JPH03292898A - Determining process for human leukocyte antigen type - Google Patents

Abstract

PURPOSE: To readily determine human leukocyte antigen type by comparing a specific DNA fragment length polymorphism (RFLP) with other DNA sample RFLP.

CONSTITUTION: The second exon nucleotide sequence of human leukocyte antigen (HLA)-DRB gene of donor DNA sample (A) such as transplant is proliferated in 25-35 cycle by polymerase chain reaction(PCR) to provide a DNA fragment (B). Then, the fragment B is separated according to the length to afford the separated fragment (C) and RFLP of the component C is determined. Then, receptor DNA sample (D) such as transplant is passed through a treating process similar to treating process to the component A to provide RFLP of the component D. Finally, RFLPs of the components A and D are compared to determine human white leukocyte antigen type.

COPYRIGHT: (C)1991,JPO

Description

[Detailed description of the invention] The present invention relates to the matching of human leukocyte antigens.

Genes of the human leukocyte antigen (HLA) class II of the human major histocompatibility complex (MBC) encode cell surface glycoproteins that have a fundamental role in presenting antigenic peptides to helper T cells in the immune response. Recognition of foreign antigens along with self-14Hc class II molecules by helper T cells triggers a cascade of immune responses, resulting in the activation of cytotoxic T and B cells, each of which kills the antigen-providing cell. , the other induces an antibody response.

At least six HLA class loci have been defined, three of which (HLA-DR, DQ and DP)
are known to express functional products. The ^ (formerly α) and B (formerly β) gene pairs within these three loci encode heterodimeric protein products that are biallelic and alloreactive. Furthermore, combinations of epitopes on DR and/or DQ molecules are recognized by alloreactive T cells. This reactivity was used to define the 0w type by a mixed lymphocyte reaction (HLR) based cellular assay.

As a result of DR and DQ alloreactivity, it has been demonstrated that matching L^-DR and DQ alleles between donor and recipient prior to allograft transplantation has a significant impact on allograft survival. . Therefore, HLA-DR and DQ matching (
matching) is now generally considered a clinical prerequisite for kidney and bone marrow transplantation.

Until recently, methods for recognizing alloreactive epitopes were limited to serum and cell typing. DR and DQ
serotyping has been established and uses antisera generated as a result of humoral responses to DR and DQ alloantigens. However, serotyping is often problematic due to the availability and cross-reactivity of alloantisera and the need for viable cells.

Recently, with the development of molecular genetic analysis of the HLA class ■ region, it has become possible to define HLA-DR, DQ, and DP alleles at the DNA level. It is now recognized that many polymorphisms detected at the DNA level cannot be defined serologically in advance. However, polymorphisms revealed by DNA probes are increasingly being confirmed to be functional, as new alloantisera capable of defining serological splits are being reported. . Therefore, it appears that polymorphisms detected at the DNA level may well reflect functional epitopes. Accordingly, DNA typing is becoming increasingly widely used as a supplement to or alternative to serological testing.

To date, the most widely used DNA typing method for recognition of these alleles has been restriction fragment length polymorphism (RFLP) analysis. Although this method is well-established as a method for HLA class I [DNA typing], it has a number of inherent drawbacks. That is, RFLP typing is too time-consuming for clinical use prior to cadaveric kidney transplantation and is best suited for living donor transplantation or anamnesis investigation. Furthermore, RFLP
generally did not detect polymorphisms within exons encoding functionally important HLA class epitopes, suggesting strong binding between allele-specific nucleotide sequences within these exons and surrounding general Non-coding D
Based on the restriction endonuclease recognition site distribution inside NA.

As a result of the efforts of many groups to sequence the HLA class ■ alleles, DRB, DQB, D
The majority of Q^ and DPB protein product product lo-reactive epitopes were found to be restricted to the membrane distal region encoded by the second exon of the respective genes. The sequences flanking these exons are locus-specific and highly conserved between alleles, and as such can be easily detected using polymerase chain reaction (PCR) techniques (Saiki et al., 5c).
ience 230.1350.1985; 5cha
rf et al., I(uman Immunol, 23.14
3.1988) can be performed. This technique facilitates the acquisition of nucleotide sequence data for nearly all of an allele, thus allowing for microheterogeneity (microheterogeneity) of allele-specific nucleotide sequences.
This enabled the construction of allele-specific oligonucleotide (^SO) probes capable of detecting erogeneity.

As a result, the PCR-ASO typing method was developed. These methods produce sufficient target DNA by PCR amplification to allow typing with ASO probes using slot or dot plot hybridization analysis. Thus, improved procedures for HLA-DR typing, HL8-DQ typing, and HLA-DP typing were developed using PCR-ASO typing.

The major methodological disadvantage of these systems is that the complexity of the technique is directly related to the number of alleles tested, i.e. at least one SO probe is used per allele, and thus One membrane with immobilized target DNA is required for each SO probe used. Another method has also been developed by Charf et al. (1988), in which immobilized SO probes are hybridized to enzyme-labeled and amplified target DNA, thus creating a single monomer containing all of the required SO probes. One membrane can be used to type each amplified DNA.

We have developed a novel and simple method, IL^-DR/lh, using PCB amplification of the second exon sequence of the HLA-DRB gene, followed by product analysis by electrophoretic separation.
We have recently developed an allotype matching method. Rapid separations are obtained with non-denaturing polyacrylamide mini gels. Unlike currently available NA typing techniques, methods such as target DNA denaturation/neutralization, immobilization on solid support membranes, hybridization with radioisotope or enzyme-labeled SO probes, or development of a hybridization signal. No post-amplification sample processing required.

Therefore, the present invention provides (i) HLA of the first DNA sample;
PCR of second exon nucleotide sequence of DRB gene
(ii) performing an amplification; and (ii) a D obtained from the amplification.
separating the NA fragments according to size;
iii) determining the length polymorphism of the DNA fragments thus separated; and <iv) PCR amplification of the length polymorphism thus determined by PCR amplification of the second exon nucleotide sequence of the HLADRB gene of a second DNA sample. and comparing length polymorphisms of DNA fragments obtained from human IIL-DR and/or Dw allotypes.

The basis of the method is the PCR amplification of the second exon sequence of the HLA-DRB gene using defined PCB primers to generate a characteristic allele-specific PCR product (PCR fin carprint). be. The method includes post-amplification enzymatic digestion, chemical treatment, DNA immobilization, target-probe hybridization, or PC
Other established D
This is different from the NA type classification method. HLA-OR and Dw PCR fingerprints can be easily recognized within 8 hours in DR/Dw homozygous or heterozygous individuals. Thus, the method of the invention for direct visual comparison of PCR fingerprints of a panel of individuals can be used, for example, for bone marrow transplantation.
^-DR/Dw matching may be appropriate for HLA-DR/DI++ allele matching in the selection of related or unrelated living volunteer donors.

When examining the DNA of HLA-DR/Du+ heterozygous individuals, the observed PCR fingerprints are similar, but do not follow the pattern expected by simply adding two corresponding HLA-DR/Dw homozygous cells. did not match. However, two HLA-DR/Dw
If DNA from homozygous cells is premixed before PCB amplification, the pattern observed in the corresponding heterozygotes is reproduced. Therefore, the method of the present invention
It can be applied to all DR/Dw allotypes. Therefore, the DR and/or Dw specificity of heterozygous individuals can be analyzed.

The method of the invention is performed on a first DNA sample. D
The NA sample is obtained from an individual or any subject having the HLA-DR and/or I)w allotype that is desired to be investigated. Solids include fetuses. HLA DNA can be extracted from whole nucleated cells. Typically, H80N^ is obtained from peripheral blood cells where appropriate. Fetal HLA DNA is obtained from placental cells or amniotic fluid. Other sources of DNA include hair follicles, mummies, etc.

DNA is isolated under conditions that prevent degradation. DNase
Cells are digested with proteases under conditions where little or no activity is expected. Extract the digest with DNA solvent. Extracted DNA can be purified, for example, by dialysis or chromatography. Appropriate N^ Isolation technique is Kan
et al., N., Eng., J., Med, 297°108
0-1084.1977 and Nature 251.3
92-393.1974 and Kan and Dozy,
Proc, Natl, ^cad, Sci.

USA75.5631-5635.1978.

Next, the second exon nucleotide sequence of the HLA-DRB gene of sample DN^ is amplified by PCR. Second
Exon flanking sequences are highly conserved among DRB alleles. Two oligonucleotide primers, Taq i , dATP, dC, were added to the HLA DNA to anneal to complementary sequences at both ends of the second exon.
Add thermostable DNA polymerases such as TP, dGTP and dTTP. The DNA is denatured, the oligonucleotide primer anneals to its complementary sequence with mutually opposing 3' ends, and the DNA polymerase extends the annealed brimer and 5
``Amplify the DNA segment defined by the terminus.

Cycles of DNA denaturation, primer annealing, and DNA segment synthesis defined by the 5' ends of the primers are performed as many times as necessary to amplify the HLA-IIRB DNA until it is sufficient for use in step (ii) of the method of the invention. repeated. Amplification may be continued for 20-40 cycles, such as 25-35 cycles.

Any suitable oligonucleotide primer can be used. Brimer is HLA-DR and/or D
It may be suitable for amplification of u+ multiple alleles or for specific amplification of such alleles. Primers suitable for multi-allele amplification are: GH46 (left): TGT to CCGGATCCCTTC
To CCCCAC8GCACGGH5 (right): CTCC
CC^^CCCCGATGTTGTGTCTGC^.

For specific amplification of 11L^-DR+a8 and -DRw5(u+12), G) 150 was used as the right-hand and left-hand brimers, PL8/12: T
TCTTGC:AC:TACTCTAC(:CG can be used.

Primers may be labeled to facilitate analysis of step (iii) of the method of the invention. The primers contain directly detectable tags, e.g. radionuclides (e.g. 32P, 3
5S, +4 (: or 12J), fluorescent compounds (eg fluorescein or rhodamine derivatives), enzymes (peroxidase or alkaline phosphatase), avidin or biotin. The labels of the two primers may be the same or different.

The second step of the HLΔ-DRB gene into the amplified DNA, i.e. the sample DNA as defined by the PCR primers.
The amplification product fragments of exonic nucleotide sequences are then separated according to size. This is achieved by electrophoresis or high pressure liquid chromatography. Separation is carried out on a support. For electrophoresis, a gel that does not denature N8 is typically used, such as a polyacrylamide gel.

The amplified DNA is separated on a gel according to the size of each fragment. Electrophoresis is performed under conditions that separate the fragments to the desired degree.

Usually, a degree of separation that separates fragments with a size difference of about 10 bp is sufficient. Size markers may also be run on the gel so that the size of the fragments can be calculated.

The size distribution, or segregation pattern, of the amplified DNA fragments is allele-specific. This separation pattern or PCR fingerprint can then be visualized. If the PCR primers are labeled, this label can be made explicit. The support carrying the separated labeled DNA fragments is contacted with a reagent that detects the presence of the label.

If the PCB primer is not labeled, the support carrying the PCR fingerprint is contacted with ethidium bromide and the DNA fingerprint is visualized under ultraviolet light.

The length polymorphism of the DNA fingerprint is thus determined. This is compared with the length polymorphism of the DNA fragment obtained from PCR amplification of the second exon nucleotide sequence of HLADRB in a second DNA sample. In this way, correspondences between HLA-DR and/or Dw allotypes of DNA samples are obtained. It can be confirmed whether length polymorphisms of the DNA fragments have been obtained and therefore whether the allotypes of the two samples are the same.

If the allotype of the second DNA sample is known, it is possible to recognize whether the allotype of the first DNA sample is the same.

The length polymorphism of the second DNA sample is obtained by using the same conditions used to obtain the length polymorphism of the first DNA sample. Typically the same brimers are used. However, it is not necessarily necessary to perform PCR amplification in the same number of cycles or under the same reaction conditions. Furthermore, if it is possible to determine the relative correspondence of length polymorphisms of DNA fragments obtained by amplification of each DNA sample, it is not necessary to similarly perform separation of the obtained DNA fragments.

According to the invention, the second DNA sample is different from the first DNA sample.
It may be analyzed simultaneously with or separately from the analysis of the sample.

In practice, the multiplicity of the RN^ samples can be analyzed.

Typically each sample is a selected sample. Samples from selected individuals can be analyzed.

The length polymorphism determined for each sample can be stored in a computer.

Thus, a computer database can be created containing size distribution patterns of different samples, different DRs and/or 0w specificities, or indeed all DRs and 0w specificities. Various molecular dimensions of fragments generated by PCR with respect to different DR and/or DW specificities determined for the sample and/or known samples can be input into the computer. Therefore, any unknown DR and/or Dw type can be determined (typed) by comparing the size of the fragments formed after PCR of the DNA of an unknown sample to a reference database. Therefore, in step (iv) of the method of the invention, length polymorphisms for the first DNA sample are determined from the length polymorphisms of the second DNA sample.
A comparison can be made to the length polymorphism stored in a computer database for the A sample.

Comparing the PCR fingerprint to another PCB fingerprint, the individual whose DNA was tested to obtain two fingerprints matched HLA-DR and 0w
It is possible to determine whether or not a person has an allotype. Therefore, the method of the invention can be applied to DNA samples from more than one individual. Alternatively, the PCR fingerprint can be compared to a previously obtained standard fingerprint.

In this way, correspondence between fingerprints can be determined.

The methods of the invention can be used to determine whether a transplant or infusion donor and a transplant or infusion recipient or applicant receptor have matching HLΔ-DR and Du+ allotypes. PCR fingerprints from the donor and acceptor or applicant acceptor can be compared.

The graft may be a tissue graft, such as a heart, lung, liver or kidney graft, or a bone marrow graft. The infusion may be a blood transfusion. Thus, HLA-DR/DW matching of related or unrelated living donors is obtained for allogeneic transplantation.

Alternatively, the methods of the invention can be used to determine the paternity of an individual. This determination can be made by comparing the PCR fingerprints obtained on the individual, the individual's mother, and the suspected father of the individual. The methods of the invention can also be used to determine whether an individual is susceptible to or suffering from a disease associated with the HLA-DR and/or DIll allotype. Such diseases are treated by Immunol, Rev.
, 70.1-218.1983.

The present invention is (a) suitable for use in PCR and HL
A test killer I.A. comprising two oligonucleotide primers capable of annealing to complementary sequences at each end of the second exon of the A-DRB gene, and (b) control DNA and/or control PCB amplification product.
Also provided.

Primers can be labeled as described above. Control DNA and control PCR amplification products also typically may be labeled. The kit also includes thermostable DNA polymerases such as Taq, dATP, dCTP, and dGTP.
and dTTP, and PC of selected DNA samples.
It may include one or more databases containing length polymorphisms of DNA fragments produced by R amplification.

The length polymorphisms stored in the database can be polymorphisms of DNA samples of known DR and/or Dw specificity. Therefore, the database contains known DR and/or DI
ll allotype, in fact may include PCR fingerprints of any such allotype available.

Figures 2.3 and 5 (DR or 0w allotype) or Figure 4
Length polymorphisms as shown in the figure (possible combinations of these allotypes) can thus be included in the database.

Hereinafter, the present invention will be described by way of examples with reference to the accompanying drawings.

9th and 10th International Histocompatibility Study Group N1ntha
nd Tenth International old st.
Laboratories participating in the CompatibilityWorkshops or, as appropriate, Porton Dow, UK.
European Co11ection located in n.
ofΔnimal Ce1l Cultures,
A well-characterized BLCL heterozygous for the DL8-Dr/Du+ allele was obtained. l(LΔ−DR
/DI11 heterozygous cells were purchased from E, Ms. Bidu+el1 (United Kin, Brstol, UK).
gdomTransplant 5erive) was obtained. )IL^-DR and 0
The w allotype is conventionally described (Bi+(uell, I+
++muno1. Today 9.18-23.19
88; Bidwell et al., Transplantati
Confirmed by RFLP typing of Taq I-digested genomic DNA using short DRB, DQB and DQAcDN^ probes according to J.D. on 45.640-646.1988).

The reaction mixture (10hffi) was a genomic DN^lu prepared as described in the literature (Bidwell et al., 198B).
g (for BLCL) or 2 μg (HLA-DR/Dw
for heterozygous cells), 10 mNTris-HCI,
pH8,3,1,5+aM MgCL, 0.01%
(,/V) Gelatin, dATP, dCTP, dGT
It contained 0.2mM each of P and dTTP, and 1μ each of the 5' (left) and 3' (right) oligonucleotide primers of the second exon of the HLA-DRB gene. Brimers used were (a) GH46 (left, nucleotide sequence 5' CCGG) for biallelic amplification;
ATCCTTCGTGTCCCCACAGCACG3'
) (Scharf et al., Human, rm+++u
no1. Kaki, 61-69.1988) and GH50 (
Right, nucleotide sequence 5' CTCCCC^^CCCC
GATGTTGTGTCTGCA3') (Schar
f et al., Human Immunol, 22.61-6
9゜1988), (b)■L^-DRw8 and DRw5
(w12) for specific amplification of PL8/12 (left, nucleotide sequence 5' TTCTTGG^GTACTCT
ACGGG3') and GH50 (right). For combinatorial amplification, GH46, PL8/12 and Gl(5
0 each 1. M was used. The mixture was heated to 94°C for 5 minutes, placed on ice for 2 minutes, and treated with 2,5 units of Taq polymerase (Pers., Norwalk CTO 6859, USA).
kin Elmer Cetus) was added.

Programmable cyclic reactor (USA, SanDi
Ego, Ericcomp I located at C^92121
DNA amplification was performed using nc, ). Amplification cycle parameters were 94°C for 60 seconds (Figure 1(d)), 9
cooling ramp for 0 seconds (Fig. 1(e)), 6 for 120 seconds
5°C (Figure 1(f)), heating ramp for 70 seconds (Figure 1(a)), 72°C for 120 seconds (Figure 1(b)) and 1
The heating ramp was performed for 60 seconds (FIG. 1(C)). After 35 amplification cycles, a 72°C heating step was performed for 10 minutes.

Polyacrylamide gel ゛Patented vertical mini gel electrophoresis system (USA,
Bio- located at Richmond, C^94804
A non-denaturing polyacrylamide mini gel (IXTBE (89+nM
Tris-borate, 89mM boric acid, 2mM ED
12%-/diacrylamide in TA pH 8, o):
P in N,N'-bisacrylamide (29:1)
An aliquot (10 μ) of CR-amplified DNA was subjected to electrophoresis at 200 volts for 95 minutes. The dimensions of the gel formed were 82 mm (w) x 72 nua (h) x 0.
75aua(d), 3mm(u+)X10+am
It contained 15 sample slots with dimensions of (h) x 0.75+am (d). After electrophoresis, the gel was immersed in l x TBE containing ethidium bromide (0.5 ml/il) for 30 minutes, and the DNA was visualized using a 302 nm ultraviolet transillumination device. DNA for reamplification experiments
was excised from polyacrylamide gel and purified by isotachophoresis and ethanol precipitation. Aliquots were reamplified using the same conditions as above.

1 To establish a simple and reproducible molecular method for the determination of the HLA-DR0w homologous PCR fin-print H^-OR and Du+ allotypes, we used PCR
Allele-specific sequences were amplified using amplification techniques.

Skin^-GH46 specific to the second exon of the DRB gene
and a series of IL^-D using the GH50 primers
When DNA is amplified from R/Du+ homozygous BLCL,
Polymorphic PCB products associated with individual HLA-DR serotypes (
We were able to form allele-specific patterns of PCR fingerprints (Figure 2). Heterosexual splits resulted in separate allele-specific PCR fingerprints. When phenotypically identical BLCL groups were tested for each HLA-DR/Dw specificity, each group exhibited identical PCB fingerprints (not shown). The PCR fingerprint was derived from the primary product (a series of IIL^-DRB gene second exon nucleotide sequences predicted from 271
bp) and a higher molecular weight multiple product (hereinafter referred to as satellite DNA). HLA-D
Besides Rw8 homozygous BLCL (see below), 290b
Allele-specific multiple satellite DNA ranging from p to 1000 bp was observed. One cluster of satellite DNA (650bp to 1000bp) is HLA-DR
4. Supertype-specific HLA-Drw related to DR7 and DR9
Figure 2, lanes 7-11, 19-21 and 24).

Experiments were performed to compare the PCR fingerprints of HLA-DRD four-fold homozygous and heterozygous HLA-DR/DIIl cells.

As a result, PCR of HLA-DR/Dw heterozygous cells
It was found that the fingerprint could not be completely predicted from a simple sum of the patterns observed in the corresponding HLA-DR/Dw homozygous cells (FIGS. 4 and 5).

However, the former PCR fingerprint is
It was reliably reproducible by premixing DNA from corresponding HLA-DR/Du+ homozygous cells before R amplification. Figure 4 shows the results of five such representative experiments. By premixing the DNA in this way, HLA-DR/
Certain combinations of DIll specificities can be constructed.

Since nothing has been described for HLA-DR...10 homozygous cells, we tested this specificity by analysis of PCR fingerprints of ITL^-DR210 heterozygous individuals. In each case considered, a unique PCR fingerprint was observed, but HLA
-No coincidence of DRwlO-specific satellite DNA bands was observed (Figure 3).

PCR fingerprinting of HLA-DRw8 homozygous BLCL showed the primary (271 bp) product but no detectable satellite DNA (Fig. 2, lanes 22 and 23), indicating that HLA-DRw8 in HLA-DR/Du+ heterozygous individuals - It was found that there are potential difficulties in recognizing the DRw8 allele. Therefore, a 5° (left) PCR primer was constructed for the purpose of allele-specific amplification. This primer PL8/12 (see Materials and Methods) is FI
L^-Dh+8 and DR...5 (w12) Complementary only to the 5'-sequence of the second exon of the DRBIIl gene. H
The specificity of PL8/12 was confirmed in an expanded panel of ^-DR/Du+ homozygous and heterozygous cells (not shown), with HLA-DRw8 and DR25 (w12) positive cells as predicted from the brimer annealing position. on 24th
A 2 bp primary amplification product is shown (Fig. 5, lanes 2 and 4). However, whether or not the PL8/12 primer was included in the PCR amplification with the GH46 and GH50 primers did not affect the resulting PCR fingerprint. All other HLΔ-DR/DI tested
For Il heterozygous cells, another unique satellite DNA band (Fig. 5, lanes 5-13) was displayed that was not observed in the corresponding homozygous cells. Thus, without relying on allele-specific PCB primers, HLA
-HLA-DRvM in Dr/D11 heterozygotes
8 specificities could be recognized.

To investigate the possible origin of the observed satellite DNA sequences of the PCR-produced satellite DNA, the second (271 bp) product band and the satellite DNA band were excised from a polyacrylamide gel and
Purified and reamplified as above.

Re-amplification of the primary product band from the HLA-DRI homozygous cell line BAF failed to reproduce the satellite DNA sequences observed after amplification to genomic DNA (6th
Figure (a)), therefore, these satellites are not considered to be due to the sequence of the second exon of the HLA-DRB gene, which is separated from the flanking DNA.

HLA-DR3 (u+17) homozygous cell line C^^-0
When the purified satellite DNA bands were reamplified, the low molecular weight bands observed in the genomic DNA PCR fingerprint were reproduced in each reamplified band. That is, the primary product (P) from the upper satellite 1~<U> band
and the lower satellite (L) band were reproduced, and the P band was reproduced from the L band (FIG. 6(b)). Judging further from this, the satellite DNA bands are each P
A set of overlapped arrays (nes
tecl set of 5 sequences).

To test whether it was possible to generate a full-length HLA-DRB gene cDN^ clone from a satellite DNA sequence, we compared the amplification products of genomic DNAΔ and cDN^ from the DR9 haplotype. Only P sequence is cDN
It is clear from this that specific flanking sequences are required to generate satellite DNA (Fig. 6(C)).

[Brief explanation of drawings]

FIG. 1 shows the sample temperature during PCB amplification. IPCR
Show the cycle. The lengths of cycle segments (a) to (f) will be explained in the examples. Figure 2 shows the PCR fingerprint of the HLA-DR/Du+ homozygous B-lymphoblastoid cell line (BLCL). The sequence of the second exon of the HLA-DRB gene was amplified using GH46 and H50 PCR primers. Ml and Ml are molecular size markers (A1 is a BstEn digest of bacteriophage λ, Ml is pBR322
The molecular size is base pair (bp).
). Cells shown in Figure 1
BAF (ECACC87033001), Lane 2 is CI (9w0201), Rou'J3 is BGE (9u+12
01), lane 4 is RML (10w9016),
'y 5ka CAA-0 (ECACC85051626)
, Lane 6 QBL (ECACC85022807),
Lane 7 is BOB-2, lane 8 is TS-10 (9w1
005), lane 9 is SSTO (9w1303), lane 10 is LS-40 (9w1403), lane 11 is R
AS-15 (9W9902), lane 12 is RAG, lane 13 is J-SIN (T29639: E, Bid
u+ell), lane 14 is HBS (9u+0
601), Lyon 15 is EMJ (9w0606),
Lane 16 is JTED (9u+0603) Rune 17ka HAG (9Iu1802), Lane 18 is BR
U (9w0901), lane 19 is PIT (9w070
4), 1i-on 20k BH13 (9u+1901) roof 21k KIJ (9wl104), lane 22 is BAE
(9w0807), 'v-n23 is LUY (9u+08
05), lane 24 is DKB (9I11 and 10w:
9th and 10th International Histocompatibility Study Group N1nth
and Tenth Internal Hi
stocompatibility 1ilorksh
op reference number, ECACC: Europe
n Co11ection or AnimaCell
Cultures reference number). HLA
-DR and 0w allotypes are shown, with the study group (w) prefix omitted for clarity. RFLP as appropriate
The split defined in is also shown. Figure 3 shows PC of HLA-DRwlO positive heterozygous cells.
Shows R fingerprint. The cells shown in the figure are CI (9w0201) in lane 1, C164 in lane 2, and C164 in lane 2.
Lane 3 is BOB-2, Lane 4 is C1103, Lane 5 is KRO (9w0905), Lane 6 is R287, Lane 7 is BUP (9u+0702), Lane 8 is R29.
It is 5. 6M, which indicates DR serological specificity, is a molecular size marker (Mspl digest of pBR322). The primers and abbreviations used are the same as in FIG. FIG. 4 shows PCR fingerprints of HLA-DR/DW heterozygous cells. The PCR fingerprints of homozygous and heterozygous cells are compared for each of the five groups of cells shown. A is DNA from two homozygous cells (0.5 uy each) mixed before PCR amplification. H is D from an HLA-DR/lh heterozygous individual
It is NA. For the cells shown in the figure, lane 1 is ΔL (9u
+0101), lane 2 is BOB-2, lane 3 is ^L
and BOB-2, lane 4 is C84, lane 5 is CI (
9w0201>, lane 6 is BOB-2, lane 7 is C
I and BOB-2, lane 8 is C650, lane 9 is C
AA-0 (ECACC85051626), Iy-n 1
0 is BOB-2, lane 11 is CAA-0 and BOB-
2. Lane 12 is C508, lane 13 is KRO (9w
0505), lane 14 is BOB-2, lane 15 is K
RO and BOB-2, lane 16ka C595, lane 1
7 is BUP (9u+0702), lane 18 is BOB
-2, lane 19 is BUP and BOB-2, lane 20
is C667. IILΔ-DR serological specificity is shown. The primers and molecular size markers M1 and M1 used are the same as in FIG. FIG. 5 shows the PCR fingerprint of HLA-DR...8 positive cells. The combinations of PCR primers used were a: GH46 and GH50, b: PL8/12 and G
H50. ).The cells shown in the figure are BAE in lanes 1 and 2.
(9w0807), lanes 3 and 4 are J-SIN (T2
9639), lane 5 is BOB-2, lanes 6 and 7 are C810, lane 8 is R to G, lanes 9 and 10 are C2
46, lane 11 is JTED (9wO603), lanes 12 and 13 are C372. ) indicates IL^-DR serological specificity. Molecular size markers M1 and M2 are the same as in FIG.

Claims (10)

[Claims] (1) (i) performing polymerase chain reaction (PCR) amplification of the second exon nucleotide sequence of the HLA-DRB gene of a first DNA sample; and (ii) converting the DNA fragments obtained from the amplification according to size. (iii) determining the length polymorphism of the DNA fragments thus separated; and (iv) converting the length polymorphism thus determined to the length polymorphism of the second DNA sample.
comparing length polymorphisms of DNA fragments obtained from PCR amplification of the second exon nucleotide sequence of the LA-DRB gene.
- DR and/or Dw allotype matching methods. 2. A method according to claim 1, characterized in that step (i) is carried out with two oligonucleotide primers each carrying a radionuclide, fluorescent, enzyme, avidin or biotin label. (3) Two oligonucleotide primers having the nucleotide sequences of [there is a gene sequence] and [there is a gene sequence] [there is a gene sequence] are used in step (i) or The method described in 2. (4) The method according to any one of claims 1 to 3, characterized in that in step (i), PCR amplification is carried out for 25 to 35 cycles. (5) The method according to any one of claims 1 to 4, characterized in that step (ii) is carried out by electrophoresis or high pressure liquid chromatography. (6) A method according to claim 5, characterized in that step (ii) is carried out by subjecting the DNA fragments to electrophoresis on a polyacrylamide gel that does not denature the DNA. (7) DNA samples from the graft or infusion donor and DNA from the graft or infusion recipient or application recipient;
Perform steps (i) to (iii) on the sample and compare the length polymorphisms thus determined in step (iv) to determine whether the donor and acceptor have matching HLA-DR and Dw allotypes. The method according to any one of claims 1 to 6, characterized in that it is checked whether the (8) (a) two oligonucleotide primers capable of annealing to complementary sequences at the respective ends of the second exon of the HLA-DRB gene; and (b) control DNA and/or control PCR amplification product. Human HLA-DR and/or Dw, characterized in that it contains
Test kit for allotype matching. (9) The kit according to claim 8, wherein each of the primers has a radionuclide, fluorescent, enzyme, avidin, or biotin label. (10) Furthermore, thermostable DNA polymerase and/or d
The kit according to claim 8 or 9, characterized in that it contains ATF, dCTP, dGTP and dTTP.