Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-24
2004-09-14
Moran, Marjorie (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S091200, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06790616
ABSTRACT:
TECHNICAL FIELD
HLA (Human Leukocyte Antigen) that is Human major histocompatibility antigen, is expressed on membranes of imuunocompetent cells, presents processed peptides derived from exogenous and endogenous antigens to T lymphocytes, and functions as a marker to recognize self and non-self. The present invention relates to a method, a reagent and a kit for typing of the HLA class I alleles. This invention is especially useful for judgement of compatibility between a donor and a recipient in organ transplantation, and for association analysis between the HLA class I genes and various types of diseases in the clinical and medical field. This invention enables us to easily automate and mechanize detection and determination of the HLA class I alleles.
BACKGROUND OF ART
Typing of the HLA antigens has been mainly performed by the serological method using human alloantibodies. By using the specific antibodies to each HLA antigen which are contained in cord blood or serum from subjects who have frequently undergone blood transfusion, complement-mediated cytotoxicity is caused in the antigen-antibody reaction. It changes permeability of positive cell membranes to take an eosinic pigment into the cell, resulting in being detected as colored and expanding cells with a microscope. It is possible to type HLA-A, HLA-B and HLA-C antigens belonging to HLA class I, and HLA-DR and HLA-DQ antigens belonging to HLA class II by this method. However, this method has problems in terms of collection, quality control and supply of the specific antibodies. Furthermore, the survival rate of cells is utilized as an indicator for judgement in this method. Therefore, poor conditions of subjects, for example, a low survival rate of cells caused by disease or influence by passage of time after blood collection, lead to decrease of credibility for results of testing.
In recent years, a development of molecular biotechnology has enabled us to analyze the region of genes encoding the HLA antigens. That has clarified the correspondence between the HLA antigens and the sequences of the HLA genes. This means it has been possible to identify the HLA antigen type by analyzing the specific sequences of the HLA genes (DNA typing). Especially, PCR (polymerase chain reaction) method which can high-sensitively detect a slight change of sequences isutilized to type the HLA-DR, -DQ, or -DP genes belonging to HLA class II. Several PCR-based typing methods for HLA class II DNA such as PCR-SSOP (Sequence-Specific Oligonucleotide Probe) method, PCR-RFLP (Restriction Fragment Length Polymorphism) method, PCR-SSP (Sequence-Specific Primers) method and PCR-SSCP (Single Strand Conformation Polymorphism) method have been developed. In all these methods, the gene region to analyze is amplified by the PCR method and then the variable region in the sequences of the amplified products is analyzed by combination with another methods in order to distinguish the genotype. The HLA class II DNA typing method makes it possible to classify the HLA type at the allele level in addition to classification by the classical serological method using human alloantisera.
Development of the PCR based-method for BLA class I DNA typing is delayed remarkably, comparing with HLA class II typing. The reasons are as follows: (1) While almost all the class II gene mutations (gene substitutions), including those which reflect the specificity of antigens, concentrate in the region of the exon 2, the class I gene mutations are interspersed among the regions of the exons 2 and 3, or the exon 4. (2) The HLA class I genes, including non-classical genes (HLA-E, -F and -G) and pseudogenes (HLA-H, -J, -K and -L), are highly homologous among them.
To date, several HLA class I DNA typing methods have been reported. However, all these methods require complicated manipulation, strict reaction condition and skill. Those are not suitable for handling a large number of samples and offer only low resolution HLA typing. Furthermore, the typing methods for each gene are not standardized.
Disclosure of Invention
The purpose of this invention is to solve problems of the manipulation of HLA class I locus antigen typing by the classical serological method, and to prodive a method, a kit and a reagent for classifing the subtype of the HLA class I antigens at the allele level (allele typing), which has not been distinguished by the classical method. Furthermore, the aim of this invention is to provide a method for typing of the HLA class I alleles which can automate and machanize easily.
As a result of intensive studies for these subjects, the inventors have established primers which can amplify all the HLA-A alleles, all the HLA-B alleles or all the HLA-C alleles and specific primers to the common sequences among all alleles in the group consisting of the specific HLA-A alleles or the specific HLA-B alleles. The inventors have established probes which can specifically hybridize with the sequence of at least one specific HLA-A allele, at least one specific BLA-B allele or at least one specific HLA-C allele. The inventors have found out that it is possible to distinguish the HLA class I antigen or allele, by hybridizing the PCR amplified products derived from the specific HLA class I allele or the specific group with the DNA probes described above which are immobilized on wells of microtiter plates, adding an enzyme-conjugate which can specifically bond to a label of the amplified products at the same time as or after the hybridization, and adding a chromogenic substrate, a luminescent substrate or a fluorescent substrate to the mixture, to detect as signals whether or not the amplified products are hybridized with the immobilized DNA probes. Thus, they have accomplished this invention.
The main embodiment of this invention is a method for typing of HLA class I alleles, which comprises the following steps from (a) to (d).
(a) A step, using HLA class I gene or nucleic acids containing their fragment for a template,
(1) To non-selectively amplify all HLA-A alleles, all HLA-B alleles or all HLA-C alleles by a PCR method using a primer pair which can amplify all the HLA-A alleles, all the HLA-B alleles or all the HLA-C alleles, or
(2) To selectively amplify a specific group consisting of specific HLA-A alleles or specific HLA-B alleles by a PCR method using a primer pair which is specific to the common sequence to alleles of the specific group consisting of the specific HLA-A alleles or the specific HLA-B alleles,
(b) A step to add the above products amplified by the PCR method to wells of microtiter plates, wherein each well is modified with a carboxyl group to covalently immobilize amino-modified DNA probes which can specifically hybridize with the sequence of at least one specific HLA-A allele, at least one specific HLA-B allele or at least one specific HLA-C allele, and to hybridize the amplified products with the immobilized DNA probes, wherein the DNA probes are selected depending on the above amplified specific HLA class I gene or group;
(c) A step to detect as signals whether or not the amplified products are hybridized with the immobilized probes; and
(d) A step to determine the type of the HLA class I allele based on the signal pattern detected at the step (c) according to the Typing Table.
The PCR amplification of the target gene at the step (a), can be classified into 2 steps. One is a step to non-selectively amplify all the HLA-A alleles, all the HLA-B alleles or all the HLA-C alleles by the PCR method using a primer pair which can amplify all the HLA-A alleles, all the HLA-B alleles or all the HLA-C alleles. The other is a step to selectively amplify the specific group consisting of the specific HLA-A allele group or the specific HLA-B allele group by the PCR method using a primer pair which is specific to the common sequences to alleles of the specific group consisting of the specific HLA-A alleles or the specific HLA-B alleles. At the former step, PCR primers are designed to be specific to the common sequences, which are within the region of all alleles belonging to
Kaneshige Toshihiko
Moribe Toyoki
Moran Marjorie
Shionogi & Co. Ltd.
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