MULTIPLEX ARMS-PCR-RFLP METHOD FOR HIGH-RESOLUTION TYPING OF HLA-DRB1

A reliable method for high-resolution HLA-DRB1 typing using the combination of group-specific amplification and RFLP analysis is described. Group-specific PCR amplification (multiplex ARMS-PCR) was carried out under the same conditions for all groups using seven different primer pairs divided into four groups: (1) DR1 and DR10; (2) DR2, DR7 and DR9; (3) DR3, DR5, DR6 and DR8, and (4) DR4. The subsequent polyacrylamide gel electrophoresis was used to determine the group(s) contained in each sample. DR1, DR2/7, DR3/5/6/8, DR4, DRB1*0901 and DRB1 * 1001 could be distinguished easily using this system. Computer analysis of the various restriction enzyme cleavage sites was carried out on 105 DRB1 allele sequences. It was shown that all DRB1 alleles, except for five allele pairs and some alleles possessing silent mutations, could be distinguished with commonly available restriction endonucleases. Computer analyses on the discrimination of the heterozygous and homozygous combinations were also carried out. Although some heterozygous combinations could not be distinguished with single digestion, double digestion using two restriction enzymes could distinguish most of such heterozygotes. The results of the typing of 100 Japanese individuals using this method showed good agreement with those obtained by other methods.     

染色体组分型分析中参数自动测量的新方法

本文中提出了一些用于染色体组分型分析中的染色体参数测量的新方法，包括染色体中轴的确定，染色体图象拉直，着丝粒位置的确定和染色体长度的测量等。由于不仅利用了染色体图象的几何信息，也利用了染色体图象的灰度信息，从而提高了参数测量的精度。     

免疫球蛋白同种异型分型技术和应用

本文介绍使用血凝抑制试验作免疫球蛋白同种异型的分型方法。该试验可在试管、玻片、或微量滴定板中进行。此技术已成功地用于群体调查、Gm和疾病的关联研究、亲子关系鉴定以及血痕鉴定。     

MOLECULAR TYPING AND HAPLOTYPIC ASSOCIATIONS OF HLA-B*44 SUBTYPES

The heterogeneity of HLA-B44 is confirmed and the sequence difference between the two major subtypes, B*4402,*4403, is attributed to one polymorphic site in the third exon. A method is described to discriminate B*4402 and B*4403, and the occurrence and linkage disequilibrium of B*44 subtypes is discussed. No example of B*4401 polymorphism in exon 2 was observed.     

DNA TYPING OF DQ AND DR ALLELES IN IgA-DEFICIENT SUBJECTS

IgA deficiency (IgA-D) represents the most common immunodeficiency syndrome of infancy. In most cases IgA-D represents an isolated immunological disorder, while sometimes it is associated with IgG subclass deficiency or with the presence of autoantibodies. We investigated the pattern of association of IgA-D with DRB1 and DQB1 loci of the HLA region by DNA molecular typing, which allows the identification of previously serologically undefined specificities. We also compared the gene frequency of DRB1 and DQB1 allelic variants between IgA-D subjects with or without serum autoantibodies. Our results indicate that the gene frequency of the DRB1*0102 subtype and of the DRBP0102, DQB1*0501 haplotype is significantly higher in IgA-D than in the general population. Furthermore, the IgA-D subjects with autoantibodies showed a positive association with DR4 and DR13 subtypes, thus supporting the hypothesis that genetic factors are also involved in the association between IgA-D and autoantibodies.     

REPORT FROM THE HLA CLASS II TYPING BY PCR-SSP MULTICENTRE STUDY

Results from 360 HLA-DR and -DQ ‘low-resolution’ typings with polymerase chain reaction sequence-specific primers (PCR-SSP), performed by nine laboratories, were analysed for their overall utility in routinely defining the HLA-DR1–DR18, DR51–DR53 and DQ1–DQ9 specificities in less than 2.5 h. Thirty EDTA blood samples and 10 DNA samples were distributed and analysed by each laboratory. DNA was extracted using a rapid bromide salt extraction protocol. Complete HLA-DR and -DQ typings were performed, three by three, on pre-aliquoted 96-tube PCR trays. When compared with reference typing, 351/360 (98%) correct DR typings were obtained, whereas 320/360 (89%) of the DQ phenotypes were correctly assigned. The time for three complete HLA-DR and -DQ ‘low-resolution’ typings, including DNA extraction, ranged from 2.0 h to 2.3 h. Unfortunately, an unusually high level of PCR amplification failures was observed (3%), probably due to diffusion and a significant volume loss from some of the pre-aliquoted primer mixes. Consequently, only 52% of the typings were without any amplification failure, and 0–2 amplification failures where found in 88% of the PCR-SSP typings performed. The number of HLA-DR–DQ retypings needed was 7 and 8%, respectively, reflecting the low number of typings where allelic identification was directly affected by the relatively high level of amplification failures in this study. Thus, a 91–98% success rate of correctly identified HLA-DR and -DQ alleles could be maintained, even under suboptimal typing conditions.     

MLC TYPING IN HL-A IDENTICAL NON-SIBLING PAIRS IN AN INBRED POPULATION

Mixed Lymphocyte Culture (MLC) tests were performed between HL-A identical non-sibling members of an inbred population. Three pairs were known to be related, and the descent of chromosomes is followed by both HL-A serology and MLC tests. The implication of a high frequency of negative MLC tests between HL-A identical non-sibling pairs in this population as compared with other populations is discussed.     

AN HLA-DR TYPING PROTOCOL USING GROUP-SPECIFIC PCR-AMPLIFICATION FOLLOWED BY RESTRICTION ENZYME DIGESTS

A simple PCR-based protocol for HLA-DR typing suitable for a routine practice is described. The method involves, first, a PCR amplification with seven different, group-specific (DR1, DR2, DR4, DR7, DR9, DR10, and DR3+5+6+8) primer-pairs, and second, typing of HLA-DR allele more exactly in DR1, DR2, DR4, and DR3+5+6+8 groups by digestion of PCR products with restriction enzymes distinguishing different HLA-DR types within each of the groups. Altogether 24 HLA-DR alleles, or any combination of these, can be typed. The whole procedure, starting from a blood sample, can be carried out during a single working-day. The method was tested by typing a set of homozygous cell lines, as well as a local panel previously typed by PCR/oligotyping. Also, 227 patients waiting for transplantation were typed to test the method in a routine setting. The results suggest that this kind of approach gives reliable HLA-DR types and works well in the routine use.     

HLA-A LOCUS DNA TYPING OF THE 4AOH CELL PANEL BY ARMS PCR

As part of the Fourth Asia-Oceania Histocompatibility (4AOH) Workshop, the authors have demonstrated a method of DNA-based tissue typing of the HLA-A locus using ARMS-designed primers in a panel of specific PCR reactions. The study was carried out blind under Workshop conditions and the results confirm the method as an accurate means of determining HLA-A locus tissue types.     

HL-A TYPING IN AN INBRED POPULATION IN A SWISS ALPINE VILLAGE: FIESCHERTAL

In a population of 164 very closely related members of a Swiss alpine village, the HL-A system and seven other immunogenetic systems were studied. The two main techniques of lymphocytotoxicity and leuco-agglutination were used in two different laboratories for the HL-A serology. The W4(4a) and W6(4b) antigens are especially emphasized, and discrepancies between the two techniques discussed. The use of large families proved valuable for the definition of several previously ill defined anitgens.     

THE COLICINE TYPING OF COLIFORM BACILLI IN THE STUDY OF CROSS-INFECTION IN UROLOGY

Three hundred and fourteen strains of coliform bacilli (79 Klebsiella and 235 E. coli), mostly isolated from urine, were investigated for their production of, or sensitivity to, certain colicines.     

HISTOCOMPATIBILITY IN RUMINANTS THE PRODUCTION AND EVALUATION OF ALLO-ANTIBODIES FOR GL-A TYPING IN GOATS

The production of goat lymphocytotoxic allo-antisera by immunization by lymphocytes or allografting is described. Analysis of typing results of several goat families and the correlation between the lengthening of allograft survival and graft exchange between compatible and incompatible animals, suggest the existence of a major histocompatibility system (GL-A) in goats.