Self-performed abo blood group determination method

METHOD^' SHAFT FOR AUTO-N CUCIO G-RUPO BLOOD ^"ABO

This invention Renere to methods and devices .for detecting antigen and antibodies determinaάltes ABO groups using immunoassays in card, so

5 Iâ same reaction leaves tm printed record of the group, therefore 'la-card where it is done is the document of the group and the test. Of esta.ínanera the intermediate stage and the error is deleted. transcription performing the annotation. It is a test of self-execution, does not require equipment^' i instrumental, needs no preparation or training and can be performed by any person, including mismo.inter^'esado.'Puede be useful for

10 doctors 'offices, blood banks, halls' emergency, military operations and underdeveloped areas.

BACKGROUND OF THE EWENCÍÓN property received a significant number of deaths from iemolítica reaction transfusioήal

15 attributed to inconxpatibiüdad "ABO". The most common errors are related to environment

'' Délitospital? Are errors in patient identification, EII its location in the identificació sample and the unit change^' transfused. Instead they are^' ost infrequent errors eή identification unit of blood. '^• . It evidenced by both the need for a method that avoids the error in the interpretación0 -from group, in order to confirm a receptor potential ABO group header^' before transfusing sick man, that is reliable, fast, secure,^' ^ simply cheap, without appliances, with. simple means, without subjecting the samples and results at pretratarαieiito - stabilized to allow delayed reading;^{• '} The antigenic determinants of Guineans sa groups present in the HematíeSj are5 gíicoproteínás or glycolipids. Main^'SOU systems^'ABO, D (? Rlι), Lewis, Kidd ,. Kelϊ, etc. The serum of most individuals contains antibodies, also called áglutiήíúas, if d l & ABO_* antígetios of reactive - a group other than their own. So people in the group O have the H antigen and have anti-A and anti-B; individuals of blood type A have anti-B in their serum, etc.0 substances of blood groups differ in sugar residues. auna added common chain which in turn depend on a glycosyltransferase enzyme or enzymes^• A, B and H. The H aαtígenos .construyen on chain ends of oligosaccharides precursors. called Type 1 and Type 2. Ei Gen H produces a fucosyltransferase that adds to the terminal Gal fiícosa chains^"Type 2 using an alpha (1-2) linkage. The genes A and B produce enzymes that add sugars glicόsiltransferasas5 lás'cadenas oligosaccharide antigens H. The N-acetilgalactosaminotransferasa encoded by the A gene and the gene galactosaminotransferasa specified GalNAc and Gal B added respectively at the same Gal residue on which acted transferase gene H? by an alpha bond (1-3). Determining the ABO group by conventional methods comprising agglutination assays and cell lysis. Agglutination antibodies produced by adding groups, anti-A or anti-B, the red blood cells of the sample, which is the direct method; or adding serum or plasma sample to known RBCs: inverse method. Agglutination can be strengthened by gentle centrifugation, adding bovine albumin polyvinylpyrrolidone, etc. Furthermore IgM antibodies are large enough to overcome the repulsive forces of RBCs and extend therebetween. These clumps can be produced in tubes, glass plates, cards with impervious surfaces, etc. There are also methods in card, where the serum with antibodies dried over plasticized nitrocellulose waterproof and serum is reconstituted with distilled water. Lysis is produced by adding the antibody and complement to the blood sample. Plug fixed on the membrane results in the rupture gives the cell membrane. The evaluation of these tests requires the subjective judgment of experienced personnel to distinguish agglutination of unspecific aggregations that cause formation of clusters. Therefore an interpretation, reading and final record of the results is necessary. This method is also based on an immunoassay in card. In 1977 Horrisberger and Rosset (J. Histochem, Cytochem, 25:.. 295-305, 1977) describe a inmυnoensayo in which gold particles used as a marker. Later Leuvering, Janssen, Valkiers and others have been refined these methods to reach inmυnoensayos side current flow in card.

BACKGROUND These immunoassays are based on the interaction between substances with specific reagents and immunological affinity, such as W / O, biotin / avidin, etc. The target substance of the sample binds to a specific receptor may be an antigen or an antibody forming an antigen-antibody complex, which in turn binds to another reagent, using the bivalence of IgG molecules allowed to react for binding site with the antigen in solution and another antigen fixed on the membrane or porous substrate. For display endpoint should produce a visible signal, for which the detector reagent is conjugated to a tracer, usually colloidal gold, either directly or through protein A that has the advantage of establishing a firm bond with the immunoglobulin IgG . This receptor reagent is absorbed as redisolvible in the membrane in the reaction field or next to the sample window area. Alternatively it can be added to the reaction. the complex antigen-antibody migrates to the solid phase or the membrane area for displaying the result where another is immobilized antibody directed to a different epitope, capturing the complex. When the target substance is an antigen is often used two types of antibodies: a) a monoclonal or affinity purified anticuepo poíiclonal conjugated with the tracer, which is in the field redisolvible or liquid phase reaction. You can also have inert material coated balls or added to the reaction; b) the other preferably monoclonal antibody directed to a different epitope, immobilized in another zone or solid phase will capture and precipitate the above complex antigen / antibody. When the target is an antibody sensitized colloidal gold capable of recognizing an antibody immunoglobulin. The complex is captured in the visible region reactive reagent which is usually the antigen. In this test called sandwich type may reverse roles between antigen and antibody, so that an antibody to detect an antigen may be immobilized. It can also be detected using an antibody an anti-human antibody conjugated to colloidal gold through Protein A as indicator reagent. There is also a competitive assay, in which is immobilized a reagent membrane with a comparable affinity for the antibody immobilized on the membrane. The substance of the sample and the conjugate compete for auxiliary antigen binding sites to the antibody. If there is antigen in the sample labeled antigen is added in the reaction; in this case the presence of color means negative. If no antigen color develops because already occupied sites by sample antigen. The signal is inversely proportional to the amount of antigen. They are used to detect the presence of a substance by binding to two receptors. Opcionahnente can increase the color reaction with intensifying co solutions or a mixture of equal parts of a solution of hydroquinone and silver nitrate. to the final stage 20 uL of this mixture is added. The reaction is stopped by washing with distilled water. There are also other immunoassays that reinforce the reaction by binding to substances such as enzymes or biotin which will bind strongly with streptavidin. Furthermore streptavidin may be coupled to latex particles. One can also use colored latex particles. These immunoassays may further comprise a second or third complex unrelated to the previous one. Furthermore the first and second zone can be separated longitudinally along the membrane. The device contains various components such as: a hard case to hold the assembly with openings or windows to see the result of the reaction and to add the sample and reagents; a solid support consisting of an elongated strip membrane or imnunociOmatográfica suitable material, disposed on a rigid support; absorbent material pads at the top end to drain the liquid sample and reagents; and a filter for separating RBCs blood and other components. The reaction zone and the detection zone of the membrane by capillarity communicate. When a few drops of the fluid sample deposited in the detection zone the specific receptor binding with antibody or antigen is dissolved. If the sample does not flow well was further added a few drops suitable buffer. The complex with the score passively diffuses, by lateral migration through the membrane being captured and precipitated in the capture zone, producing a signal. The rest of the complex conjugated antibodies advanced to the far end for the control of the reaction which is immobilized an appropriate antibody. This way you can control the reation immobilizing in a particular area of ​​the membrane, reagents that will bind the conjugate regardless of the presence or absence of the substance to be detected agent. These reagents can be for example human Ig or Protein A.

SUMMARY The invention is an immunoassay for autorun embodiment of ABO blood, so that the same reaction to produce letters of the group. By removing the outer intervenpión the human factor is discarded. The method is based on the detection of substances that identify the group and antibodies by a reaction immunochromatographic card type "lateral flow" using a marker such as colloidal gold.

DESCRIPTION The present invention incorporates the immunoassay known card type "lateral flow" to detect substances or antibodies in liquid samples, adapting to the determination of the ABO group in order to avoid the error resulting from the intervention of the human factor, in addition to allowing a direct, quick and simple diagnosis. With this method when making the determination the group is printed on the card because the same reaction produces or "writing" letters of the group. The assay is based on detection of agglutinins or substances ABO through said reaction immunochromatography with a marker such as colloidal gold. The end result will produce one or more lines or bands forming the letter or letters of the group. Detecting agglutinins or substances of groups is based upon their binding to a specific receptor, forming an antigen-antibody complex. In the solid phase or the membrane area for displaying the result, they are immobilized as capture reagents antigens or substances of the groups and their antibodies as specific graphics to draw the corresponding letter. This procedure can also express the ABO group producing the reaction, a + or - sign (plus or minus), with preimpresan letters on the cards. The preferred technique refers to a colloidal gold immunoassay, but may serve other bookmarks. Detecting group substances includes substances that identify which can also be enzymes A, B and H. The invention is not restricted to the materials and procedures described, being able to make changes or variations in the procedure and reagents to reach object thereof.

Antibody detection For detection of agglutinins noncompetitive sandwich type assays is preferred. It is used as reagent or receptor binding reagent, type antibodies anti-human Ig conjugated with protein A labeled with colloidal gold, or attached directly to the marker. These reagents are incorporated into the reaction field in contact with the sample.

Area caprine substances of groups including derivatives, extracts, powders erythrocyte membranes erythrocytes or immobilized. These substances are fixed in various graphics as will be shown later. Erythrocyte membranes can be obtained from blood samples which is withdrawn plasma, red cells by centrifugation and a volume of erythrocytes nine volumes of digitonin added to 0.1% in saline are collected. The resulting membranes are washed by centrifugation and collected until a white grain is obtained.

Detecting substances noncompetitive sandwich assays it is also preferred. The objective of the detection of substances of the groups is not isolate these substances but free them from the wall of erythrocytes since as these immunoassays are great sensitivity, can detect small amounts to release may use any hemolysing substance. Furthermore 80% of those substances present in plasma. To release the substances are deposited in the sample chamber 100-300 uL sample (2- 6 drops) to the sample chamber to be tested, and you add one or two solvents gotasde suitable as aqueous dilutions type of inert substances DMSO, dioxane or other. HE. will use as reagent or reagent receptor binding substances, antibodies or polyclonal ono, with Antuã specific, anti-B. These antibodies are conjugated with protein A labeled with colloidal gold or can be conjugated directly to the scoreboard. These antibodies are incorporated into the reaction field absorbed in the membrane. Alternatively they can be. adhered to introduce inert material balls or added and lyophilized matrix protein to the reaction. It will be used as a reagent or reagent receptor binding substances, mono or polyclonal antibodies, with specific anti-A, anti-B. These antibodies are conjugated with protein A colloidal gold labeled or can be conjugated directly to the scoreboard. These antibodies are incorporated into the reaction field absorbed in the membrane. Alternatively they can be introduced attached to inert material balls or lyophilisates are added and the reaction protein matrix. Capture antibody complex / substances is performed in Souda phase in result display area by means of monoclonal or polyclonal antibodies specific purified anti-A or anti-B immobilized in the desired shape. As indicated are admissible other combinations of detection reagents or capture and modifications or variations to the described processes easily understood by one skilled in these techniques.

COMPONENTS OF THE DEVICE The device contains: a hard case to hold the assembly with openings or windows to add the sample and reagents and to see the result of the reaction; a solid support consisting of an elongated strip immunochromatographic membrane or suitable material disposed on a rigid support; absorbent material pads at the top end to drain the liquid sample and reagents and a filter to separate red blood cells from blood and other components. As the area where the sample is applied is coated by the antibody or conjugated with a marker such as colloidal gold, with affinity for the antigen substance testal- it said. The capture area is immobilized an antibody to precipitate the complex. The reaction zone and the detection zone of the membrane can communicate by capilaiϊdad. In the device (Figure 1) are distributed sequentially one (or two, one on each end) reaction chambers where the sample and the detection field where the reaction will consist of areas or windows displayed is added, meaning horizontal, one after other divided or not by partitions. Among the sample chamber and the windows will be placed, a filter to prevent passage of red blood cells, for example 0.25 um. They can replace the two sample chambers by one but with different membranes, as two or more taijetas one.

Case or outer packaging The device is housed in a suitable container to be comfortable, easy to use and safe to protect materials and reagents from the solid phase and avoid spillage when used. It must be of inert material as any variety of plastic that can be molded. There are available on the market similar large variety polyethylene, polypropylene, polyacrylate, and polystyrene. Any design is appropriate provided that can be integrated and properly align the components. The dimensions are not critical and must allow assembly of the components and leaving openings for the sample and visualization. You can have a length of approximately 2-5 cm. solid support or membrane. It is a suitable material for immunochromatography or immunoassay card as membranes or sheets of inert materials with suitable affinity and sufficient to allow passive diffusion of the reactive side of the test surface porosity sense. porous membrane material is used in the form of flat, thin, elongated strips of opaque white. The preferred solid substrate is a polystyrene polymer or also a microporous cellulose ester especially ester of nitrocellulose that several market. They could also use other microporous and fibrous materials as polymers of natural carbohydrates gelatin, agar, agarose, natural gums, dextran, starches; synthetic polymers nitrocellulose, polyethylene, polyvinyl, polyesters, polyamides, polyacrylamides, polymethacrylates, hydrated gels, etc. There are many commercially available membranes. They are also offered with the treated surface suitable for biological joints as antigen / antibody. They can also be prepared with a treatment based substance with carboxyl and amino groups. The materials used, such as cellulose nitrate, contain negative charges on the surface due to the nitrite groups, thus a more specific binding is achieved. The pore size is important for proper interaction between reactive molecules. They are offered in various sizes. The selection of pore size is not critical and can vary widely in a range for example between 1 and 10 nm. These membranes must have adequate capillarity to aqueous solutions, which in turn depends on the thickness. The larger the thickness is greater availability capture detector reagent and thus more sensitivity but an excessive thickness slows the flow. The thickness of the strip will affect the amount of antibody solution that need be applied to the substrate. It can be 0.5 to 1.0 mm. At the end of the strip can be placed a band of cellulose acetate, cotton or other material to soak it with excess reaction liquid. The membrane was_. cut into strips and mounted on a plastic support using a suitable adhesive. Since the dimensions can be modified depending on the design of the device is indicated. The exposed area can be delimited with hydrophobic or similarly placed and sealed with plastic membrane material. Its extension may be about 150 mm2 (4x4 mm), but other measures are acceptable.

Immobilization of reagents in the membrane. It is performed by covalent bonding or absorption, membrane coating or impregnating. Covalent attachment is by means of substances such as glutaraldehyde. But it is preferable to use printing machines directly sprayed the membrane. There are machines available of this type containing different widths jet to print multiple lines. To prevent nonspecific binding of antigenic sites not occupied an inert protein or immunologically nonreactive with other assay components such as albumin and casein is used. This treatment locking is performed after immobilizing the capture reagent and assemble the device. incorporating reagents it is also provided with the surface coating with sugars for which the detection zone in the indicator reagent impregnating porous material thickness resolubilized. Mobility tetando reagent facilitates the region in which the reagent is applied with ma aqueous sugar or cellulose. Sucrose is preferred but may be other as glucose, lactose or trehalose. They are also found in the pre-treated membranes market. The format of these devices is well known in the sector. Experts in this field know that various components can be changed as porosity, thickness or type of material, etc.

Reagent buffer is a buffer used to dilute the sample to resolubilize reagent conjugated desiccated and for the transport and washing of reagents. Resolubilization physiological saline buffer requires. It must also carry albumin or gelatin for blocking and washing. To reduce background staining and non-specific binding surfactactes, dispersants and detergents, as well as a neutral pH and an ionic salt are used. It must also contain a pH stabilizer, one presenvante and calcium chelator. The pH can be between 7- 10 are acceptable nonionic at a concentration of 0.01 -0.50% (w / v) detergents. Preferred salts NaCl at concentrations of 0-300 mM, preferably 50- 200 mM. As dispersing high molecular weight polymers are used. polyvinyl pyrrolidone (PVP) -40 to 1.4% concentration is preferred. As the etilendíaminotetraacítico calcium chelator (EDTA) it is preferred to mine 5-100 mM, preferably 10-50 and more

20 mM. As pH stabilizer used trizmahidroclorídrico 20- to 30 mM concentration. You can use a conventional phosphate buffer, MES, BIS-TRIS, CLH-TRIS, citrate buffer, borate buffer with concentrations of 5-100 mM, preferably 5-30 mM. a buffer monobasic sodium phosphate dibasic is preferred.

Indicator reagent. The preferred indicator reagent is colloidal gold. Solutions on the market colloidal gold. Furthermore preparing the colloidal gold particles is well known (shown in Frens, N ature 241, 20-1973). It is achieved valias ways and through reduction tetraáurico reacting acid gold chloride with sodium citrate in water producing particles of various sizes. 15-20 nm particles are preferred. These particles are conjugated molecules or coated with antibody or antigen either directly or through intermediate linking reagent such as protein A or protein G. The absorption adapting these substances is accomplished by controlling the concentrations, ionic strength and pH of the mixture. After differential centrifugation or filtration is performed to control the particle size. The method is well known in the sector. To prepare the colloid should be treated glass surfaces with a 2% solution of dimetilclorosilane in chloroform for 30 min. Then wash in absolute ethanol and distilled water. Prepare 1 liter of a 1% solution of gold chloride in water. Heat to 100 °, 10 minutes. Add tannic acid to 1% and 50 ml of trisodium citrate. Boil for 5-10 minutes. Cool slowly. gold colloid is formed with homogeneously dispersed particles of size between

10-15mm. Centrifuge the solution at 20,000 g for 30 min and resuspended in tris buffer pH 8.2.

Add 1% bovine serum albumin and 0.05% sodium azide. Centrifuge and resuspend twice in the same buffer to a final optical density of 1.5 at 540 nm. For conjugation to protein A (can be seen in Horisberger, 1979, Leuvering et al), the solution to pH 6.5 with C03K2 is adjusted 0.1 M. a dilution of 1 mg / ml protein A is prepared in NaCl 0.005 M . each liter of colloidal gold PEG 20,000 is added to 1%. Centrifuge in a gradient of 5% glycerol for 45 minutes at 30,000 g. Discard the supernatant and repeat. Resuspend in a ratio of 1/10 of the initial dilution. Filter membrane 0.45 um. Adjust to a final concentration of 1 OD with 540 nm wavelength with phosphate buffer 1% bovine serum albumin and 0.05% sodium azide. For attachment to antibodies is added to one liter of colloidal gold at a pH of 6, a gram of bovine serum albumin prediluted in 10 ml of distilled water. Stir 2 hours. Membrane filter of 0.2 um. Centrifuge 45 minutes at 4 ° 35,000 g. Discard the supernatant. Repeat twice with distilled water. Adjust the concentration to an optical density of 1 with a wavelength of 540 lira. Add to each 100 ml to 1% glutaraldehyde. Mix 2 hours. Incubate overnight at 2 ° - 5 ° with phosphate buffer, pH 7.4 containing 1 mg of purified polyclonal antibodies. Stabilizing covalent bonds with 1 mg of sodium borohidrado. After incubating 30 minutes stirring solution of bovine serum albumin buffer with glycine at pH 8 and the mixture was centrifuged at 5,000 g for 45 min is added. Continue with two washes with PBS 0.1 M APH 8. Resuspend the final pellet in PBS containing 0.05 M triethanolamine with 0.2% bovine serum albumin and 0.1% sodium azide adjusting at OD 5 with wavelength of 540 n. Save at 2-4 °.

Antigens. Polypeptide antigens and polysaccharides of these groups may be chemically synthesized characteristic doubling antigenic determinants. For this immunoassay substances of natural or synthetic affordable groups are used commercially. Also they can be used extactos substance of affordable groups also on the market. OTA paite can be isolated extracts of these substances of red cell membranes. The substances are diluted in lampón phosphate, borate or carbonatobicarbonato to pH 8-9, at a concentration of 10 ug / ul. These substances are deposited on the field reaction window the sample. To fix the membrane can be coated with bovine seroalbunina support chromic chloride or other fixatives as fibrinogen, polylysine, phytohemagglutinin, concanavalin A, antibodies, etc. Alternatively they may be added to the reaction in vials. A simple method is to incubate overnight at room temperature and washing with PBS-Tween 20. 40 ul of the solution of chemicals are needed to cover the reaction field. It can also be incorporated by known means of printing devices.

Anti-A, anti-B, anti-AB, IgG, IgM or both antibodies are used; may be monoclonal or polyclonal and purified determinants with specificity for blood group so not react with other substances or antigens of other groups present in erythrocytes. These antibodies are commercially available. Addition can be produced by cell culture techniques and purified by affinity using corresponding antigens A, B and D. There methodologies available for the purification of antibodies and for production of hybridomas. They can be diluted in various mediums. phosphate buffers or sodium borate buffer 0.05 M pH 8. The concentrations of antibodies can vary between 100 and 150 ug per ml of solution are preferred. preferably using 120 ug / ml. Are applied to the porous support by direct contact with capillary tubes, special pens, pipettes or syringes or atomizers automatic propelling gases or liquids with a propellant by known processes miniaturized and microelectronic lithography. You can also use templates or strips impregnated with the antibody. To deposit an antibody in a line about 10 mm width and 0.5 may be needed about 20 mu mouse monoclonal antibody.

PROCEDURE The sample is deposited through the opening or reservoir intended for this purpose. the least amount of blood may be used, two or three drops (180 uL). To resolubilized indicator desiccated buffer reagent is added. about 10-15 drops of buffer are necessary. To enter the buffer can be placed in the reservoir a mark equivalent to a certain amount of buffer, but it is preferable to add a sterile pipette. By adding the reservoir drops to contact the redisolvible antibody. Free sample RBCs flows laterally through the absorbent pad contacting the free antigen binds to the antibody as spurious components diffuse beneath the absorbent pad.

DESCRIPTION OF THE DRAWINGS Figure 1: standard card with compartments cited: one or two sample chambers and areas or windows to display or not by partitions separate reaction. Figure 2: diagram showing the arrangement of the antigenic substances to form the letter O. The reagents are immobilized on the windows and are presented in faint traces of course not seen. Figure 3: diagram showing the arrangement of the antigenic substances to form the letters B and A with the overlapping letters forming an unreadable graphic on the left side, and separate right. Figure 4: diagram of the device with three windows showing the arrangement of substances B and A to identify the groups A and B with the expected result: one of the letters and a semicircle. Figure 5: diagram of the device with three windows showing the arrangement of substances

B and A to identify the group or with the expected result: an unreadable graphic and a circle. Figure 6: diagram of the device with three windows showing the arrangement of antibodies for the detection of substances of the groups enn the left. Thus the groups are identified

A, B and AB. In the right diagram for the detection of agglutinins shown to identify the group O. Figure 7: Scheme of the device having the arrangement of the substances A and B to identify the groups A, B and O by the method of detecting agglutinins, showing prerecorded common sections of the letters A and B overlap.

Embodiments As already indicated, with this device the substances that define the group identified

ABO by detecting group antigens and antibodies or agglutinins so that the reaction reveals the group. an embodiment based on detection of antibodies and ota based on detection of substances are proposed. As noted, it is also possible to identify this group group detecting enzymes, enzymes A, B and H, using anti-enzyme antibodies. In this case the solution is the same as the proposal for the detection of substances or antigens of the group.

DETECTION OF GROUP ABO AGGLUTININS The reaction will produce the corresponding letters in the presence of agglutinins group. This method has the disadvantage that the AB group would be blank and the O group appear as an AB. To avoid this it is proposed to have a card with two windows after the other laterally. It is proposed to leave in the case or rigid support a split between the two windows in order from the card and discard a part. Optionally you can use two etas tai, or taijetas with separate membranes. As mentioned, it is used as detecting reagent conjugated anti-human Ig colloidal gold incorporated redisolvible to the area adjacent to the sample chamber. To identify the group O substances A and B are used as capture reagents are immobilized in complementary graphics to draw a paite of mine or with each. These graphics can be in the form alternate lines, alternate quadrants or semicircles vertically or horizontally, so that the positivity separately from only one of them will produce an unreadable chart. Preferred semicircles horizontally (Figure 2). To produce the group A is set to the next window, substance B as A preferably with rounded upper paite (Figure 3). To produce the B group is fixed or immobilized substance A in the form of letter B on the letter

A so that the three lines are parallel lines transverse to different heights to the transverse line A (Figure 3); the side lines are also parallel to the A. It is preferred to round the right angle formed by the transverse lines and the right sideline slightly. As indicated these reagents are immobilized on the membrane by spray printing machines directly membrane. There are machines available of this type containing different widths jet to print multiple lines. The result of the superposition of these two letters unreadable produce a graph. To avoid the interpretation or the intervention of the human factor, objectives of the present invention may be valid any other design. Expected results: the O group appears as a circle or circle and an illegible next figure. In this case you can separate the card through the slot and discard the window with the unreadable chart. Groups A and B will appear with the corresponding letter and a semicfrculo or adjacent arc (Figure 4). Although these two groups to present a semicircle there can be no confusion and in any case the unreadable graphic can be taken as a control. Optionally you can also split the card and discard this part. The AB group is obtained by exclusion. Alternatively it can be performed in separate card by the method of determining substances mentioned below. Must take into account the frequency of this group is usually less than 5%. Unreadable graphic produced by superimposing the letters A and B have common sections on the sidelines; therefore also it proposes another design consisting leave prerecorded these lines in the common part, in a color as close as possible to the reaction. These lines can be recorded by printing methods or screen printing (Figure 7).

DETECTION OF SUBSTANCES AND AGLUT1MΪMAS GROUP ABO With this method the group O, having no substance A or B, is blank, so it is proposed to have a card or device with windows or zones of successive display following others destmadas the letters a, B and O. It is also proposed to introduce in the case or rigid support weaker between the window for the letter B and O, in order to discard the card and from a party band. Optionally cards can be used with separate membranes destining one for groups A, B and AB, and one for the O group (Figure 6). Logically order among the last group and the above can be reversed. Receptor reagents are incorporated into the membrane as redisolvible absorbed in the area adjacent to the sample chamber. preferably using monoclonal antibodies anti-A and anti-B with colloidal gold conjugated directly or through protein A is applied by means of pipettes or automated spray as indicated above. Optionally you can add lyophilised in vials or ampoules. In areas or windows to display the result capture, purified monoclonal or polyclonal, anti-A and anti-B in the form of the corresponding letter antibodies are immobilized. They are incorporated into the membrane by printing machines as discussed. Both windows must be adjacent one after another. With this part they are identified and expressed groups A, B and AB. The group O is performed through the detection of agglutinins, in the last window or separate card as^" It indicated. For it is used as a reagent detector anti-human Ig conjugated with colloidal gold, which is incorporated redisolvible to the area adjacent to the sample chamber. As capture reagents A and B substances are immobilized complementary graphics to draw a part of a O each. These graphics can be in the form alternate lines, semicircles altemos quadrants or vertically or horizontally, so that the positivity separately only one-they will produce an unreadable chart. Preferred semicircles horizontally (Figure 4). Expected results: Group A will produce an A in the window for A and B equally in the window. In the part for the group or groups A or B will produce an unreadable graphic in a semicircle. The AB group will be identified by the union of the two letters A and B. Those skilled in the art will recognize that changes, modifications or variations are supported in the form, details, method and device, various combinations, techniques, additions or omissions it can be done without departing from the spirit and scope of the invention. Therefore such modifications are included in the claims of the present invention.

PERFORMANCE OF GROUP ABO and Rh by signs positive or negative determination of these and other groups using the plus or minus signs is also proposed (+ / -). • The letters would be printed in adjacent windows. The Rh.se identified by antigen detection D. The methodology would be similar to that described above but a vertical line instead of a point on a horizontal control line derived from monitoring occurs.