles within the mixture often occurs. 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 method variant for the detection of antibodies and / or antigens, such as for blood group determination, antibody search test, serum cross-test and for 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 is unbound and / or bound to a carrier and the sample mixture thus produced is subjected to an incubation step, 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 is optical as a sedimentation picture is detectable, the same is that a predetermined amount of the incubated sample mixture is added to a predetermined amount of a mixture of a buffer, embedding buffer, with particles, buffer-particle mixture, abbreviated PP mixture, which is located in a detection 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 centrifugation in order to visualize the reaction of the sample mixture in the detection vessel, the PP mixture promoting the positive reaction. The embedding buffer is made from an aqueous solution of dextran [(C ₆ HιoO ₅ ) _n ] and / or polyethylene glycol [HO (C ₂ H ₄ O) _n H], the embedding buffer being Glycocoll (H ₂ NCH ₂ COOH) (glycine) and / or trisodium citrate (C ₆ H ₅ O ₇ Na ₃ ) can be added. Beads, which are preferably dextran gel SEPHADEX G-50 superfine with a dry diameter of 20 μm to 50 μm, are added as particles to the embedding buffer, with the centrifuging step then preferably at an acceleration of 1900 to 2,600 × g at about 2 to visualize a reaction Minutes centrifugation time is carried out.

The sample mixture is first subjected to an incubation step, which is preferably carried out in an incubator and lasts between one minute and 40 minutes, with the test liquid being mixed thoroughly during the incubation. Permanent forced mixing is not the state of the art in immune hematology. This forced mixing is preferably achieved by shaking or stirring the test liquid. The forced mixing shortens the incubation time and thereby accelerates the reaction time of the antigen-antibody reaction, as does the forced mixing increases the sensitivity of the method. An incubation time of 5 to 7 minutes has preferably been found to be advantageous in the antibody detection test, that of 1 to 5 minutes in the blood group determination and 2 to 3 minutes in the serum cross-test. This offers a standardization time of 5 minutes, so that blood group determination, antibody test and serum cross-test can be carried out with the same PP mixture and the same gravitation number and the same centrifugation time as with one and the same vessel, e.g. microtiter plates.

To carry out the incubation step and the forced mixing, Liss and / or modified Liss can be added to the sample mixture or the erythrocytes. Liss can be used without or modified with bovine albumin. The following Liss based on laboratory water (H ₂ O Bidest) is preferred: Sodium chloride, preferably 1, 788 g / l disodium hydrogen phosphate Na ₂ HPO ₄ , preferably 0.204 g / l potassium dihydrogen phosphate KH ₂ PO ₄ , preferably 0.233 g / l glycine H ₂ NCH ₂ COOH, preferably 18 g / l

Thymerosal C ₉ H ₉ HgO ₂ SNA preferably 0.20 g / l (preservative) gives Liss with 0.03 mol at a pH of about 6.7.

For example, 4 ml plus 0.5 ml laboratory water plus 0.5 ml 22% bovine albumin are taken from this Liss buffer and result in modified Liss. The pH is around 7.1.

In a further embodiment of the process, albumin, such as bovine serum albumin, is added to the PP mixture in order to fine-tune the reaction, and the dextran concentration can be adjusted accordingly simultaneously or independently of this. In the previous table, for example in column 3, the PP mixture was fine-tuned using bovine albumin.

In a further embodiment of the invention, when the same is applied to the infection serology, instead of erythrocytes, colored beads which are encoded as predetermined specific binding partners with proteins are used, which have approximately the specific weight of the erythrocytes. Standard particles - also in contrast to the prior art of EP 0849595B1 - with a diameter of 3 μm to 7.5 μm are advantageously used for this purpose, which have been colored in an advantageous manner. This reaction mixture, which contains the analyte to be determined, is then added to make the reaction to the PP mixture visible.

In this preferred embodiment, the carriers (colored beads) are coupled to binding partners, for example antigens. Such antigens are preferably antigens that play a role in blood analysis. If, for example, an antigen of the hepatitis C virus (HCV) is involved, a test can also be carried out to determine whether the serum to be examined contains antibodies against this pathogen. In this case, an antibody which is directed against HCV will bind to the antigen coupled to the colored beads and if the detection vessel is encoded with anti-antibodies, the colored beads will stick to the walls of the detection vessel and will not form after centrifugation Button in the tip of the detection vessel that would speak for a negative reaction. Portions of the HCV antigen exemplified above can also be coupled to the beads of other antigens, for example antigens from 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 then the sample mixture is applied to the PP mixture inside the detection vessel, the PP mixture being able to be individually filled into the detection vessel as required during processing and thereby an even greater flexibility is guaranteed. Due to the use of two vessels, namely reaction vessel and detection vessel, the flexibility of the method is greater, and the permanent forced movement of the sample mixture can also be carried out. This configuration is particularly suitable for automatic processing of samples, because during the first incubation, in an emergency, individual loading can take place in this plate, which sample can then be placed in the second plate or a strip after a short incubation period of 5 minutes the centrifuge step can be processed further, whereas the extended incubation time acting on the remaining samples of the first plate has no negative effects on the subsequent results.

Of course, it is also possible, when using coded tubes, to add the sample mixture directly to the PP mixture in the tube before incubation; there is no forced movement of the sample mixture. The detection vessels used to carry out the method for receiving the PP mixture are preferably pointed-bottom vessels.

For the antibody test using the PP mixture, it has proven to be advantageous if the inner walls of the detection vessels are encoded with proteins, in particular with anti-IgG, anti-IgA, anti-IgM, protein A, protein G or anti-C3d and / or with a mixture of the same. The lawn formed in a known manner with a positive reaction is extremely stable according to the invention, the reaction is extremely sensitive. It has also been shown that plates prepared in this way can be vacuum-packed or packed under protective gas in the simplest manner and can be stored in the refrigerator for a long time.

In a further preferred embodiment of the method, it has proven to be advantageous if the capture proteins, for example anti-IgG, are stabilized. Stabilizing substances, such as Triton X-100 or albumin or casein or gelatin or Tween 20 or mixtures thereof within a buffer, are used to stabilize the proteins located on the inner walls of the detection vessels. These known buffers with the substances mentioned, which are used in ELISA technology for Blocking non-specific reactions (false positive or increased background) are used here to mechanically stabilize the proteins and thus to visualize the positive reaction. One explanation is that the stabilizing substances envelop the capture proteins, so that the capture proteins cannot be mechanically bent or moved at the high number of centrifugations. This stabilization can either be carried out immediately after coding or only before the coded vessels are used. It has been shown that if the capture proteins are not stabilized, no reasonable positive reaction can be demonstrated when the method is carried out, although such a reaction would have been expected due to the test conditions.

In a further preferred embodiment of the method, the separate reaction vessel for reacting the sample mixture during the incubation is closed with a membrane at the bottom, the reaction vessel and the detection vessel in which the PP mixture is located being able to be added to one another or to one another and to make the reaction visible the reaction vessel is inserted into or onto the detection vessel and both are centrifuged together, the membrane of the reaction vessel becoming at least partially permeable to the sample mixture during centrifugation of the two vessels and this entering the detection vessel.

This configuration using a membrane, which forms the bottom of the reaction vessel, is particularly advantageous for manual processing of samples. Because when the samples are processed manually, 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 for carrying out the incubation and reaction as well as for the detection of the reaction, the vessel being divided into two parts and the PP mixture which is in the lower part of the vessel is covered by a membrane, and above it in the upper part is the reaction space for the reaction of the sample mixture during the incubation. During the subsequent centrifugation of the vessel, the membrane becomes at least partially permeable to the sample mixture. This configuration has the general advantages that the gel or PP mixture used can already be filled into the vessel and is initially securely closed by the membrane. The further advantage is that the incubation step with the forced movement of the sample mixture can now be carried out with one and the same vessel. In this embodiment of a sample vessel with a membrane, the vessel can be practically arbitrarily shaped, for example a pointed-bottom or round-bottom or flat-bottom microtiter plate or a column. A pointed-bottom or round-bottom microtiter plate or strip is evaluated from above or below; a column or flat-bottom plate or strip is evaluated laterally.

The application of a membrane can also be applied to any particle tests, for example gel tests. Such a container for carrying out a method for the detection of antibodies and / or antigens, such as for blood group determination, antibody search test, serum cross-test and for 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 are unbound and / or bound to a carrier and the sample mixture thus produced is subjected to an incubation step, an agglutinate being formed from antigens or antibodies to be detected or the specific binding partners and the carriers in the event of a positive antigen-antibody reaction, which is optically detectable as a sedimentation image is characterized in that a container, which is a round-bottom or pointed-bottom or flat-bottom vessel or a column, is separated by a membrane in two chambers, namely an upper and an upper one lower chamber, the membrane covering the detection layer located in the lower chamber, for example a gel, for the visual visualization of a reaction that has taken place and the upper chamber representing the reaction space for the uptake and reaction of the sample mixture, the membrane being at least partially permeable by a centrifugation step and thereby the sample mixture from the reaction space reaches the detection space.

In the general case, the container can also consist of two separate vessels, the first vessel being closed at the bottom by the membrane and used to hold the sample mixture and possibly carrying out an incubation and the reaction, and the second vessel is the detection layer, for example a gel , contains and serves to visualize the reaction and the two vessels can be joined together or one above the other and both vessels are centrifuged together, the membrane of the reaction vessel becoming at least partially permeable to the sample mixture during centrifugation of the two vessels and thereby entering the detection vessel , In both cases, of course, the sample mixture can be forced to move during the incubation. The membrane can be made of plastic or rubber or a rubber mixture or be a latex or a gel.

Examples for carrying out the method are described below. The drawing shows:

1 shows a V-bottom vessel, for example in a microtiter plate, with a

Membrane Figure 2 shows a round bottom vessel of a microtiter plate with a membrane Figure 3 shows a V-bottom vessel with a spike located beneath a membrane for piercing the membrane and Figure 4 shows a V-bottom vessel with a spike which is arranged above a membrane and can be moved downwards Piercing the membrane.

The following example assumes coded vessels. Commercial V-bottom ELISA plates, which can be divided into 12 or 8 bars, are preferably used. A coding buffer is diluted with specific proteins, such as anti-antibodies, in a ratio of approximately 1: 800 with PbS buffer and a solution is prepared. About 50 μ \ of the solution mentioned are pipetted into each vessel. The vessel is subjected to overnight incubation at room temperature. The solution or remaining solution of coding buffer and specific proteins is removed. A repeated wash step is not necessary. It is then added to each vessel from a PbS buffer containing approximately 1% bovine serum albumin solution. This last step with bovine serum albumin is also beneficial for the uncoded V-bottom plates for examining the blood groups. Because a coating with proteins is generally conducive to adhesion. This means that when the PP mixture is filled, the PP mixture is distributed evenly over the bottom wall of the microtiter plate, making the plate easier to fill.

The vessel is incubated for 1 to 2 hours at room temperature, after which, for example, the vessel is washed out using a washing buffer, which preferably contains Tween 20 and bovine albumin. The vessel is now prepared as a coded vessel and is available for further use as a detection vessel. These have a very long shelf life after vacuum or inert gas packaging. With a plate coded in this way, a workflow for an antibody search test, for example antibody search cells of 1, 2 and 3, can be carried out as follows: In separate reaction vessels, which can be untreated, commercially available V-bottom vessels, microtiter plates, 3 times 25 μ \ serum or plasma plus 50 μ \ search cell suspension 1, 2.3 are entered at 0.3% + 0.1%, the Working dilution is carried out with a modified Liss buffer. The search cell suspension can be prepared from a commercially available 3% +/- 1% stem search cell suspension as required. This has the advantage that these search cells have a very long shelf life in their special buffer, which can be problematic in Liss, and they are easier to manufacture on an industrial scale due to the higher proportion of search cells. For example, in an extra microtiter plate (or tube for a larger series) 270 μl mod. Liss plus 30 μl stem search cell suspension are given.

The tubes are incubated at about 37 ° C and at the same time at a shaking frequency between 700 + 25 rpm for 5 minutes and a sample mixture is generated. During the incubation, the buffer-gel mixture is pipetted into the detection vessels using a 50 μl stepper; 50 μl of the sample mixture are then pipetted onto the PP mixture in the detection vessels. The detection vessels are subjected to a centrifugation step for approximately 2 minutes at approximately 2054 g. The reaction result within the detection vessels is then optically evaluated either visually or automatically. A positive reaction is shown as a monolayer, turf, a negative as a button. When buttons are formed, the non-aggregated erythrocytes collect at the deepest point of the well in the microtiter plate. As this is a microtiter plate, the result can be read either from above or from below.

The plate encoded with anti-IgG once shows IgG antibodies and, due to the PP mixture, also shows any IgM antibodies present. In the event of a positive reaction, a selective distinction as to whether IgG and / or IgM antibodies are present can be made as follows by using a second, uncoded plate with a PP mixture and performing the test again. If a negative reaction with the second plate is obtained, then an IgG antibody is present. If a positive reaction appears similar to the coded plate, then an IgM antibody is present.

If the reaction in the second plate is weaker than that in the first plate, then 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 complement-dependent IgG antibodies that form the C3d antigen on the erythrocyte are sufficient for detection. If no IgM antibodies because of any unspecific reactions are desired, reducing, known agents, for example sodium dithionite, can be used to reduce or lower the IgM antibodies.

For example, an untreated or bovine albumin-treated microtiter plate is again used as the reaction vessel for ABO-RH determination and in addition to performing the serum cross-test. In these are:

a) 50 μl AntiA (Biotest Clone: A003 IgM) of a 1: 100 solution prepared with a Liss buffer or with isotonic NaCI

50 μl AntiB (Biotest Clone: B005 IgM) one using a Liss buffer or with an isotonic

NaCI prepared solution of 1: 100

50 μl AntiD (Immucor, Clone: RUM-1 IgM) one using Liss buffer or with isotonic

NaCI prepared solution of 1:30

50 μl RH mono-Control one made with a Liss buffer or with isotonic NaCI

Solution 1: 30 is pipetted in, b) 25 μl of a 0.6 + 0.2% erythrocyte patient suspension, in which Liss buffer or isotonic NaCI is contained, are pipetted onto the four previously charged vessels (this is for the ABO-RH erythrocyte side determination necessary) c) 50 μl plasma (serum cross-sample) is pipetted 4 times into another four reaction vessels, d) 25 μl each of a 0.6 ± 0.2% suspension containing Liss buffer is produced with A1 cells, A2 cells, B cells and 0 cells and pipetted into the reaction vessels.

The iso search cells can be prepared from a commercially available 3% +/- 1% stem cell suspension as required. This has the advantage that these iso search cells can be stored well in their special buffer, which can be problematic in Liss, and they are easier to manufacture on an industrial scale due to the higher proportion of search cells. For example, in an extra microtiter plate (or tube for a larger series) 240 μl mod. Liss plus 60 μl of stem cell suspension are taken; e) the sample mixtures are incubated for about 2 to 5 minutes at room temperature between 18 to 25 degrees Celsius and at the same time at a shaking frequency between 700 ± 25 rpm, f) in eight untreated or albumin-treated, uncoded detection vessels, 8 times each 25 μl PP- Mixture pipetted in using a stepper, g) 50 μl of the sample mixtures are now pipetted into the detection vessels onto the PP mixture, h) the detection vessels are subjected to a centrifugation step for approximately 2 minutes at approximately 2054 g and the reaction results are evaluated visually or automatically. A negative reaction shows up as a button; a positive reaction can be seen either on or within the PP mixture, but this gives the impression of a lawn. As this is a microtiter plate, the result can be read either from above or from below.

For example, a test kit consists of the following components:

Anti-IgG coded plate (detection plate),

Bottle with PP mixture, blood group plate treated with albumin,

Bottle with modified Liss,

3 bottles with test cells 1, 2, 3, commercially available reaction plate,

4 bottles with iso cells A ^ A ₂ , B, O

A particular advantage of such a test kit is its ease of dispatch and its flexibility.

FIGS. 1 and 2 each show a V-bottom vessel (FIG. 1) and a round-bottom vessel (FIG. 2) with a membrane 2 or 4, which on the wall of the vessel above the V-bottom or the round bottom over the entire Cross section is fixed and the vessel separates into two separate parts. The PP mixture 5 is located below the membrane 2, 4 within the vessel 1 or 3.

With regard to the membrane 2, 4 used, the following design examples are given. A membrane 2, 4 made of latex can be made permeable by a previous perforation, preferably with a diameter of 6 to 8 μm. Since latex is very flexible, these holes are closed at rest. When centrifuging, for example, 2,500xg of a vessel which has previously been filled with a sample mixture of 50 μl, this sample mixture has a weight of approximately 125 g, which is sufficient to widen the membrane openings and to let the sample mixture or the erythrocytes pass. Or a latex membrane 2, 4 has a small membrane thickness and is pre-stressed in such a way that it tears when the vessel 1, 3 is centrifuged even with a weight of a few 10 g.

Figures 3 and 4 show two other options for execution. Within a V-bottom vessel 6, a latex membrane 7 is arranged over the entire cross-section of the vessel 6, namely pre-stressed, above the V-bottom. Beneath the latex membrane 7 there is - in addition to the PP mixture 5 - a spike 8 on the sloping wall of the V-bottom, which protrudes in the direction of the membrane and extends to the membrane 7.

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

During centrifugation, the membrane 7, due to the centrifugal force, cushions downward in the direction of the bottom of the vessel and penetrates the spike 8 or presses on the edge and is thus caused to tear.

FIG. 4 shows a further V-bottom vessel 9 with a membrane 10, which is stretched above the V-bottom over the entire cross section of the vessel 9. Above the membrane 10, a movable spike 11 is arranged on the wall of the vessel 9, which extends to the membrane 10. If, for example, it has a weight of 0.5 g, a centrifugation of 2,500 x g results in a spiked weight of 1,250 g, so that the membrane is pierced and brought 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 test and the cross test. It is a one-step centrifugation process that does not require any washing steps. A complete blood group determination and an antibody search test is possible within 7 minutes, which is why the procedure is particularly suitable for emergencies. Due to the variable incubation times, the procedure is suitable for emergencies and very well suited for semi and full automation. Due to the identical basic reagents, incubation times and centrifugation times, the complete blood group determination with serum cross-test, antibody test and cross-test is possible on a microtiter plate. It is also possible to work through infection serology, such as hepatitis B, on the same microtiter plate.

Claims

 claims: 1. A method for the detection of antibodies and / or antigens in a test liquid by reaction with a predetermined specific binding partner, the Antigens are bound to a support and the sample mixture produced in this way is exposed to an incubation step and then centrifuged through a particle-buffer mixture, an agglutinate from which is to be detected if the antigen-antibody reaction is positive Antigen, antibody and carrier is formed, which is optically detectable as a sedimentation image, characterized in that a predetermined amount of the incubated Sample mixture is added to a predetermined amount of a buffer-particle mixture, which is located in a detection vessel, wherein the Buffer-particle mixture has an embedding buffer which consists of an aqueous solution of dextran and / or polyethylene glycol and glycine and / or trisodium citrate. 2. The method according to claim 1, characterized in that the embedding buffer is an aqueous solution containing 0.1 to 500 g / l dextran and / or 1 to 100 g / l polyethylene glycol, as well 1 to 100 g / l glycine and / or 0.01 to 10 g / l trisodium citrate. 3. The method according to claim 2, characterized in that the dextran has a molecular weight of approximately 100 to 40,000,000, preferably of 2,000,000 (2 10 ⁶ ) g / mol, and in a range from 5 g / l to 40 g / l, preferably 20 g / l is present in the embedding buffer. 4. The method according to claim 2 or 3, characterized in that the polyethylene glycol has a molecular weight of about 50 to 40,000 g / mol, preferably 3500-4500 g / mol, and in a range of 10 g / l to 70 g / l , preferably 40 g / l, is present in the embedding buffer. 5. The method according to claim 2, 3 or 4, characterized in that the glycine is present in a range between 10 g / l to 25 g / l, preferably 18.0168 g / l, in the embedding buffer. 6. The method according to claim 2, 3, 4 and 5, characterized in that the trisodium citrate (C ₆ H ₅ O ₇ Na ₃ ) in a range between 1 g / l to 5 g / l, preferably 2.581 g / l, in Embedding buffer is present. 7. The method according to any one of the preceding claims, characterized in that the embedding buffer has a pH of 4 to 9, preferably from 6.9 to 7.1. 8. The method according to any one of the preceding claims, characterized in that beads are used as particles within the buffer-particle mixture. 9. The method according to claim 8, characterized in that the beads used are acrylic, gelatin, polyacrylamides, solid nets, silica, Ficoll, Percoll, phthalate esters, agarose or dextran gel. 10. The method according to claim 9, characterized in that beads with a dry diameter between 1 micron, to 300 microns, in particular SEPHADEX G-50 superfine, in particular 20 microns to 50 microns is used to produce the PP mixture. 1 1. The method according to any one of the preceding claims, characterized in that for fine adjustment of the reaction, the particle-buffer mixture albumin, such as BSA, is added and / or the dextran concentration is adjusted accordingly. 12. The method according to any one of the preceding claims, characterized in that the particle-buffer mixture has such a low viscosity that it is in liquid or pasty form, which allows the pipetting of the PP mixture by means of a stepper or pipette into the detection vessel. 13. 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. 15. The method according to claim 14, characterized in that the forced mixing of the test liquid is carried out by shaking or stirring. 16. The method according to any one of the preceding claims, characterized in that this Liss and / or modified Liss is added to carry out the incubation step and the forced mixing of the test liquid. 17. The method according to claim 1, characterized in that the centrifugation at an acceleration of 80xg to 20,000xg and during a centrifugation time of 0.1 minutes to <10 minutes, preferably LOOOxg to 4,000xg, and during a preferred centrifugation time between 1 minute and 5 minutes, in particular 1900 to 2,600xg at 2 minutes. 18. The method according to claim 1, characterized in that the separate reaction vessel for reacting the sample mixture during the incubation with a membrane is closed down, wherein the reaction vessel and the detection vessel in which the PP mixture is located can be added to one another and to one another and to visualize the reaction, the reaction vessel is inserted into or onto the detection vessel and both are centrifuged together, the membrane of the reaction vessel becoming at least partially permeable to the sample mixture during centrifugation of the two vessels and this entering the detection vessel. 19. The method according to claim 18, characterized in that the reaction vessel and the detection vessel are one and the same vessel for carrying out the incubation and reaction as well as the detection of the reaction, which is divided into two parts, the PP mixture in the lower part of the vessel is located, which is covered by a membrane, and above it in the upper part is the reaction space for the reaction of the sample mixture during the incubation, the membrane becoming at least partially permeable to the sample mixture during the subsequent centrifugation of the vessel. 20. The method according to claim 18 or 19, characterized in that the membrane consists of plastic or rubber or rubber mixture or latex or gel. 21. The method according to any one of the preceding claims, characterized in that the detection vessels for receiving the particle-buffer mixture are pointed bottom vessels or columns. 22. The method according to any one of the preceding claims, characterized in that the inner walls of the detection vessel are coded with proteins, in particular with anti-IgG, anti-IgA, anti-IgM, protein A, protein G and / or with a mixture thereof. 23. The method according to claim 22, characterized in that stabilizing substances, such as Triton X-100 or albumin or casein or gelatin or Tween 20 or with mixtures thereof, are used to stabilize the proteins located on the inner walls of the detection vessels. 24. The method according to claim 1, characterized in that proteins are either mixed into the particle-buffer mixture or the beads are encoded with proteins. 25. The method according to claim 24 for use in infection serology, characterized in that the antigens bound to a carrier are protein-encoded, colored beads which have approximately the specific weight of erythrocytes.