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1.10.1. Cellular typing

HLA-D specificities represent functional epitopes

recognised by the TCR and are typed by the mixed lymphocyte reaction (MLR). The MLR consists of looking for a

proliferative response (blast cell transformation) in a mixed culture of lymphocytes (Festenstein and Ollier 1988). The test uses homozygous typing cells (HTCs), which are cell cultures of lymphocytes that are homozygous for a

known Dw specificity, and that have been irradiated so that they can no longer respond to stimulation, but are still able to stimulate non-irradiated or "responder"

lymphocytes. The HTC or "stimulator" and responder cells are cultured together and if no blast cell formation is observed (negative MLR), then the responder and stimulator cells must share an HLA-D specificity. If blast cells are seen, a proliferative response has occurred (positive MLR) and the responder and stimulator cells do not share HLA-D specificities.

The DP antigens cannot be studied by the MLR as HLA-DP molecules are expressed at lower levels that DR and DQ

(Bodmer et al 1987) and do not stimulate HTCs. DP

polymorphisms are detected cellularly by PLT, also called the secondary mixed lymphocyte reaction. The PLT relies on the accelerated secondary response of primed lymphocytes; the lymphocytes used to type DP molecules, or responder

cells, are produced by culturing with irradiated stimulator cells that are identical to the responder cells at every

locus except at the DP locus. After 10 days culturing the responder cells have proliferated in response to a known DP type on the stimulator cell. The cells to be typed are first irradiated then cultured with the primed responder cells. In contrast to the MLR, if a proliferative response occurs then the cells being typed share a DP type, and if there is no proliferative response, the cells being typed do not share a DP specificity with the responder cells

(Festenstein and Ollier, 1987).

1.10.2. Serologic typing

Lymphocytes can be assayed for class II alleles by using a panel of antibodies directed against allele specific

epitopes. Anti-HLA-class II antibody can be obtained from antisera produced by multiparous women, or by individuals that received blood transfusions (Bodmer et al 1987).

Due to the low expression of HLA-DP, DP specific sera has been difficult to obtain. However, with the advent of monoclonal antibody technology, there has been an

improvement in the availability and specificity of typing sera (Bodmer et al 1987). Monoclonal antibodies against DPwl and w3 (Nadler et al, 1981) and DPw2 and w4 (Heyes et al 1986) have been produced, where DP had been previously untypeable by conventional serology. Although serological typing is relatively simple to perform, until such time as

there is a complete repertoire of HLA-DP typing antisera available, serological typing will not be a method of choice.

1.10.3. Restriction fragment length polymorphism typing

Restriction fragment length polymorphism (RFLP) analysis schemes have been presented for the DR (Kohonen-Corish and Serjeantson 1986, Riisom et al 1988, Bidwell et al 1988), DQ (Bidwell et al 1988, Guardiola et al 1988) and DP

(Easteal et al 1989, Hyldig-Nielsen and Svejgaard 1989, Mitsuishi et al 1989) regions. RFLP typing of HLA-DP is carried out using three restriction enzymes and two probes

(Easteal et al 1989) and can distinguish between all PLT defined type except DPw3 and DPw6. RFLP analysis is easier

to perform, faster and more concise than PLT typing, and does not rely on expression of the DP molecule. RFLP analysis allows better characterization of new variants since heterozygotes can be detected. However, RFLP relies on complete association between polymorphic restriction sites and coding sequence variation. The mutations leading to altered RFLP patterns may not be necessarily linked to novel DP sequence variation.

1-10.4. Sequence specific oligonucleotide typing

A ngelini et al (1989) d e s cr i be a method of HLA-DP t yp i n a

amplified DPB1 second exon PCR product is hybridized with sequence-specific oligonucleotides (SSOs) designed to hybridize to regions containing the hypervariable

sequences. The pattern of hybridization of the panel of probes to the PCR product allows the assigning of an allele to the PCR product. This method is more accurate,

sensitive and efficient than PLT and RFLP typing and only the variation within the polymorphic domain of DPB1 is analysed. This method also takes advantage of the fact there are only a limited number of sequences at the HVRs, and alleles differ in the combination of HVRs rather than allele specific sequences. DPB1 typing schemes using this strategy have been published (Bugawan et al 1990,

Fernandez-Vina et al 1991).

1.10.5. Amplified fragment length polymorphism typing

Amplified fragment length polymorphism (AFLP) typing is based on restriction site analysis of polymorphic enzyme recognition sequences occurring in HVRs. PCR product is digested by restriction enzymes and the fragments separated by gel electrophoresis and visualised by ethidium bromide staining. The development of an AFLP typing scheme for DPB1 alleles is the subject of this thesis. During the course of this work, AFLP schemes for HLA-DR (Uryu et al 1990) , -DQ (Maeda et al 1989, Uryu et al 1990, Ota et al 1991) and -DP (Maeda et al 1990, Ota et al 1991) loci have been published.