Techniques used to define human MHC antigens: monoclonals, class I and class II biochemistry

Lanchbury, Jerry S.

doi:10.1016/0165-2478(91)90194-f

Immunology Letters

1991

DOI: 10.1016/0165-2478(91)90194-f

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Techniques used to define human MHC antigens: monoclonals, class I and class II biochemistry

Jerry S. Lanchbury

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1993

1997

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Cited by 4 publications

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“…With respect to the multiple functions of the HLA class I1 antigens, adequate typing protocols are extremely important. Since the elucidation of their nucleotide sequences, analyses combining PCR amplification (9,10) and restriction fragment length polymorphism (RFLP) (1 1-1 3), sequencing, or SSO hybridization (14-17) protocols have partially replaced the originally described serological (18), cellular (19), biochemical (20) and RFLP (21) methods. Typing small quantities of material at the genomic level therefore became possible for the different loci.…”

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confidence: 99%

Rapid DNA typing of class II HLA antigens using the polymerase chain reaction and reverse dot blot hybridization

et al. 1993

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A nonisotopic oligotyping method using reverse dot blot hybridization was developed for HLA class II DQA1, DQB1, DPB1, DRB1, DRB3, DRB4, DRB5 alleles. The polymorphic second exon of the different genes was amplified by the polymerase chain reaction (PCR). For each gene the amplified DNA was hybridized at stringent conditions to membrane-bound sequence-specific oligonucleotides (SSOs) and visualization of positive signals was done by chemiluminescence. A combination of 11, 18, 23 and 31 SSOs was designed to identify 9/13 DQA1, 16/17 DQB1, 23/24 DPB1 and 50/55 DRB1, 4 DRB3, 1 DRB4, 3/4 DRB5 alleles respectively. For the DRB1 locus, an additional DRB1*04 group-specific PCR was developed to make discrimination between the DR4 alleles possible in different heterozygous combinations. The procedure described here provides rapid and nonisotopic genotyping of heterozygous samples from a variety of sources and can be applied for tissue typing, disease susceptibility studies and forensic medicine.

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Rapid DNA typing of class II HLA antigens using the polymerase chain reaction and reverse dot blot hybridization

et al. 1993

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Immunologic Considerations in Tissue Grafting

Chan

1993

Oral and Maxillofacial Surgery Clinics of North America

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A three‐step allele‐level DRB1‐DRB3‐DRB4‐DRB5 genotyping assay using polymerase chain reaction with immobilized sequence‐specific oligoprobes

et al. 1997

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A three-step reverse hybridization assay for allele level-resolution DRB1-DRB3-DRB4-DRB5 genotyping is described. Samples are initially amplified using a generic primer pair for all DRB1-DRB3-DRB4-DRB5 alleles and PCR products are hybridized to generic typing membranes. An intermediate-resolution level genotyping is obtained at this point. Depending on the phenotype, samples are then subjected to a DR1, DR2, DR4, DR52A, DRB3 and/or DRB5 type-specific amplification and hybridization. A third step, involving sequence-specific PCR followed by type-specific hybridization, is only performed to solve certain DR4 and DR52A heterozygous combinations. The assay allows 100% allele level-resolution DRB genotyping. Hybridization membranes contain panels of SSO probes that were optimized to all react specifically under identical stringency conditions. A computer program was written to assist in analysis of the hybridization patterns. The assay was throughly evaluated and has been used to type over 10,000 donors from the Canadian Unrelated Bone Marrow Donor Registry (UBMDR) at allele level-resolution. This method proved to be flexible, easy to update for newly described alleles, easy to perform, fast, and safe. It is also reliable and specific, as 9 novel DRB alleles so far have been detected as aberrant hybridization patterns.

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Techniques used to define human MHC antigens: monoclonals, class I and class II biochemistry

Cited by 4 publications

References 11 publications

Rapid DNA typing of class II HLA antigens using the polymerase chain reaction and reverse dot blot hybridization

Rapid DNA typing of class II HLA antigens using the polymerase chain reaction and reverse dot blot hybridization

Immunologic Considerations in Tissue Grafting

A three‐step allele‐level DRB1‐DRB3‐DRB4‐DRB5 genotyping assay using polymerase chain reaction with immobilized sequence‐specific oligoprobes

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