JP4505014B2 - Oligonucleotide composition for analyzing HLA genotype and test method thereof - Google Patents

Description

The present invention relates to an oligonucleotide chip.
The present invention relates to a composition and a method for producing the same, and more particularly, an oligonucleotide chip composition for analyzing an HLA genotype for conducting a histocompatibility test necessary for tissue transplantation, a method for producing the same and a method for detecting the result About.

Major Histocompatibility Complex (MHC) is a complex antigen system controlled at the gene level, and a series of genes related to rejection (immune reaction) when transplanting organ tissue Refers to a group and is present in all mammals. Human MHC was first discovered as an anti-leukocyte factor that recognizes antigens on the surface of leukocytes, called human leukocyte antigen (HLA), and is located on the short arm of chromosome 6. This HLA locus is classified into three groups (HLA-class I, II, III) according to immunological characteristics and molecular structure, gene location and cell type to be distributed. The HLA-A, -B, and -Cw loci and HLA-DR at the second group of antigen sites are considered to be closely related to tissue rejection.

Typical characteristics of the HLA gene are as follows. First, since the HLA gene has more than several tens of alleles, it has a large number of genetic markers in the human body.
In particular, it represents the highest genetic polymorphism. Second, genetic inheritance at the haplotype level. Many genes belonging to the HLA gene group are located very close together on chromosome 6, so when inherited from parent to child, they are inherited as a single unit, and specific combinations of such alleles Is referred to as the HLA haploid type. Third, HLA genetic polymorphism between races can be mentioned. The distribution of HLA alleles shows significantly greater differences between races than other genes. Such an aspect shows further racial differences in the HLA haploid form. Some HLA genes have strong linkage disequilibrium (linkage disequilib)
specific species of HLA haploid forms are characteristically present in certain races. Due to the genetic polymorphism of HLA, the genotype frequency and the haploid frequency of HLA genes between races.
cy), it is used as a powerful marker to study genetic background differences between races. Fourth, yet another important function of the HLA gene is related to immune responses. The immune response is presented on the cell surface after the HLA-class I and class II molecules bind to the antigen in the cell, and the T cell antigen receptor on the surface of the T lymphocyte recognizes the antigen bound to the HLA molecule. To be expressed.

Because of the high genetic polymorphism of the HLA gene and the characteristics that are deeply involved in the immune response, it has been widely applied to forensic applications such as organ transplantation, disease relevance, blood transfusion, parentage testing, and anthropological research. Applied. Currently, HLA testing is essential in kidney and bone marrow transplant programs. ABO blood group tests, HLA-A, -B, -Cw, and DR tests are performed on donors and recipients, and cross-match tests are performed before transplantation. If the HLA types between donor and recipient are different from each other when transplanting bone marrow, kidney, etc., rejection of the transplanted organ will occur. Since bone marrow transplantation is usually an immune cell transplantation, perfect genetic matching is required between the patient and the donor, and a donor having the same type of HLA as the recipient is selected in advance. It is important that you have to do. In particular, in bone marrow transplants attempted in acute or chronic leukemia, immunodeficiency, or aplastic anemia, the HLA genotype must be perfectly matched. For this reason, siblings or sisters who inherit the same HLA type genetically are best suited as bone marrow donors. But if you don't have a family, you have to find someone to offer you among unrelated people. At this time, it is essential to examine many people and identify the HLA genotype.

  As the base sequence of the allele that controls the HLA antigen becomes clear in detail as a result of progress in molecular biology, it has become possible to perform HLA gene analysis that can find the diversity of HLA through the difference in base sequence. In particular, with the introduction of the PCR method, the DNA typing field of HLA has developed rapidly, and polymorphisms of HLA antigens have become almost clear at the DNA level. Classification of HLA alleles using the PCR method requires separation of T lymphocytes and B lymphocytes because all nucleated cells can be used as samples regardless of the expression level of HLA molecules on the cell surface. In addition, there is an advantage that the genotype can be analyzed even in a body fluid or tissue that does not contain lymphocytes. Further, since DNA is a relatively stable molecule, it can be stored for several months if stored refrigerated and almost permanently if stored frozen. Among the many advantages that DNA typing methods have, the most important advantage is that a large number of HLA alleles can be categorized.

Looking at the currently used HLA DNA typing methods, first of all PCR
-There is an SSP method. Since this method synthesizes primers specific to the base sequence of each allele and performs PCR, tens of pairs of primers are required to discriminate tens of alleles. This method is a simple method with a short analysis time. However, since careful attention, knowledge and time are required when synthesizing such primers, there is a limit to analysis of a large amount of samples. In addition, since the number of alleles increases the number of PCR-SSPs, it is difficult to handle a large number of specimens at once. The classification of HLA genotypes using the second PCR-RFLP method is a relatively simple method for examining the reaction pattern of alleles to restriction enzymes. Alleles by size of DNA fragments cleaved by various restriction enzymes. Classify the genotype. Although this method is easy to judge the results and simple, it becomes difficult to distinguish when the sites that can be recognized by restriction enzymes are limited, and a polyacrylamide gel must be used. It is difficult to classify alleles at the same time. For this reason, this method is suitable for the classification of genotypes with a small number of alleles. As a third method, the PCR-SSCP method is a method in which a PCR product is denatured to single-stranded DNA (ssDNA) by adding a denaturing agent such as formamide and then electrophoresed on a non-denaturing polyacrylamide gel. is there. In electrophoresis, ssDNA has a peculiar structure based on its base sequence, and shows different migration speeds during electrophoresis to form different types of bands. It is a method for categorizing HLA genotypes using such properties and requires a complicated and highly difficult technique, and experience and knowledge are also required for analysis of the results. Fourth, the sequence-based typing method is to determine and examine the DNA base sequence after PCR for each HLA gene. As a kit using this method, there is HLA Sequence Based Typing (SBT) of ABI (USA), but the cost of the kit is very high, an expensive apparatus is required, and the experimental process is complicated. The fifth PCR-SSOP method is a method of synthesizing a probe complementary to the base sequence of the region showing genetic polymorphism and analyzing the possibility of hybridization between the probe and the PCR amplification product. This requires a lot of probes and is prone to hybridization reactions with other alleles that have similar base sequences. This is a method that requires experience and technology to set the appropriate temperature and reaction conditions. It is. Although this method can obtain an accurate result depending on the type of probe, there is a limit to fixing a large number of probes in order to use a filter. Since only one type can be analyzed with one filter, it takes a lot of time and effort to process and analyze many samples. Products that are commercialized using this method include HLA cl
Line probe assay (reverse hybridization) using assorted SSOP for assI and class II
(probe assay), INNOGENetics (Belgium) IN
There is a NO LiPA kit and a Dynal Biotech (USA) Dynal RELI ^™ SSO HLA Typing Kit.

  As described above, various typing methods are used to analyze the HLA gene and commercialized. However, the common drawback of these methods is that HLA-A, -B, -Cw, -DR Typing at the same time requires a great deal of time and effort, and is particularly difficult to analyze a large number of samples.

  Very recently, gene expression has been achieved through DNA chip technology that allows very small amounts of DNA to be attached at high density onto a substrate such as a small glass slide or silicon, and at the same time search many. A new dimension of analysis system has been developed that can be used for analysis, genetic diagnosis, gene mutation search, drug testing and disease diagnosis. The present invention is possible by simultaneously analyzing HLA-A, -B, -Cw, and -DR on one slide using this DNA chip technology. That is, each probe is accumulated in five areas on one aldehyde glass slide. The probes accumulated on the slide are hybridized with the HLA gene amplified using the asymmetric PCR method. HLA genotypes are categorized by examining the probes in which the reaction appeared using the HLA typing program.

  The present invention solves the problems of the above-described method and has been devised in response to the above-described necessity. An object of the present invention is to provide an oligonucleotide composition comprising a composition for analyzing HLA genotype.

Another object of the present invention is to provide an improved method of analyzing HLA genotype using the above composition.
In order to achieve the above object, the present invention provides a probe selected from the group consisting of the base sequences represented by sequence information 1 to sequence information 41, and two or more probes for determining an HLA-A genotype A group consisting of probes; a probe selected from the group consisting of the base sequences shown in sequence information 42 to sequence information 89, and a group consisting of two or more probes for determining the HLA-B genotype; sequence information 90 to a probe consisting of two or more probes for determining the HLA-Cw genotype, and a group selected from the group consisting of the base sequences shown in the sequence information 112; A probe selected from the group consisting of the base sequences shown, and consisting of a group consisting of two or more probes for determining the HLA-DR genotype Including one or more probe group selected from the group, to provide a composition having a plurality of oligonucleotide probes to determine the HLA genotypes.

The present invention also provides
A probe selected from the group consisting of the base sequences shown in the sequence information 1 to the sequence information 41, a group consisting of two or more probes for determining the HLA-A genotype; from the sequence information 42 to the sequence information 89 A probe selected from the group consisting of the base sequences shown in the above, and a group consisting of two or more probes for determining the HLA-B genotype; consisting of the base sequences shown in the sequence information 90 to the sequence information 112 A probe selected from the group consisting of two or more probes for determining the HLA-Cw genotype; and a group consisting of the base sequence shown in the base sequence shown in the sequence information 113 to the sequence information 140 Selected from the group consisting of two or more probes for determining the HLA-DR genotype The containing one or more probe group, provided the composition is tested support having immobilized thereon having a plurality of oligonucleotide probes to determine the HLA genotypes (test support).

  The support of the present invention may be a support selected from the group consisting of a filter, a strip, a microsphere, a chip, a slide, a multiwell plate, a membrane, and an optical fiber. desirable.

  The present invention also includes a primer for determining one or more HLA genotypes selected from the group consisting of the base sequences represented by sequence information 141 to sequence information 157, which comprises the probe and the target gene. It is a primer characterized by being used for detecting a hybridization reaction between.

  Among the primers of the present invention, biotin (rhodamine) or biotin (rhodamine) is attached as a preferable example of the antisense primer shown in the sequence information 142, 144, 146, 148, 150, 152 or 154. Among these primers, the antisense primer to which biotin is attached is characterized in that it interacts with streptavidin-cyanine.

The present invention also provides
a) preparing HLA DNA from blood, cells and tissues;
b) performing asymmetric PCR with the prepared DNA;
c) binding the product of the asymmetric PCR to the first or second probe; and d) confirming the binding;
A method for analyzing an HLA genotype is provided.

  In the present invention, the above confirmation step is performed by using rhodamine or streptavidin-cyanine that binds to biotin, confirming the result using a microarray scanner, and analyzing the result using an HLA typing program. It is characterized by confirming conveniently. In the present invention, an HLA oligonucleotide chip capable of simultaneously analyzing HLA-A, -B, Cw, and DR on one slide using a recently developed DNA microarray technology was developed. That is, to determine the HLA genotype, 38 oligonucleotides that can specifically react with 21 types of HLA-A and 46 oligos that can specifically react with 36 types of HLA-B 20 oligonucleotides that can specifically react with 14 types of nucleotides, HLA-Cw, 22 oligonucleotides that can specifically react with 16 types of HLA-DRB1 / 3/4/5, 17 oligonucleotides that can specifically react with 8 types of HLA-DRB1, B1 * 3, * 8, * 11, * 12, * 13, * 14, * 15, and * 16 Were collected in 5 areas on a single aldehyde glass slide. The HLA gene amplified using an asymmetric PCR method was subjected to a hybridization reaction with an oligonucleotide probe accumulated on the slide. The probe in which the reaction appeared by using the fluorescence reaction method was analyzed, and 87 genotypes for total HLA were classified.

  FIG. 1 is a photograph showing an entire slide of an HLA oligonucleotide chip. That is, it consists of five “chambers” designated by the displayed numbers to simultaneously analyze HLA-A, -B, -Cw, -DRB genotypes on one slide .

On this slide, as shown in the table, 38 probes for categorizing HLA-A genotypes, 2 probes used as positive controls and 2 negative controls 1 probe, 46 probes to classify HLA-B genotype and 1 probe each used as positive and negative control, 20 probes to classify HLA-Cw genotype and positive 2 probes used as controls and 1 probe used as negative controls, 22 probes for classifying HLA-DRB1 / 3/4/5 genotypes and 1 each used as negative control and positive control Pieces Robe, HLA-DRB1 / 3/4/5 genotypes are affected by the DRB3 / 4/5 genotype, so the HLA-DRB1 genotype analysis is partially ambiguous, making up for HLA- 17 probes to improve the ability to discriminate genotypes of DRB1 and 1 probe each used as negative control and positive control, each gene group is divided into a total of 155 probes and fixed in duplicate Turned into.

Then, Coverwell Perfusion Chamber (SIGMA, USA) was attached on this probe, and the probes for discriminating each gene group were separately mixed and reacted so that an HLA oligonucleotide chip was produced.

  FIG. 2 to FIG. 6 are photographs showing the results of fluorescence reactions related to HLA-A * 11 / * 31, B * 27, Cw * 06, DRB1 * 01 / * 11 / DRB3 types of HLA oligonucleotide chips, FIG. 2 is an enlarged image of reactions occurring in five chambers in the slide of FIG.

Fluorescent primers and probes are used for PCR and hybridization reactions, and the results are analyzed for GenePiX4000 scanner (Axon inst.
and the HLA genotype was analyzed using the HLA typing program.

  Hereinafter, the present invention will be further described through examples that should not be construed as limiting. These examples are merely illustrative of the present invention, and it is obvious to those skilled in the art that the scope of the present invention should not be construed as being limited by these examples. I will.

Synthesis of HLA primer and its nucleotide sequence HLA PCR primers are HLA-A, -B and -Cw as shown in Table 1, exons 2 and 3, and HLA-DRB1 / 3/4/5 Is the site that can react in common with exon 2, HLA-DRB1 is exon 2 and B1 * 3, * 8, * 11, * 12, * 13, * 14, * 15, * 16 This was used in asymmetric PCR by analyzing sites that can specifically amplify only certain genotypes.

Rhodamine was attached to the 5 ′ end of the antisense primer used in asymmetric PCR so that it could be confirmed by a fluorescent reaction after the hybridization reaction, or biotin was attached after the hybridization reaction so as to bind to streptavidin-cyanine. The primer used here was commissioned to Metabion, Germany, and the molecular cloning version 3 (Sambrook and Rusell, Cold Sprint)
g Harbor Laboratory Press, New York, USA, 2001) was synthesized using the oligonucleotide synthesis method described in 10.42.

Extraction of HLA DNA and Asymmetric PCR Reaction 1) HLA DNA was isolated using the PUREGENE ^™ DNA isolation kit from Gentra systems. RBC lysis solution, 900 μl, and 300 μl of blood were mixed and allowed to react at room temperature for 1 minute while shaking about 10 times, followed by centrifugation at 13,000 rpm for 20 seconds, and the supernatant was removed leaving about 20 μl.

2) The remaining supernatant was vortexed for 10 seconds to resuspend leukocytes, and then 300 μl of cell lysis solution was added, and the cells were lysed with a pipette.

3) 100 μl of protein precipitation solution was added to the solution in which the cells were lysed, mixed by vortexing for 20 seconds, and then centrifuged at 13,000 rpm for 1 minute.
4) After mixing the supernatant containing DNA with 100% isopropanol, the tube was shaken 50 times to mix, and then centrifuged at 13,000 rpm for 1 minute.

5) After discarding the supernatant, the pellet was washed with 300 μl of 70% ethanol and centrifuged at 13,000 rpm for 1 minute.
6) After discarding the supernatant and drying the pellet, the pellet was resuspended with 100 μl of DNA hydration solution.

7) The asymmetric PCR reaction is carried out by the method shown in Table 2 and the GeneAmp PCR system.
9600 thermal cycler (Perkin Elmer Cetus, USA).

8) 5 μl of asymmetric PCR finished product was added to gel loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 15% Ficoll 4)
00) 1 μl was added and mixed. 2 containing 1 μg / ml ethidium bromide (EtBr)
Electrophoresis was performed on a% agarose gel. The PCR band was confirmed with an image analyzer (Vilber Lourmat, France) equipped with a UV transducer.

Synthesis of probes to make HLA oligonucleotide chips and their base sequences Aldehyde glass to covalently attach amino links to each 5 'end of all probes to facilitate hybridization reactions After attaching 10 to 20 oligos (dT), the base sequence shown in Table 3 was changed to Ami.
It was attached to _{no link-Oligo (dT) 10} ~ 20. In other words, “Amino link
-Oligo (dT) ₁₀ ~ ₂₀ - a primer consisting of the order of the probe nucleotide sequence ", was synthesized by requesting the Metabion German company. The nucleotide sequences of the HLA genotypes identified at this time are 21 genes for HLA-A, 36 species for HLA-B, 14 species for HLA-Cw, and 16 genes for HLA-DR as shown in Table 3. Was determined by analysis.

  In the base sequences shown in Tables 4 to 7, the base sequence indicated by capital letters in the center is the most important part. A primer having a size of about 13 bp to 30 bp centering on this was synthesized so as to be about 60 to 65 ° C. in consideration of Tm value and GC%.

Production of HLA oligonucleotide chip 1) In order to produce an oligonucleotide chip, an aldehyde glass slide of Nucellell (Korea) or Cell (USA) was used. The probe with amino group attached, 100 pmole / μl and the same amount of 3X SSC were mixed well and fixed on a slide, and reacted at room temperature for 16 hours. The slides were then washed twice with 0.2% SDS for 5 minutes and then twice with distilled water for 5 minutes. The slide was then washed once with distilled water heated to 95 ° C. for 2 minutes and then once with distilled water for 5 minutes at room temperature.

2) Sodium borohydride (1.3 g NaBH ₄ , 375 ml PBS, 125 ml
The slide was reacted with 100% EtOH for 5 minutes, then washed with 0.2% SDS three times for 1 minute, washed twice with distilled water for 1 minute, and then dried at room temperature.

3) To separate and react 5 HLA PCR reactions, SIGMA Cov
An erwell Perfusion Chamber was attached to the slide to separate the chamber. The completed HLA oligonucleotide chip was stored in a dark place at room temperature until use.

Hybridization reaction with HLA PCR product 1) The HLA PCR product bound with rhodamine or biotin and the hybridization solution (3X SSC, 0.3% SDS) were mixed at a ratio of 1: 9. When biotin was used to cause a fluorescent reaction, streptavidin-cyanine was added at a concentration of 1 μg / ml for reaction.

2) With the chamber covered, 90 μl of hybridization buffer was dropped into the chamber on the slide and reacted at 62 ° C. for 1 hour.
3) Wash the slides with 1X SSC for 5 minutes at room temperature, then 0.1X SSC
For 2 minutes and then dried at room temperature.

4) Confirmation of results Using a scanner (GenePiX4000, Axon instruments, USA), the HLA genotype was analyzed using the HLA typing program developed by the present inventor for probes that exhibit fluorescence.

  In the base sequences shown in Tables 4 to 7, the base sequence indicated by capital letters at the center is the most important part, and a primer having a size of about 13 bp to 30 bp centering on this is used as a Tm value and GC% Was taken into consideration so that the temperature would be 60 to 65 ° C.

  The results are shown in FIGS. In these figures, fluorescent primers and probes are used in the PCR and hybridization reaction, and the results are analyzed using a GenePiX4000 scanner (Axon instruments, USA) as a probe that shows fluorescence. HLA genotypes were analyzed using the HLA typing program developed by the company.

The HLA typing program analyzes the results of the probes located in each “chamber” of the HLA oligonucleotide chip of the present invention and produces a method of normalization appropriately. The program produces an intermediate zone that incorporates concepts such as boundary values that can discriminate positive and negative. The negative positive of each probe is determined based on the boundary value. It was developed so that the matching HLA genotype could be examined by comparing the negative and positive forms of the probe with the negative positive form control table stored in the program.

In the present invention, an oligonucleotide chip for analyzing the HLA gene and diagnosing HLA-A, -B, -Cw, -DR genotypes has been developed. Analysis of HLA genotypes using this can save labor costs, material costs and time significantly compared to existing commercialized kits. In other words, the existing inspection methods and kits are HLA-A, -B in HLA typing.
, -Cw, -DR genotypes cannot be analyzed at the same time, so there is a problem that separate tests must be performed separately. However, using the HLA DNA chip of the present invention, HLA- Not only can all A, -B, -Cw, and -DR genotypes be identified, but even if the number of HLA alleles increases, the probe is immobilized on the slide at a high density, so only one slide is sufficient. It is. Further, since the method of the present invention uses a fluorescent substance, not only a coloring process is not required, but also the steps required in the test of the conventional method, that is, washing, coloring reaction, etc. are omitted, so the results are seen. The time until is greatly shortened.

FIG. 1 is a photograph showing an entire slide of an HLA oligonucleotide chip. FIG. 2a is a photograph showing the result of the fluorescence reaction of the HLA oligonucleotide chip for a sample having both HLA-A * 11 type and HLA-A * 31 type simultaneously. Among 41 probes, as shown in “Reaction HLA type” in Table 4, No. 13, No. 14, No. 15, No. 17, No. 18, No. 23, No. 29, No. 30 related to A * 11 type No.33, No.33 probe, and No.11, No.13, No.15, No.18, No.21, No.25, No.26, 29, No.32, No.39, No.40 related to the A * 31 type At the same time, a positive reaction occurred. In addition, the accuracy of the experiment can be confirmed by making probes 01 and 41 appear as positive controls in all reactions, and probe 06 as negative controls so that they do not appear in any reaction. . The reason why two identical points appear is that the same probe is double-fixed in order to reduce errors and improve accuracy. FIG. 2b shows the position of the probe immobilized on the slide. FIG. 3a shows the result of the fluorescence reaction of the HLA oligonucleotide chip against a sample having HLA-B * 27 type. Among the 48 probes, as shown in “Reaction HLA type” in Table 5, No. 09, No. 12, No. 21, No. 30, No. 31, No. 32, No. 36, and No. 37 related to the B * 27 type. No. 42, No. 47, and No. 47 positive reaction occurred. In addition, the accuracy of the experiment can be confirmed by making the probe 01 appear in all reactions as a positive control and the probe 06 not appear in any reaction as a negative control. The reason why two identical points appear is that the same probe is double-fixed in order to reduce errors and improve accuracy. FIG. 3b shows the position of the probe immobilized on the slide. FIG. 4a shows the results of the fluorescence reaction of the HLA oligonucleotide chip against the sample with HLA-Cw * 06. Among the 23 probes, as shown in “Reaction HLA type” in Table 6, positive reactions occurred with the 06, 08, 13, and 17 probes related to Cw * 06. In addition, the accuracy of the experiment can be confirmed by making probes 01 and 23 appear as positive controls in all reactions and probe 04 as negative controls so that they do not appear in any reaction. . The reason why two identical points appear is that the same probe is double-fixed in order to reduce errors and improve accuracy. FIG. 4b shows the position of the probe immobilized on the slide. FIG. 5a is a photograph showing the result of the fluorescence reaction of the HLA oligonucleotide chip for the sample having the HLA-DRB1 * 01 type and the HLA-DRB1 * 11 type simultaneously. Among the 41 probes, as shown in “Reaction HLA type” in Table 7, the 07th and 10th probes related to the DRB1 * 01 type and the 04th, 10th, 13th related to the DRB1 * 11 type No. 16, No. 16 probe and the No. 16 and No. 24 probes related to DRB3 type inherited together with DRB1 * 11 type were simultaneously positive. In addition, the accuracy of the experiment can be confirmed by making the probe 01 appear in all reactions as a positive control and the probe 05 not appear in any reaction as a negative control. The reason why two identical points appear is that the same probe is double-fixed in order to reduce errors and improve accuracy. FIG. 5b shows the position of the probe immobilized on the slide. FIG. 6a shows the result of the fluorescence reaction of the HLA oligonucleotide chip against a sample having both HLA-DRB1 * 01 type and HLA-DRB1 * 11 type simultaneously. Among 19 probes, as shown in “Reaction HLA type” in Table 8, positive reactions occurred simultaneously with the 07th, 10th, and 13th probes related to the DRB1 * 11 type that is selectively amplified. It was. In addition, the accuracy of the experiment can be confirmed by making the probe number 01 appear in all reactions as a positive control and the probe number 04 not appear in any reaction as a negative control. The reason why two identical points appear is that the same probe is double-fixed in order to reduce errors and improve accuracy. FIG. 6b shows the position of the probe immobilized on the slide.

Claims (3)

A group of probes for determining the HLA-A genotype, comprising the base sequences represented by sequence information 1 to sequence information 41;
A group of probes for determining the HLA-B genotype, comprising the base sequences shown in the sequence information 42 to the sequence information 89;
A probe group for determining the HLA-Cw genotype consisting of the base sequence shown in the sequence information 90 to the sequence information 112; and the HLA-DR genotype consisting of the base sequence shown in the sequence information 140 from the sequence information 113 A composition having a plurality of oligonucleotide probes for determining HLA genotype, comprising all groups of probes for determination.   A test support comprising the composition of claim 1 wherein the probe is immobilized.   The support according to claim 2, characterized in that the support is selected from the group consisting of filters, strips, microspheres, chips, slides, multiwell plates, membranes and optical fibers.

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