HLA-DRB1 Shared epitope
QKRAA/QRRAA/RRRAA

RDB2035

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

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

Test procedure:

1. 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..
   
   
2. 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).
   
   
3. 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.