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HLA-DRB1 Shared epitope
QKRAA/QRRAA/RRRAA
Reverse hybridization kit for the detection
of the shared epitope QKRAA/QRRAA/RRRAA of rheumatoid arthritis in
all known HLA-DRB1 alleles; differentiation between homozygot and
heterozygot
RDB2035
>> Nomenclature
and clinical relevance, References
>> Examples
for the detection
Principles of the test
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Principles of the Test
The "HLA-DRB1 Shared epitope QKRAA/QRRAA/RRRAA" kit from the
company AID enables a reliable and quick detection of the amino
acid motives QKRAA, QRRAA and RRRAA in third hypervariable region
of all yet known HLA-DRB1 alleles. A low resolution typing of DR1
or DR4 isn�t necessary.
The kit enables
- a low resolution typing of DRB1 alleles DRB1*01 (DR1), DRB1*04
(DR4) and DRB1*1001
- a high resolution differentiation of DRB1*01 and DRB1*04 alleles
in shared epitope-positiv or shared epitope negativ,
- typing of the "shared epitope" aminoacid sequences QKRAA, QRRAA
or RRRAA,
- differentiation between homozygot and heterozygot shared epitope
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Test procedure:
- Polymerase chain reaction
With the DNA of a genomic DNA preparation from whole blood, four
polylmerase chain reactions (PCR) are carried out. In this way,
the alleles of the HLA-DRB1 gene are selectively amplified with
the PN mixes PN-SE1 to PN-SE4 with biotin-marked primers (see
chart).
| PN-mix |
amplified DRB1 alleles |
amplified control |
| PN-SE1 |
DR1 |
La/SS-B pseudogene1 |
| PN-SE2 |
DR4 |
Alpha-1-antitrypsin |
| PN-SE3 |
DR7, 10 |
La/SS-B pseudogene1 |
| PN-SE4 |
DR3, 8, 9, 11-16 |
Alpha-1-antitrypsin |
Chart 1: Specificity of the PN mixes
The PCR-products are analysed by reverse hybridization on gene
probes for the presence of the shared epitope. By the detection
of positive controls, the successful DNA isolation and PCR is
documented..
- First reverse hybridization: PN-SE1 and PN-SE2
The PCR-products from the amplifications with PN-SE1 and PN-SE2
are mixed together and analysed by reverse hybridization on gene
probes for the presence of the "shared epitope" in the alleles
of the DRB1*01 and DRB1*04 groups. By the detection of positive
controls, the successful DNA isolation and PCR is documented,
even in DR1 or DR4 negative samples.
In this first hybridization, not only all carriers of a DRB1*01
or DRB1*04 shared epitope (DRB1*04-QKRAA, DRB1*04-QRRAA and DRB1*01-QRRAA)
can be identified, but also heterozygous combinations of different
DRB1*04 alleles, e.g. DRB1*04-QKRAA and, at the same time, DRB1*04-QRRAA,
and also all heterozygous combinations of DRB1*01- and DRB1*04.
In order to distinguish homozygous positive carriers of DRB1*04-QKRAA
(or DRB1*04-QRRAA) from those with only one DRB1*04 "shared epitope"
allele, a second hybridization with a mixture of the amplification
products of PN-SE3 and PN-SE4 is necessary (alternatively, the
PCR products of PN-SE3 and PN-SE4 can be examined by electrophoresis
in agarose gel).
- Second reverse hybridization: PN-SE3 and PN-SE4
The PCR products from PN-SE3 and PN-SE4 are mixed together and
hybridized on another shared epitope strip. Again, the successful
DNA isolation and PCR is documented by the positive controls,
even in samples which have no further non-DR1 or non-DR4 allele
(which are for example homozygous DRB1*04-QKRAA).
The second hybridization enables a distinction between homozygous
and heterozygous positive carriers of a DR4 "shared epitope".
In addition, the allele DRB1*1001 with the motive RRRAA can be
identified.
Several subtypes from the groups DRB1*11 and DRB1*14 also contain
the "shared epitope" QKRAA or QRRAA. This group of non-DRB1*01
or non-DRB1*04 "shared epitope" positive alleles is also detected.
>> Nomenclature
and clinical relevance, References
>> Examples
for the detection |
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