Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-10-20
2003-03-04
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024330, C536S024310
Reexamination Certificate
active
06528261
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the typing of human leukocyte antigen (HLA) alleles. More particularly, the present invention relates to the typing of new HLA alleles
BACKGROUND OF THE INVENTION
The human major histocompatibility complex (MHC) is contained within about 4 Mbp of DNA on the short arm of chromosome 6 at 6p21.3 (Campbell and Trowsdale, 1993). The human MHC is divided into class I, class II and class III regions. The genes of class I and class II encode highly polymorphic cell-surface molecules that bind and present processed antigens in the form of peptides to T-lymphocytes, initiating both cellular and humoral immune responses. The class I molecules, HLA-A, -B, and -C, are found on most nucleated cells. They are cell-surface glycoproteins that bind and present processed peptides derived from endogenously synthesized proteins to CD8+ T-cells. These heterodimers consist of an HLA-encoded &agr;-chain associated with a non-MHC encoded monomorphic polypeptide, &bgr;
2
-microglobulin (Townsend and Bodmer, 1989; Spencer and Parham, 1996). The class II molecules are encoded in the HLA-D region. These cell-surface glycoproteins consist of HLA-encoded &agr;-, and &bgr;-chains, associated as heterodimers on the cell surface of antigen-presenting cells such as B-cells and macrophages. Class II molecules serve as receptors for processed peptides. However, these peptides are derived predominantly from membrane and extracellular proteins and are presented to CD4+ T-cells. The HLA-D region contains several class II genes and has three main subregions: HLA-DR, -DQ, and -DP. Both the HLA-DQ and -DP regions contain one functional gene for each of their &agr;- and &bgr;-chains. The HLA-DR subregion contains one functional gene for the &agr;-chain; the number of functional genes for the &bgr;-chain varies from one to two according to the haplotype (Andersson et al., 1987; Apple and Erlich, 1996).
A variety of techniques are currently used to detect HLA polymorphism, including serological, biochemical, T-cell recognition and, most recently, molecular biological methods.
Serology remains the mainstay method for HLA typing—especially for class I—for many routine histocompatibility laboratories. The micro-lymphocytotoxicity assay (Kissmeyer et al., 1969; Terasaki and McClelland, 1964) is the standard approach: viable peripheral blood mononuclear cells (class I) or separate B-cells (class II) are mixed with antisera (polyclonal or monoclonal) of known HLA specificity.
Detection of polymorphism can be achieved by looking at the different amino acid composition of HLA molecules through biochemical techniques such as one-dimensional isoelectric focusing (IEF; Yang, 1987). This method relies on amino acid substitutions contributing to changes in charge of the HLA molecule.
Another HLA typing method is the mixed lymphocyte reaction (MLR). Concurrent to observations being made using HLA-specific antisera, it was noted that lymphocytes from two unrelated sources, when mixed in culture, would proliferate (Hirschorn et al., 1963).
Analysis of HLA specificities from DNA provided a new approach to defining their polymorphic differences. Rather than looking at differences in the expressed molecule, polymorphism is characterized at the nucleotide level.
An important and powerful development in the field of molecular biology has been the polymerase chain reaction (PCR, Mullis et al., 1986; Mullis and Faloona, 1987). In tissue typing, PCR is used to amplify the polymorphic regions of HLA genes. This HLA PCR product can then be analysed for its polymorphic differences, to establish the tissue type. A number of such approaches have been developed, including hetero duplex analysis of PCR products (Clay et al., 1994), single-stranded conformational polymorphism analysis of the PCR product (PCR-SSCP; Yoshida et al., 1992), sequence-based typing (SBT; Santamaria et al., 1992 and 1993), the use of sequence specific primers in PCR reaction (PCR-SSP; Olerup and Zetterquist, 1991), the use of PCR in combination with sequence-specific oligonucleotide probing (PCR-SSOP; Saiki et al., 1986) or probing by reverse dot-blot (Saiki et al., 1989). These approaches, used singly or in combination, have all been applied as DNA-based methods for tissue-typing of class I and class II HLA specificities.
For class I alleles, hypervariable regions are found at different degrees in both exon 2 and exon 3, which encode the peptide binding groove of the class I molecule. Polymorphism within class II is contained mainly within defined hypervariable regions in exon 2. These polymorphisms make differentiation between alleles achievable through hybridization with relevant probes.
Aims of the Invention
It is an aim of the present invention to provide a method for typing of the alleles HLA-DRB1*0820, HLA-DRB1*04new, HLA-DRB4*01new, HLA-B*3913, HLA-B*1406 and/or HLA-B*51new.
It is a more specific aim of the present invention to provide a method for typing of said alleles, with said method comprising an amplification step and a hybridization step.
It is also an aim of the present invention to provide primers for said amplification step.
It is also an aim of the present invention to provide probes for said hybridization step.
It is also an aim of the present invention to provide a diagnostic kit enabling said method for typing.
It is another aim of the present invention to provide a method for detection of the protein fragments encode by the HLA-DRB1*0820, HLA-DRB1*04new, HLA-DRB4*01new, HLA-B*3913, HLA-B*1406 and/or HLA-B*51new genes.
It is another aim of the present invention to provide an antiserum or a ligand for use in the detection of said protein fragments.
It is another aim of the present invention to provide a diagnostic kit for the detection of said protein fragment.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses the sequence of exon 2 of the HLA allele DRB1*0820. This sequence is identified by SEQ ID NO 1 and is shown below.
5 10 15 20
(47)
(SEQ ID NO 1)
CA CGT TTC
T
TG
GA
G
TAC
T
CT ACG TCT GAG TGT CA
T
TTC TTC AAT GGG
25 30 35
(92)
ACG GAG CGG GTG CGG TTC CTG GAC AGA
TA
C TTC
T
AT
A
AC CA
A
GAG
40 45 50
(137)
GAG
TA
C
GT
G CGC T
T
C GAC AGC GAC GTG GGG GAG T
A
C CGG GCG G
T
G
55 60 65
(182)
ACG GAG CT
G
GGG CGG CCT
GAT
GCC GAG
TA
C TGG AAC AGC CAG AAG
70 75 80
(227)
&emsp
De Canck Ilse
Mersch Guy
Rossau Rudi
Einsmann Juliet C.
Innogenetics N.V.
Jones W. Gary
Lahive & Cockfield LLP
Nand, Esquire Richa
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