A simple, sensitive ELISA that is performed in 96-well microtiter plates and that requires less than 90 minutes to complete was developed for HLA-DRB oligotyping. The second exon of HLA-DRB1 was amplified using an unlabeled forward primer and a biotinylated reverse primer and the PCR product was immobilized in avidin-coated wells. Subsequent treatment included exposure to 0.4 N NaOH to remove the nonbiotinylated sense strand, addition of a fluorescein-labeled oligonucleotide probe, one or more 5-minute stringency washes, addition of an alkaline-phosphatase-labeled anti-FL FAB, and then alkaline-phosphatase substrate and amplifier. An intense red-violet color developed within 15 minutes in positive wells and could be quantitated by OD readings at 490–495 nm. To control for stringency and to establish threshold OD values for positive reactions, biotin-labeled antisense oligos that were complementary to the probe or that differed by one or more bases were immobilized in wells in place of PCR products. The assay was sensitive to τ0.05 pmol (∼4 ng)/ well and required only standard incubators and waterbaths and an optional microplate reader. All reagents were commercially available. The method should facilitate oligotyping of both class I and class II alleles and is adaptable for analysis of other polymorphic gene products.

The HLA-B5,B35 cross-reacting group is a large and serologically complex antigen family which includes the World Health Organization-recognized specificities. HLA-B5,B51,Bw52,B35,B18,Bw70,Bw71, and Bw72. In addition, several variants of antigens in this cross-reacting group have been described in the past but have not yet gained official recognition. A genetic basis for the complexity and the protein and molecular bases of this highly cross-reactive and polymorphic cross-reacting group have yet to be established. The potential contributions of shared amino acid sequences, the occurrence of multiple epitopes on a single HLA-B molecule, and the presence of new HLA-C antigens have been difficult to resolve. To address this issue, we have carefully examined the serologic reactions of more than 900 allo- and monoclonal antibodies (Tenth International Workshop, Third Asia-Oceanic Workshop, and local reagents) versus lymphocytes from 92 individuals of diverse ethnic origin (North American Caucasians, North American blacks, Amerindians, Middle Eastern Caucasians), 84 of whom were informative for the HLA-B5,B35 cross-reacting group and related antigens. Our results demonstrate that the HLA-B5,B35 gene products share different combinations of distinct epitopes. We have constructed a model for the evolution of this cross-reacting group by assigning polarity to distinct diversification steps utilizing principles of maximum parsimony.

We describe here two monoclonal antibodies with HLA-DR7 serologic specificity. The antibodies, SFR16-DR7M, a cytotoxic rat IgM antibody of high affinity, and SFR16-DR7G, a noncytotoxic antibody of the rat IgG 2a class, react with only DR7-positive cells in radioimmunoassay. The cytotoxic activity of SFR16-DR7M correlates completely with the presence of the DR7 specificity, and segregates with the DR7-bearing haplotype in a family. SFR16-DR7M precipitates a class II molecule with the electrophoretic characteristics of DR molecules from LG-10, an HLA-DR7 homozygous cell line. SFR16-DR7G completely inhibits the cytotoxicity of SFR16-DR7M, but only partially inhibits the cytotoxicity of a chimpanzee antiserum with DR7 specificity, Gay/Swei. In binding-inhibition studies, binding of SFR16-DR7M to LG-10 cells is only partially inhibited by the chimpanzee antiserum and vice versa. Both SFR16-DR7M and Gay/Swei reciprocally deplete the same class II molecules from a 35S-methionine-labeled detergent-solubilized membrane preparation of the LG-10 cell line. The chimpanzee serum Gay contains antibodies reactive with epitopes on separated DR7 beta chains, while both SFR16-DR7M and SFR16-DR7G bind only to DR7 alpha-beta complexes. These data suggest that at least two allogeneic epitopes exist which result in the same serologic specificity, and that these epitopes differ in their requirement for alpha-beta complex formation.

A general breeding protocol useful in the construction of congenic lines of mice disparate in the 15 cMT-H-2 region of chromosome 17 in non-t chromosomes is described. Two such congenic lines, B6.TC2/Rn and B6.TC3/Rn, were derived from the C57BL/6J and B6.C-H-2d/By strains using this protocol. Both B6.TC2 and B6.TC3 dissociate the quantitative activity locus for glyoxalase I (Qglo-1) from theH-2 complex, and hence possess BALB/cBy DNA centromeric toH-2. However, neither new strain is able to map anyH-2-associated restriction fragment length polymorphism with anH-2 cDNA probe even though both strains are recombinant in the 2 cMQglo-1-H-2K interval.

Two hybrid clones from a fusion of C57BL/6 anti-DBA/2 spleen cells and the myeloma line Sp2/0 secrete antibodies reactive with a product of the murine major histocompatibility complex (MHC). The two antibodies are provisionally designated 513.11 and 513.29. Both react in rabbit-complementmediated cytotoxicity with spleen cells of H-2 d , H-2 f , H-2 r , and H-2 p strains. In addition, both antibodies hemagglutinate red blood cells from these strains. S13.11 is also cytotoxic for H-2 a , H-2 k , H-2 u , and H-2 v spleen cells but does not hemagglutinate red blood cells from mice bearing these haplotypes. With the exception of H-2 v , this strain pattern mimics the public specificity H-2.8. Quantitative absorption of S13.11 shows that H-2 d cells are twice as efficient as H-2 k cells in their ability to remove the 513.11 antibody. 513.29 reacts weakly in cytotoxicity with H-2 kv spleen cells and does not react with cells from H-2 u or H-2 v . Blocking studies indicate that 513.11 and S13.29 react with the same or a closely related molecule on the cell surface.

TheHLA complex codes for three distinct 44000 dalton molecules associated withΒ2 microglobulin — HLA-A, B and C —each with its own multiallelic series of private antigens. The HLA-B molecule is exceptional in that it also carries a diallelic system,Bw4 andBw6. One of these,Bw4, is often associated with the A- locus specificity A9. This finding has usually been ascribed to linkage disequilibrium between A-and B-locus antigens. We have shown, however, that an epitope called LHe is actually shared by A-locus and B-locus molecules. This epitope is found on all HLA-B molecules bearing the Bw4 determinant and is also found on all HLA-A molecules carrying the A9 (Aw23 and Aw24) or Aw32 specificities. We consider this a “public” HLA antigen; the possible molecular basis for both subtypic and public antigens on a single glycoprotein is discussed.

Development of information about the HLA system, although still incomplete, has been greatly accelerated by international workshops. Some of these are bench type, some involve scientists from many countries and some are regional. This report summarizes a workshop‐type conference of a different kind—between scientists of two major racial groups, Caucasian and Oriental. Some specificities are common to both groups, as are the diseases they are associated with. Some diseases are associated with given specificities in Caucasians and with other specificities in Japanese, while in other instances neither diseases nor specificities are shared. The principal features of disease associations in the two groups are contrasted here. Both similarities and differences offer new approaches to studies of the nature of disease association.

Abstract H-2 antigens from EL4 (H-2b) and RL♂ 1 (H-2d) leukemias were solubilized with deoxycholate and partially purified based on their adherence to a Lens culinaris hemagglutinin column. In double reciprocal experiments we have shown that these preparations specifically inhibit conjugate formation between target cells and alloimmune peritoneal lymphocytes, a preparation rich in cytotoxic T lymphocytes.

Publisher Summary The human genetic histocompatibility complex controls the expression of HL-A as well as antigens detected in mixed lymphocyte reactions. This chapter discusses an experiment in which sera from multiparas from an inbred population were screened for cytotoxic activity to B and T lymphoid cells maintained in long-term tissue culture to define non-HL-A alloantigens serologically. Selected cytotoxicity to cultured B lymphoid cells was observed with no detectable reactivity to T cells. These sera were then used in studies on peripheral blood lymphocytes separated into T and B cell subpopulations. The B and T cell isolates were tested against the above antisera in complement-dependent dye exclusion assay. Thus, human B lymphocyte specific antigens were detected and appeared to be allotypic.

A nomenclature committee composed of geneticists and immunologists, including specialists in tissue typing, has met after each of the histocompatibility workshops beginning with the third workshop in 1967. The Committee in part under the auspices of World Health Organization and International Union of Immunological Societies (WHO‐IUIS), met after the sixth workshop in Århus in July 1975. The expanding knowledge of the genetics of the major histocompatibility system of man has necessitated a revision of the terminology for the HLA region following the principles established in previous reports. This has been done with as few changes as possible.

To analyze the nature of the target cell determinants recognized and bound by killer lymphocytes during lymphocyte-mediated cytolysis (LMC), the specific binding of serologically active tumor cell membrane fractions to cytotoxic T lymphocytes has been investigated. Particulate membrane fractions and soluble antigen preparations (extracted by papain or 3 M KCl) from tumor target cells were tested for their ability to inhibit the destruction of intact 51Cr-labeled target cells by killer lymphocytes in vitro. The effect of papain-solubilized tumor cell antigen on the binding of killer lymphocytes to tumor cell monolayers was also evaluated. Direct assays to determine the extent of binding of unlabeled or radioiodinated soluble antigen (extracted by papain or deoxycholate) to cytotoxic lymphocytes were carried out. In marked contrast to their serological activity, all of these particulate and soluble preparations failed to inhibit LMC or bind to killer lymphocytes in an immunologically specific way. It is suggested that killer lymphocytes recognize and bind to an antigenic complex whose organization is dependent upon the integrity of the target cell membrane.

To analyze the nature of the target cell determinants recognized and bound by killer lymphocytes during lymphocyte-mediated cytolysis (LMC), the specific binding of serologically active tumor cell membrane fractions to cytotoxic T lymphocytes has been investigated. Particulate membrane fractions and soluble antigen preparations (extracted by papain or 3 M KCl) from tumor target cells were tested for their ability to inhibit the destruction of intact 51 Cr-labeled target cells by killer lymphocytes in vitro . The effect of papain-solubilized tumor cell antigen on the binding of killer lymphocytes to tumor cell monolayers was also evaluated. Direct assays to determine the extent of binding of unlabeled or radioiodinated soluble antigen (extracted by papain or deoxycholate) to cytotoxic lymphocytes were carried out. In marked contrast to their serological activity, all of these particulate and soluble preparations failed to inhibit LMC or bind to killer lymphocytes in an immunologically specific way. It is suggested that killer lymphocytes recognize and bind to an antigenic complex whose organization is dependent upon the integrity of the target cell membrane.