MXPA00005608A - A method for identifying a mycobacterium - Google Patents

Abstract

The invention relates to a method for identifying a Mycobacterium species comprising the steps of:a) contacting at least one immuno-cross reactive antigen component of a mycobacterial species with a sample of a body fluid of a human or animal individual;b) contacting at least one antibody, which is capable of reacting with a mycobacterial antigen, with said body fluid sample;c) detecting the presence of antigen-antibody complexes, and identifying the Mycobacterium species present in said body fluid sample.

Description

METHOD FOR THE IDENTIFICATION OF MICOBACTERIUM SPECIES The invention relates to a method for identifying Micobacterium species responsible for a mycobacterial infection in a human or animal, and to diagnostic kits for use in said method.

The genus Mycobacterium. It contains approximately 50 species. It is responsible for a number of diseases which are collectively known as mycobacteriosis. The most known and the most widely spread is leprosy, caused by M. leprae, and tuberculosis caused by M. tuberculosis. Both diseases affect more than ten million people worldwide. Most other mycobacteria occur only as environmental saprophytes. However, they can also cause opportunistic diseases, which occurs frequently, but not exclusively, in organisms that suffer from problems with their immune system, such as patients with AIDS or people suffering from immunosuppression. Opportunistic types include the slow-growing species M. avium, and their close relative M. intracellulare and M. scrofulaceum (usually referred to collectively as the MAIS complex), M. kansai, M. marinum and M. ulcerans, and the species of rapid growth M. chelonae and M. fortui tum. Despite being quite rare in REF .: 120786 the Western world for several decades, the occurrence of opportunistic mycobacterial diseases and tuberculosis have shown a significant increase with the incidence of AIDS. In addition, it has been reported that mycobacteria or antimicrobial agents of mycobacteria play a role in the etiology of a plurality of other diseases, such as sarcoidosis and Crohn's disease, as well as different autoimmune diseases, such as autoimmune dermatitis, rheumatoid arthritis. and diabetes. It has been suggested that this role can be attributed to a structural mimicry between epitopes of mycobacteria and those of the host organism.

The cell walls of mycobacteria are very complex and contain many different lipids, some of which have structures unique to the genus. These structures comprise mycolinic acids and esters, peptide-glycolipid, arabino-galactane and lipo-arabinomanana. The lipid-rich cell walls of a mycobacterial cell are responsible for the remarkable coloring properties of mycobacteria. They also allow mycobacteria to counteract an attack of the immune system of a host organism. A number of species, after being taken up by macrophages, surround themselves with a thick layer of secreted lipids.

Several of the different components of mycobacteria interact with the immune system of a host organism. These components comprise proteins and hydrocarbon antigens, which can be actively secreted by the mycobacterium or form part of the cell wall or cell membrane. In addition, they can be present in the cytoplasm, for example in the cytoplasmic matrix, ribosomes and enzymes. Mycobacteria also possess immunomodulatory components, such as immunosuppression components and adjuvants. As a consequence, a simple mycobacterial species can induce a wide variety of immune responses in different forms with diverse specificities. This makes it very difficult to obtain protein antigens which are susceptible to the detection of species-specific humoral responses as a basis for a sensitive and specific diagnostic test for the above-mentioned diseases, particularly tuberculosis. Because mycobacteria have a frequent occurrence, the fluids of the human and animal bodies almost always have antimicobacterial antibodies.

In the past, researchers have tried to develop a diagnostic test sensitive enough for icobacteriosis. The focus of these attempts has been on the discovery of species-specific glycolipid antigens for the detection of specific humoral immune responses, due to problems with the specificity of protein antigens.

In the application of the international patent 94/14069, the use of the antibody response of an organism to cross-reactive immunodominant mycobacterial antigens (hereinafter referred to as Im-CRAC) to develop a diagnostic test for infections has been described. mycobacterials. It is believed that Im-CRACs provide indirect information about the nature of the immune recognition of, and that they respond to, the specific, mycobacterial pathogen.

The method proposed in WO-A-94/14069 is based on the discovery that the clinical manifestation of mycobacterial diseases is related to the capacity of variation of an individual host to produce a humoral response to different immuno-cross reactive antigen components (Im -CRAC). Each mycobacterial infection generates its own response to a number of specific antigens. The analysis of the response to the antibody, for example by immunoblotting tests, has shown that immunodominant Im-CRAC varies according to the in-pathological manifestation of mycobacterial diseases. Such analyzes result in different and distinguishable pattern bands of mycobacterial antigens for different individuals which are infected with different species of Mycobacterium. The band pattern, which is obtained after an immunoblot, can be referred to as an Antigen Bar Code. The antigen-antibody reactions which are shown in the bar code are, when taken together, unique to certain species of Mycobacterium.

The present invention has the purpose of providing an improved method for identifying the Mycobacterium species in a diagnostic test. Despite being satisfactory in most aspects, it is still desirable to possess a diagnostic test which is even more sensitive than the method described in WO-A-94/14069. In addition, there is a need for a diagnostic test that can be used in the determination of a previous vaccination, and to monitor the therapy of organisms infected with Mycobacterium species. During therapy, there are situations where low levels of certain antibodies occur, which can alter the accuracy and / or sensitivity of the test by using the antibody-antigen cross-reactions, as mentioned above.

It has now been found that a highly sensitive diagnostic test can be performed by contacting a sample of a body fluid with antibodies and antigens. It has been found that, in addition to antibodies, several mycobacterial components are present in animal and human body fluids, whose presence can be determined by the use of cross-reactions with a selected set of antibodies in a reliable manner.

Therefore, the invention provides a method for identifying My cobacterium species comprising the steps of: a) contacting at least one immuno-cross reactive antigen component of a mycobacterial species with a sample of a body fluid from a human or animal individual; b) contacting at least one antibody, which is capable of reacting with a mycobacterial antigen, with said body fluid sample; c) detect the presence of antigen-antibody complexes, and identify the Mycobacterium species present in said sample of body fluid.

Surprisingly, a highly reliable test based on the concept has been developed as mentioned above. By using the method of the invention it is possible to monitor the different stages of a treatment of a mycobacterial disease.

Also, it is possible to determine if an individual has been vaccinated for a mycobacterial disease, and for which mycobacterial disease. As each organism infected with a species of Mycobacterium does not show the same reaction to such an infection, a very reliable test is provided, in which a wide area of antigens and antibodies that may be present can be covered by the test method.

The present method can be applied to any body fluid sample of any animal or human individual. Of course, the most reliable results are obtained with a sample of a body fluid in which the presence of a species of Mycobacterium is found more frequently. Examples of suitable body fluids include serum, blood and excretion fluids, such as sputum, saliva, CSF (cerebrospinal fluid), or tear fluid. These body fluids may be subjected either directly according to a method of the invention, or may undergo some type of pretreatment. Usually, bodily fluids will be diluted by dissolution in a buffer solution prior to the diagnostic test.

In a preferred embodiment, the saliva or a solution or its preparations is subject to a diagnostic test to identify the Mycobacterium species. If necessary, the mucus-like structure of the saliva can be removed by treatment with, for example, xylometasoline or by another known method. The invention also encompasses a diagnostic test for identifying Mycobacterium species wherein a saliva sample is contacted with at least one immuno-cross reactive antigen component of a mycobacterial species, and / or at least one antibody which is capable of reacting with a mycobacterial antigen, detecting the presence of antigen-antibody complexes, and identifying the Mycobacterium species present in the saliva sample. Surprisingly, the high content of several enzymes in saliva does not alter the reliability of the diagnostic test to any substantial degree. Alternatively, the effect of the enzymes on the test results can be conveniently canceled.

The realization of a test in the saliva has proved surprisingly, to be very successful and reliable. Apparently, mycobacterial infections are sufficiently manifested in saliva. The accumulation and use of saliva has several advantages over the use of more conventional body fluids, such as serum or blood. It is relatively easy to accumulate, even under the difficult conditions of activity, which are frequently found in Third World countries where both tuberculosis and leprosy have high incidence. In addition, saliva is non-aggressive, which increases compliance by individuals to be evaluated, requires minimal training to store it and has a minimal biological risk in its collection, transportation and evaluation, particularly in areas where there is incidence of HIV infections.

As stated above, according to the present invention a sample of a body fluid is contacted with antigen or its preparation and an antibody or its preparation. Very good results have also been obtained with a test in which a sample of a body fluid is contacted with only one antibody, which is capable of reacting with a mycobacterial antigen, or its preparation. The inventions also encompass a method for the identification of Mycobacterium species comprising the steps of contacting at least one antibody, which is capable of reacting with a mycobacterial antigen, detecting the presence of antibody-antigen complexes, which have been formed between said antibody and one or more antigen components of the Micobacterium species, and identifying the Mycobacterium species present in said body fluid sample.

According to the invention, it is possible to contact a body fluid sample first with the antigen and subsequently with the antibody or vice versa. In any case, it is preferred that the body fluid sample is contacted with the antigen and the antibody simultaneously. This can, for example, be carried out by passing the sample of body fluid over two surfaces, one of which the antigen or its preparation has been circumscribed, and to another of which the antibody or its preparation has been circumscribed. Based on this principle, the skilled person will be able to develop several ways to perform the diagnostic test. It will be clear that when the method is chosen such that the antigen and its preparation and the antibody or its preparation are in contact with each other, the antigens and antibodies used have to be selected in such a way that they do not react with one another.

The at least one immunoreacting reactive antigen component may comprise the whole or a fraction of the components of Mycobacterium or the total or a fraction of the culture medium of Mycobacterium. In principle, any fraction of Mycobacterium or its culture medium can be used. Such a fraction can be obtained in a conventional manner, and is advantageously separated by electrophoresis prior to the method according to the invention as described in WO-A-94/14069 to provide a strip pattern after the test has been performed. Usually, the antigen component will mainly comprise lipo-polysaccharides. However, proteins will often also be present. According to the invention, lipo-polysaccharides and proteins having antigenic action can be used separately or in combination.

Preferred antigen components are chosen from a non-exhaustive group of KATG, MPT63 (= 18 kD), MPT64 (= 24 kD), MPT51, MTC28, Ag85a (= 30-31 kD), Ag85b (= Ag6), Ag85c, Ag5 (= CIE Ag78 e = 38 kD), DES, MPB70, 80 (= 22/23 kD), Lipoologosaccharides (LOS), lipoarabinomannan (LAM), PMB67 (67 kD), isocitrated Dehydrogenase, Malate Dehydrogenase, 2,3-diacyl -trehalosa (DAT), phenoliglycolipid (PGL), ESAT6 (= 6 kD), hsp70 = Dnak = Ag63 (= 71 kD), CIÉ Ag82 = GroEL and homologs (= 65 kD), GroES and homologs = BCGA (= 10 kD) ), Antigen 60, as well as those with molecular weights of 6 kD, 10/12 kD, 16 kD (usually referred to as 14 kD), 18 kD, 19 kD, 21 kD, 22 kD, 23 kD, 24 kD , 28 kD, 29 kD, 30 kD, 32 kD region, 33 kD, 34 kD, 36 kD, 38 kD, 42 kD, 50-55 kD, 60 kD, 65 kD, 67 kD, 71 kD, 88 kD, and 95 kD.

Preferably, the at least one immuno-cross reactive antigen component comprises a KP90, KS90, antigen, KP100, or SP100 fraction, or a culture medium fraction of a preparation of Mycobacterium species. It has been found that these antigen preparations provide very reliable test results. In addition, they are stable enough to be stored for a long period of time without affecting the reliability of the diagnostic test.

The at least one unique antibody for mycobacterial species with which the sample of a body fluid is contacted according to the invention, can be any antibody that is capable of reacting with a certain mycobacterial antigen. Such an antibody or its preparation can be obtained in a manner known to the skilled artisan. According to the invention, it is preferable to use monoclonal antibodies, although the use of polyclonal antibodies has also proven to be adequate. Preferably, IgG, IgA, IgM or its combination is used as the antibody. These antibodies have proven to be very convenient to handle in a diagnostic test and to lead to very satisfactory results.

In a preferred embodiment, the immuno-cross reactive antigen component or at least the only antibody for mycobacterial species is immobilized on a support. Both non-solid supports and solid supports can be used. Preferably, a solid support is used.

Particularly preferred is the embodiment wherein the antigen component and the antibody are immobilized on a support. When steps a) and b) are performed simultaneously, a particularly useful test has proven to be based on an embodiment of the invention wherein the antigen component and the antibody are immobilized on the same support. As stated above, in such a case it is necessary that the antibody does not react with the at least one immuno-crossreactive reactive antigen component. It is advantageous to provide a layer of the at least one immuno-crossreactive reactive antigen component in the upper part of the layer of the at least one antibody, or vice versa. Preferably, a layer of the at least one reactive antigen component in one crosslinked to the solid support is first applied, and then a layer of the at least the single antibody is applied thereon.

The at least one immuno-cross reactive antigen component will generally be used at a concentration of 0.1 to 20 μg / ml, and preferably 1 to 10 μg / ml. When the antigen component is applied to a support, the amount of said component will depend on the test and support chosen. The at least one antibody will generally be used at a concentration of 0.1 to 20 μg / ml, and preferably 1 to 10 μg / ml. When the antibody component is applied to a support, the amount of such component will depend on the test and support chosen. The ratio wherein the at least one immuno-crossed reactive antigen component and the at least one antibody are used, will usually be from 1:10 to 10: 1, preferably from 1: 2 to 2: 1.

Preferred solid supports are chosen from the non-exhaustive group consisting of membranes, such as nitrocellulose membranes, indicator rods, filters, beads, granules and microtitre plates. The antigen component and or the antibody can be immobilized on these supports in a manner known in the art.

After the body fluid sample has been contacted with at least one immuno-cross reactive antigen component and at least one antibody, antibody-antigen complexes may or may not have formed. These complexes can be of two types; both the antibody and the antigen component of these complexes will be from the body fluid. Both or only one type of these complexes may be present. The detection of both types of complexes can be carried out separately or together. Preferably, they are performed simultaneously. Appropriate detection methods are immunoblotting, as has been described in WO-A-94/14069, as well as any of the usual methods of direct or indirect labeling known in the art. Appropriate markers may be chosen from the non-exhaustive group of biotin, biocytin, iminobiotin, digoxigenin, avidin, streptavidin, colloidal coloring substances, fluorochromes, such as rhodamine, reducing substances, such as eosin or erythrosin, colored latex soles, carbon sols, metals, metal sols or other particulate sols, dansyl lysine, infrared dyes, coumarins, enzymes, iodine markers. Particularly preferred is the use of gold labels, colloidal labels, latex solutions, and enzymes. The use of these marks allows the development of a method according to the invention in a so-called rapid test.

After the analysis, the species Micobacterz can be identified appropriately. Although any method of ancemal identification can be used, it is preferred that the species Micobacterium be devised. _: icae on the basis of one or more reference standards.

Of course, the invention also comprises a diagnostic equipment for use in a method as described above. The equipment comprises a support, on which are circumscribed at least one immuno-cross reactive antigen component of a mycobacterial species and of at least one antibody, which is capable of reacting with a mycobacterial antigen and which does not react with said antigen component. immuno-crossed reagent, and means to detect the presence of antigen-antibody complexes. The support is preferably chosen from a group of membranes, indicator rods, filters, spheres, granules and microtiter plates. The means for detection may comprise means for detecting the form as described herein.

The invention will now be illustrated with the following non-restrictive examples.

EXAMPLES Preparation of KP90 / KS90 KP90 was prepared from mycobacterial bulk materials without purification as described in WO-A-94/14069 (the contents of which are incorporated herein by reference). The starting material prepared from 1 gram of lyophilized bacteria was centrifuged at 90,000 x g at 4 ° C for 2 hours. The pellet was washed twice with PBS. Between washing steps, the sample was sonicated on ice for 6x30 seconds with interruptions of 10 seconds and centrifuged at 90,000 x g at 4 ° C for 2 hours. The pellet was collected and resuspended in 10 ml of Tris buffer solution (TBS) 0.05 M of pH 7.4. The floating matter, designated as KS90, can also be used as an antigen. The pellet was sonicated for 6x15 seconds on ice with interruptions of 10 seconds. This preparation is designated by the name of KP90. After the concentration of the protein was determined, amounts of 1.7 ml were frozen at concentrations of 1 mg / ml in 25 mM TBS / 50% glycerol and stored at -20 ° C. The following components have been shown to be present in KP90: LAM (++), 10 kD (+), 16 kD (+), 21 kD (+), 24 kD (), 30 kD (+/-), 31 kD (-), 34 kD (+/-), 38 kD (-), 65 kD (++) , and 95 kD (+).

Preparation of a culture fluid fraction After culturing Mycobacterium tuberculosis for 3 weeks, the bacterium was removed by centrifugation and filtration. The components of the culture fluid were precipitated with 0-45% ammonium sulfate. After centrifugation, the precipitate was dialyzed and further purified by ion exchange chromatography. This fraction is called antigen6. Further purification can be carried out by hydrophobic interaction chromatography.

Preparation of monoclonal antibodies (MoAb) IgG monoclonal antibodies were prepared according to the literature (Clin. Exp. In unol. (1984) 58: 511-521) Saliva preparation Saliva was collected using the Omni-Saltm collection container (Saliva Diagnostic Systems) and stored at -20 ° C. The tube contains 1 ml of a preservative solution. The material holder is designed to hold 1 ml of fluid when saturated, resulting in a 2: 1 dilution of saliva. Before using the saliva, the sample was pretreated with protease inhibitor and triton X and nonidet P40 (0.01%).

Serum preparation The blood was obtained by venipuncture and processed to serum using standard methods.

EIA Coating Prior to coating, KP90 was sonicated for 5x10 seconds on ice with 10 second interruptions.

The microtiter plates were coated with KP90 in successive dilutions in PBS pH 8.0 for 22 hrs a 37 ° C, or with one or more MoAbs (IgG or IgM) against components of M. tuberculosis in successive dilutions in PBS pH 8.0 overnight at 4 ° C. The MoAbs that were evaluated were: a MoAb against a 38kD protein of M. tuberculosis, and a MoAb against LAM. The combination of MoAbs and KP90 coatings was performed by first coating with KP90 and then with one or more MoAbs, or by first coating with KP90 on one half of the well of the microtitre plate, while the plate was placed under an angle followed by coating with one or more MoAbs after the plate was turned 180 °.

After coating, the plates were blocked with 3% BSA for 1 hour at room temperature, dried at 37 ° C and stored at 4 ° C.

Test The serum was evaluated at a dilution of 1: 200 and 1: 400. The saliva samples were evaluated in successive dilutions from 1: 1 to 1: 100. 100 μl were pipetted into the wells coated in a microtitre plate and incubated for 1 hour at 37 ° C. The excess conjugate was removed by washing.

The indication of the presence of human antibodies of the IgA or IgM subtype binding specifically to KP90 and / or the presence of M. tuberculosis components in the sample takes place by the addition of TMB (tetramethylbenzidine) to the wells.

The binding of the enzyme results in the advent of a blue color, after the addition of a color blocking solution, it changes to yellow. This yellow color has an absorption maximum of 450 nm.

The interpretation of the results of the test takes place on the basis of the so-called cut sample. A test sample can be considered positive when the result found in the test has a higher score than the cut sample. The sample cut is based on the results with a large panel of positive or negative arguments.

Results After performing the procedures described above under "Test" and "Coating," the results as shown in Table I were obtained with respect to the presence of antibodies in the saliva. To determine anti-KP90-IgA, it was found that a dilution of saliva of 1: 100 is adequate. To determine anti-KP90-IgC, a dilution of the saliva of 1:20 seemed appropriate. In Table I, samples 38, 39, 40, 44, 4, 18, 25, 31, and 33 were TB-positive, while the other samples were TB-negative.

A combination of a detection of anti-Mycobacterium IgA and IgG antibodies in saliva, where different cuts are used, has an additional value in the diagnosis. In addition, it has been found that the test with saliva in combination with serum has additional value in the diagnoses of Mycobacterium.

Table I: detection of anti-Mycobacterium IgA and IgG in saliva samples. Saliva IgA (saliva +/- bij OD IgG (saliva +/- bij OD 1: 100 OD450) CO = 0.7 1:20 OD 450) CO = 0.22 1 0.689 0.074 2 0.345 0.064 3 0.302 0.050 5 0.321 0.065 6 0.462 0.063 7 0.141 0.071 9 0.509 0.075 10 0.609 0.066"l2 0.446 0.162 15 0.173 0.054 16 0.335 0.108 22 0.561 0.100 23 0.389 0.096 28 0.177 0.064 32 0.305 0.178 34 0.683 0.173 35 0.55 0.213 36 0.567 0.070 38 0.742 0.234 + 39 0.351 0. 364 + 40 0.555 0. 301 + 44 0.801 + 0. 235 + 4 1,074 + 1. 152 + 18 0.833 + 0. 106 25 1.064 + 0. 678 + 31 0.059 2. 204 33 1.945 + 0. 077 To determine the presence of LAM in the saliva, the saliva was first prepared as described above. Additionally, samples were dialyzed to remove triton and salts, which were subsequently frozen-dried and placed in 200 μl of PBS. In the samples thus obtained, 200 μl was pipetted into each well of a microtiter plate provided with a coating as described above. Detection took place as described above using anti-W antibodies. rabbit tuberculosis, which were detected at the time using rabbit anti-rabbit peroxidase-IgG conjugate. The results are shown in Table II.

Table II: detection of LAM in saliva Saliva 0D1 0D2 average 1 TB-positive 0.924 0.97 0.947 2 TB-positive 0.737 0.79 0.764 3 TB-positive 0.472 0.453 0.463 4 TB-positive 0.722 0.734 0.728 TB-positive 0.439 0.431 0.435 6 TB-positive 0.599 0.603 0.601 7 TB-positive 0.72 0.693 0.707 8 TB-positive 0.69 0.565 0.628 9 TB-positive 0.643 0.669 0.656 average 0.659 TB-negative 0.289 0.285 0.287 11 TB-negative 0.353 0.338 0.346 12 TB-negative 0.294 0.293 0.294 13 TB-negative 0.296 0.294 0.295 14 TB-negative 0.298 0.297 0.298 TB-negative 0.315 0.309 0.312 16 TB-negative 0.35 0.34 0.345 17 TB-negative 0.353 0.344 0.349 18 TB-negative 0.253 0.261 0.257 average 0.309 In addition, IgG, IgA, and LAM were detected in sputum samples analogous to those described above for saliva. The anti-W antibodies. tuberculosis were determined in the sputum using KP90.

To detect antibodies in the sputum, the sputum samples were diluted 1:20 or 1:25 (for IgG and IgM) or 1: 100 (for IgA) in a serum dilution buffer from the KREATECH IgA EIA pool. The samples were vigorously shaken for at least one hour. Subsequently, the samples were evaluated for the presence of anti-tuberculosis antibodies by ELISA as described above.

KP90 was coated on a microtiter plate. The cut-off ratio was 0.8 for IgG and IgM and 0.6 for IgA with serum 8-59 as a reference to calculate the ratio. The results are shown in Table III.

Table III: detection of antibodies in sputum Sputum IgG IgA IgM 1 TB-negative negative positive negative 2 TB negative negative negative 3 TB negative negative negative 4 TB negative negative negative negative TB negative negative positive negative 6 TB negative negative negative negative 7 TB negative negative negative negative 8 TB negative negative negative negative 9 TB positive positive positive positive TB-positive positive negative positive 11 TB-positive positive negative negative 12 TB-positive negative positive positive Western Blot Gel-electrophoresis and membrane preparation for the assays were performed as described in WO-A-94/14069, page 13 under item 2.

The tapes were incubated with serum or human saliva diluted 1: 100 or 1: 200 collected with an Omni-Sal container with dilutions ranging from 1: 1 to 1: 100.

For the detection of antibodies in serum and saliva, immunodetection was performed as described in WO-A-94/14069.

Spot Blot Test The antigen (KP90) 1-10 μg / ml and MoAb 0.5-5 μg / ml were dripped separately onto the nitrocellulose membrane. The membrane (blocked or unblocked with BSA) was incubated with serum (1: 200 dilution) or saliva sample (1: 1-1: 100 dilution) in PBS / between / BSA for 1 hour at room temperature. After washing the membrane with PBS / between the membrane was incubated with conjugate: 1. indirect labeling: a combination of anti-IgA or anti-Hu-IgG labeled with peroxidase and a MoAb (IgG or IgM) against the same component of M. tuberculosis, which is labeled for peroxidase monitoring. Detection was performed using AEC substrate (0.8% 3-amino-9-ethylcarbazole in dimethylformamide) diluted 1:10 in AEC buffer (50 mM Buffer Acetate, pH 5.0, 0.1% ureppoxide), DAB (3, 3'-day ino-benzidine tetrahydrochloride) using standard methods, or the ECL • detection system from Amersham. 2. direct labeling: a combination of IgA-antihuman or IgG-antihuman marked with gold and a MoAb (IgG) against the same component of M. tuberculosis, which is monitored, marked with gold.

The IgA-gold conjugate was obtained commercially or obtained as described in, for example: WO-A-96/35696. The MoAb-gold conjugate was obtained as follows: The gold particles were obtained commercially. The conjugate was obtained according to standard procedures or in the case of the platinum-base linkage as described in, for example, WO-A-96/35696.

Rapid Tape Test The antigen (KP90) 1-10 μg / ml and one or more 0.5-5 μg / ml MoAbs (IgG) are immobilized in different lanes on the nitrocellulose membrane. A control lane that depends on the detected Ab (anti-IgA or anti-IgG / protein) is also applied above the other lines. Nitrocellulose is used blocked with 0.1% BSA or unblocked. The anti-IgA-gold conjugate and the MoAb-gold conjugate (MoAb against the same antigen as the immobilized MoAb but recognizing another determinant) are dried on an attached absorbent pad. The IgA-gold conjugate was obtained commercially or obtained as described above. The sample (serum or saliva) is diluted in 1 ml of PBS (pH 7.4) in a glass tube. The test tape is then placed inside the tube with the absorbent pad facing up. The results were determined after 15 minutes to 2 hours, when the control line was colored.

Second procedure: The antigen (KP90) 1.10 μg / ml and one or more MoAbs (IgG) 0.5-5 μg / ml are immobilized on different lanes in the nitrocellulose membrane (see Figure 1). A control lane that depends on the detected Ab (anti-IgA or anti-IgG / protein) is also applied above the other lines. An absorbent pad was attached. The entire sample was labeled with a gold conjugate through a platinum-based link (see e.g. WO-A-96/35696). Then the tape was introduced into the sample with the absorbent pad up. The results were determined after 15 minutes to 2 hours, when the control line was colored.

Agglutination test Coverage The latex particles are washed several times with borate (pH 8.5) or PBS, by filtration, or by the use of ion exchange resins depending on the quality that is needed. The latex particles are covered with antigen (KP90 or antigen6) or with antibody incubating, for 1-16 hours at 37 ° C, 20 ° C or 4 ° C, 1 ml of 1% suspension of the appropriate particles in Borate ( pH 8.5) or PBS containing 0.01% tween-20 and a final protein concentration up to 5 mg / ml. Subsequently the latex is centrifuged and the floating matter containing any non-absorbed ligand is discarded. After several washing steps (see above) the coated latex is resuspended in MES buffer containing 0.1% BSA and can be used in a latex agglutination test or stored at + 4 ° C.

Agglutination Coated latex particles are mixed in several dilutions with serum or saliva sample in a drop on a glass slide. The agglutination test is carried out at room temperature from 1 to 16 hours at an incubation time of + 4 ° C. The agglutination can be described ocularly (appearance of flocculent) or after filtering the mixture. In the latter case, a pore size of the membrane is selected which allows the non-agglutinated latex particles to pass and the agglutinated particles to be retained. When using colored particles the membrane is colored in the case of a positive reaction.

By using different colored latex particles for the antigen (KP90 or antigen6) and the antibody, both reactions can be monitored separately.

Results The serum was tested according to the above procedure using KP90 and polystyrene particles. The incubation time of the test was 5 minutes at room temperature. The results are shown in Figure 1. Figure IA shows a positive reaction, while Figure IB shows a negative reaction.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content is claimed as property - in the following.

Claims (1)

1. CLAIMS A method for the identification of Mycobacterium species characterized in that it comprises the steps of: a) contacting - at least one antigenic component of immunocruced reactivity of mycobacterial species with a sample of a body fluid from a human or animal individual; b) contact with at least one antibody, which is capable of reacting with a mycobacterial antigen, with said body fluid sample; c) detection of the presence of antigen-antibody complexes, and identification of Micobacterium species present in said body fluid sample. A method according to claim 1, characterized in that the sample of a body fluid is selected from the group consisting of serum, blood and excretion fluids, such as sputum, saliva, CSF (cerebrospinal fluid) tear fluid, and its solutions or preparations A method according to any of the preceding claims, characterized in that the at least one immunoreactive cross antigen component is circumscribed in a support. A method according to any of the preceding claims, characterized in that the at least one antibody for the mycobacterial species is circumscribed on a support. A method according to any of the preceding claims, characterized in that steps a) and b) are carried out simultaneously. A method according to claim 5, characterized in that the at least one cross-immunoreactive antigen component and the at least one antibody for mycobacterial species are circumscribed in the same solid support, and in that said antibody does not react with the at least one cross-reactive immunoreactive component . A method according to claim 6, characterized in that the support is selected from the group consisting of membranes, indicator rods, filters, spheres, granules and microtitre plates. A method according to any of the preceding claims, characterized in that the at least one antibody for mycobacterial species is a monoclonal antibody. A method according to any of the preceding claims, characterized in that the detection of the presence of antigen-antibody complexes is carried out using an indirect or direct labeling method. . A method according to claim 9, characterized in that the detection is performed using a marker selected from the group consisting of biotin, biocytin, iminobiotin, digoxigenin, avidin, streptavidin, colloidal coloring substances, fluorochromes, such as rhodamine, reducing substances, as eosin or erythrosin, latex soles (colored), carbon sun, metal sol or other particulate sun, dansyl lysine, infra red dyes, coumarins, enzymes, and iodide markers. A method according to any of the preceding claims, characterized in that the species Micobacterium is identified based on one or more reference standards. . A method according to any of the preceding claims, characterized in that the at least one cross immunoreactive antigen component comprises the total of a preparation of Mycobacterium species, or the total of the culture medium of said species. . A method according to claims 1-11, characterized in that the at least one cross-immunoreactive antigen component comprises a fraction of KP90, KS90, antigend, KP100 or SP100 of the total of a preparation of a Mycobacterium species, or an appropriate fraction of a culture medium of said species. . A method according to any of the preceding claims, characterized in that the at least one antibody for mycobacterial species comprises IgG, IgA, IgM or any combination thereof. A diagnostic device, characterized in that it comprises a support, on which at least one cross-immunoreactive antigen component of a mycobacterial species and at least one antibody, which is capable of reacting with a mycobacterial antigen and which does not react with said antigen, is circumscribed. less an antigen component, and means to detect the presence of antigen-antibody complexes. . A diagnostic equipment according to claim 15, characterized in that the support is selected from the group consisting of membranes, indicator rods, filters, spheres, granules and microtiter plates.