WO2004019038A2 - Procede pour identifier des anticorps et/ou des antigenes dans un liquide a tester, notamment lors d'une determination de groupe sanguin - Google Patents

Procede pour identifier des anticorps et/ou des antigenes dans un liquide a tester, notamment lors d'une determination de groupe sanguin Download PDF

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Publication number
WO2004019038A2
WO2004019038A2 PCT/EP2003/008995 EP0308995W WO2004019038A2 WO 2004019038 A2 WO2004019038 A2 WO 2004019038A2 EP 0308995 W EP0308995 W EP 0308995W WO 2004019038 A2 WO2004019038 A2 WO 2004019038A2
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Prior art keywords
mixture
buffer
vessel
reaction
detection
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PCT/EP2003/008995
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German (de)
English (en)
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WO2004019038A3 (fr
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Jörg Spindler
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Deutsches Rotes Kreuz Blutspendedienst Baden-Württemberg-Hessen Gemeinnützige Gesellschaft Mbh
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Priority to AU2003260413A priority Critical patent/AU2003260413A1/en
Priority to EP03792313A priority patent/EP1532454A2/fr
Priority to CA2495728A priority patent/CA2495728C/fr
Publication of WO2004019038A2 publication Critical patent/WO2004019038A2/fr
Publication of WO2004019038A3 publication Critical patent/WO2004019038A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells

Definitions

  • 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 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.
  • 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.
  • 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.
  • test liquid for example serum
  • isoagglutinins antibodies
  • 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.
  • ID-gel technique 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.
  • 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.
  • 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 ⁇ m 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the embedding buffer can be in a pH range from 4 to 9, preferably from 6.9 to 7.1.
  • 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%).
  • 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.
  • an embedding buffer is produced as follows:
  • 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.
  • 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 ⁇ m and 300 ⁇ m, preferably a dry bead diameter between 1 ⁇ m and 300 ⁇ m.
  • the use of the dextran gel SEPHADEX G-50 superfine, in particular between 20 ⁇ m to 50 ⁇ , has become Production of the PP mixture proved to be advantageous.
  • SEPHADEX beads between 20 ⁇ m and 300 ⁇ m 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.
  • 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 ⁇ ⁇ 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.
  • 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.
  • 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.
  • 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.
  • the monolayer layer is formed quickly and is relatively stable, which enables a short centrifugation time with a high g number.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the PP mixture is pipetted into the detection vessel shortly before the examination.
  • the standard gels known from the prior art are highly viscous and are therefore difficult to fill into plates or columns.
  • 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.
  • 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 H ⁇ oO 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 ⁇ m to 50 ⁇ m, 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.
  • 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
  • albumin such as bovine serum albumin
  • the dextran concentration can be adjusted accordingly simultaneously or independently of this.
  • the PP mixture was fine-tuned using bovine albumin.
  • the carriers are coupled to binding partners, for example antigens.
  • antigens are preferably antigens that play a role in blood analysis.
  • 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.
  • 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.
  • 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.
  • the detection vessels used to carry out the method for receiving the PP mixture are preferably pointed-bottom vessels.
  • 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.
  • 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.
  • the capture proteins for example anti-IgG
  • 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.
  • 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.
  • 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.
  • 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.
  • the membrane becomes at least partially permeable to the sample mixture.
  • 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.
  • a membrane can also be applied to any particle tests, for example gel tests.
  • 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.
  • 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
  • Figure 4 shows a V-bottom vessel with a spike which is arranged above a membrane and can be moved downwards Piercing the membrane.
  • 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 ⁇ ⁇ 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.
  • 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.
  • 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 ⁇ ⁇ serum or plasma plus 50 ⁇ ⁇ 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 ⁇ l mod. Liss plus 30 ⁇ l 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.
  • the buffer-gel mixture is pipetted into the detection vessels using a 50 ⁇ l stepper; 50 ⁇ l 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.
  • 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.
  • 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.
  • 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.
  • these are:
  • Solution 1 30 is pipetted in, b) 25 ⁇ l 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 ⁇ l plasma (serum cross-sample) is pipetted 4 times into another four reaction vessels, d) 25 ⁇ l 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 ⁇ l mod.
  • Liss plus 60 ⁇ l 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 ⁇ l PP- Mixture pipetted in using a stepper, g) 50 ⁇ l 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.
  • test kit consists of the following components:
  • Anti-IgG coded plate (detection plate)
  • test kit 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.
  • a membrane 2, 4 made of latex can be made permeable by a previous perforation, preferably with a diameter of 6 to 8 ⁇ m. 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 ⁇ l, 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.
  • FIGS. 3 and 4 show two other options for execution.
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne un procédé pour identifier des anticorps ou des antigènes dans un liquide à tester, par mise en réaction avec un partenaire de liaison spécifique prédéfini. Les antigènes sont liés à un support et le mélange d'échantillon ainsi établi est mis en incubation, puis est centrifugé à travers un mélange particule-tampon. Une agglutination d'antigène à identifier, support et anticorps se forme en cas de réaction antigène-anticorps positive. Une quantité prédéfinie du mélange d'échantillon incubé est ajoutée à une quantité prédéfinie d'un mélange particule-tampon se trouvant à l'intérieur d'un récipient d'identification. Ce mélange particule-tampon présente un tampon d'incorporation constitué d'une solution aqueuse de dextrane et/ou de polyéthylèneglycol, de glycine et/ou de citrate trisodique.
PCT/EP2003/008995 2002-08-23 2003-08-13 Procede pour identifier des anticorps et/ou des antigenes dans un liquide a tester, notamment lors d'une determination de groupe sanguin WO2004019038A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU2003260413A AU2003260413A1 (en) 2002-08-23 2003-08-13 Method for the detection of antibodies and/or antigens in a test liquid, particularly for determining the blood type
EP03792313A EP1532454A2 (fr) 2002-08-23 2003-08-13 Procede pour identifier des anticorps et/ou des antigenes dans un liquide a tester, notamment lors d'une determination de groupe sanguin
CA2495728A CA2495728C (fr) 2002-08-23 2003-08-13 Procede pour identifier des anticorps et/ou des antigenes dans un liquide a tester, notamment lors d'une determination de groupe sanguin

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DE2002139568 DE10239568A1 (de) 2002-08-23 2002-08-23 Verfahren zum Nachweis von Antikörpern und/oder Antigenen in einer Testflüssigkeit sowie zur Blutgruppenbestimmung
DE10239568.3 2002-08-23

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WO2004019038A3 WO2004019038A3 (fr) 2004-04-22

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Cited By (1)

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CN103926415A (zh) * 2014-03-24 2014-07-16 上海市血液中心 人血液中血型抗体联合快速检测体系

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DE102006024927B4 (de) * 2006-05-25 2008-12-11 DRK-Blutspendedienst Baden-Württemberg-Hessen gemeinnützige GmbH Verfahren zum Nachweis von Antikörpern und/oder Antigenen sowie zur Blutgruppenbestimmung in einer Testsubstanz
CN102445550B (zh) * 2010-10-09 2014-02-05 苏州苏大赛尔免疫生物技术有限公司 一种ABO、RhD血型定型试剂卡及其制备方法
CN105628940A (zh) * 2015-12-30 2016-06-01 合肥天一生物技术研究所 一种abo亚型血型检测卡
DE102017114537A1 (de) * 2017-06-29 2019-01-03 Endress+Hauser Conducta Gmbh+Co. Kg Sensormembran, Sensorkappe und optischer Sensor

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US5552064A (en) * 1993-02-26 1996-09-03 Ortho Diagnostic Systems, Inc. Column agglutination assay and device using biphasic centrifugation
EP0849595A1 (fr) * 1996-12-18 1998-06-24 Stiftung Für Diagnostische Forschung Utilisations des particules synthétiques comme réactif d'agglutination
US5905028A (en) * 1994-05-17 1999-05-18 Gamma Biologicals, Inc. Method and apparatus useful for detecting bloodgroup antigens and antibodies

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ES2126521B1 (es) * 1997-06-05 1999-11-16 Transfusion De La Comunidad Va Metodo para la deteccion de antigenos presentes en la membrana de hematies, propios o acoplados y de anticuerpos irregulares en muestras de suero.

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Publication number Priority date Publication date Assignee Title
US5552064A (en) * 1993-02-26 1996-09-03 Ortho Diagnostic Systems, Inc. Column agglutination assay and device using biphasic centrifugation
US5905028A (en) * 1994-05-17 1999-05-18 Gamma Biologicals, Inc. Method and apparatus useful for detecting bloodgroup antigens and antibodies
EP0849595A1 (fr) * 1996-12-18 1998-06-24 Stiftung Für Diagnostische Forschung Utilisations des particules synthétiques comme réactif d'agglutination

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103926415A (zh) * 2014-03-24 2014-07-16 上海市血液中心 人血液中血型抗体联合快速检测体系

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EP1532454A2 (fr) 2005-05-25
AU2003260413A8 (en) 2004-03-11
DE10239568A1 (de) 2004-03-04
CA2495728C (fr) 2012-08-21
CA2495728A1 (fr) 2004-03-04
AU2003260413A1 (en) 2004-03-11

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