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Method for typing of HLA class I alleles

a typing method and allele technology, applied in the field of methods, reagents and kits for typing of hla class i alleles, can solve the problems of reducing the credibility of testing results, affecting the quality of testing, and the collection of specific antibodies, and requiring complicated manipulation of methods

Inactive Publication Date: 2005-05-26
SHIONOGI & CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is a method for typing HLA class I alleles at the allele level (allele typing) using a PCR amplification method. The method can automate and machine-ize the typing process easily. The inventors have developed primers that can amplify all HLA-A, HLA-B, and HLA-C alleles, as well as specific primers for the common sequences of these alleles. The PCR-amplified products are then immobilized on microtiter plates and hybridized with specific DNA probes. The signals generated by the hybridization reaction are detected and used to determine the type of HLA class I allele. The invention provides a solution for the problem of difficulty in typing HLA class I alleles using the classical serological method.

Problems solved by technology

However, this method has problems in terms of collection, quality control and supply of the specific antibodies.
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.
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.

Method used

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  • Method for typing of HLA class I alleles
  • Method for typing of HLA class I alleles
  • Method for typing of HLA class I alleles

Examples

Experimental program
Comparison scheme
Effect test

example 1

HLA-A2 Allele Typing

[0064] Leukocytes (Samples 1-4) which were isolated from peripheral blood (about 10 ml) of normal subjects according to usual methods, were lysed in 500 μl of guanidine thiocyanate buffer (4M guanidine thiocyanate, 25 mM sodium citrate(pH7.0), 0.5% sodium N-lauroylsarcosinate, 1% mercaptoethanol). The solution was extracted twice with phenol to eliminate proteins. After mixing with 3M sodium acetate buffer (pH 5.2), genome DNAs were obtained by adding twice volume of chilled ethanol. By using this DNAs, typing of the HLA-A2 alleles was performed as follows.

[0065] By using A2-5T and 5′-biotinylated A3-273T for a primer pair, amplification of the region containing the exon 2, the intron 2 and the exon 3 of the HLA-A2 alleles from DNAs described above was performed by the PCR method. Likewise, by using A4-8C and 5′-biotinylated A4-254G for a primer pair, amplification of the region containing the exon 4 of the HLA-A alleles was also performed by the PCR method. Th...

example 2

HLA-B40 Allele Typing

[0069] Leukocytes (Samples 5-8) which were isolated from peripheral blood (about 10 ml) of normal subjects according to usual methods, were lysed in 500 μl of guanidine thiocyanate buffer(4M guanidine thiocyanate, 25 mM sodium citrate(pH7.0), 0.5% sodium N-lauroylsarcosinate, 1% mercaptoethanol). The solution was extracted twice with phenol to eliminate proteins. After mixing with 3M sodium acetate buffer (pH 5.2, genome DNAs were obtained by adding twice volume of chilled ethanol. By using this DNAs, typing of the HLA-B40 alleles was performed as follows.

[0070] By using BASF-1 and 5′-biotinylated BASR-1 for a primer pair, amplification of the region containing the exon 2, the intron 2 and the exon 3 of the HLA-B40 alleles from DNAs described above was performed by the PCR method. The reaction solution was composed of genomic DNAs (100 ng), 1.4 units of thermostable DNA which was pretreated with Taq Start™Antibody for 5 min at room temperature, 33.5 mM Tris-HC...

example 3

HLA-A Antigen and Allele Typing

[0074] Leukocytes (Samples 9-12) which were isolated from peripheral blood (about 10 ml) of normal subjects according to usual methods, were lysed in 500 μl of guanidine thiocyanate buffer (4M guanidine thiocyanate, 25 mM sodium citrate(pH7.0), 0.5% sodium N-lauroylsarcosinate, 1% mercaptoethanol). The solution was extracted twice with phenol to eliminate proteins. After mixing with 3 M sodium acetate buffer (pH5.2), genome DNAs were obtained by adding twice volume of chilled ethanol. By using this DNAs, typing of the HLA-A antigens and alleles was performed as follows.

[0075] By using CGA011, CGA012 and 5′-biotinylated AIn3-66C for a primer pair, amplification of the region containing the exon 2, the intron 2 and the exon 3 of the HLA-A alleles from DNAs described above was performed by the PCR method. The reaction solution was composed of genomic DNAs (100 ng), 1.4 units of thermostable DNA polymerase which was pretreated with Taq Start™Antibody for...

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Abstract

This invention provides a method, a kit and a reagent for typing of the HLA class I alleles. Explaining concretely, a single HLA class I antigen or allele is determined by combining PCR amplification using a primer pair which can amplify all HLA-A alleles, all HLA-B alleles or all HLA-C alleles, or 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, with reverse hybridization analysis using DNA probes capable of specifically hybridizing with the sequence of al least a specific HLA-A allele, at least a specific HLA-B allele or at least a specific HLA-C allele, which are covalently immobilized on wells of microtiter plates.

Description

TECHNICAL FIELD [0001] 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 [0002] 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...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12Q1/6834C12Q1/6881
CPCC12Q1/6834C12Q2531/113C12Q1/6881C12Q2600/156
Inventor MORIBE, TOYOKIKANESHIGE, TOSHIHIKO
Owner SHIONOGI & CO LTD
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