Serological analysis of xenogeneic anti-lymphoblastoid cell-line sera with specificity against HLA-B12

In view of the importance of potent anti-HLA sera with narrow reaction patterns against defined HLA antigens, two xenogeneic antisera were raised in rabbits following immunization with human lymphoblastoid cell lines from HLA-nonidentical donors homozygous for HLA-B12. After absorption with lymphoblastoid cell lines of an appropriate HLA phenotype, the antisera were purified over DEAE-cellulose ion exchange chromatography and reconcentrated. Both antisera recognized HLA-B12-positive peripheral blood cells of unrelated donors tested in the microcytotoxicity assay. The two rabbit antisera revealed a high degree of similarity in their anti-HLA-B12 antibody specificity. One antiserum showed some cross reactivity with HLA-B13 as has been reported in allo-anti-HLA-B12 sera. The other antiserum revealed some activity against HLA-DRw7-positive donors. Antibody activity could be removed completely from two further rabbit anti-HLA antisera by absorption with lymphoblastoid cell lines from related and unrelated HLA-identical donors. The advantages of using lymphoblastoid cell lines as immunogens and absorption material for the production of heterologous anti-HLA typing sera are discussed.     

Serological identification of HLA-B13 subtypes.

A serological approach is used to confirm subtypes of HLA-B13 originally observed by one-dimensional isoelectric focusing (1D-IEF). Sixty anti-B13 alloantibody sera were screened against Chinese panel cells. Two clusters of sera showing distinct reactive patterns were identified. One is a shorter reactive pattern than the other. Using these serological reaction patterns, the B13 antigen can be divided into two subtypes, B13.1 and B13.2, in the Chinese population. These serological subtypes appear to correlate well with the 1D-IEF patterns of B13 subtypes. The serological subtyping is also in agreement with the differences in nucleotide sequence previously determined to exist in B13 antigen subtypes. Family studies show that both B13.1 and B13.2 segregate as HLA-B locus alleles. Gene frequencies for B13.1 and B13.2 were 0.0676 and 0.0612, respectively, in our study population of 337 southern Han Chinese.     

HLA-B27 antigenicity: antibodies against the chemically synthesized 63-84 peptide from HLA-B27.1 display alloantigenic specificity and discriminate among HLA-B27 subtypes

A chemically synthesized peptide with an amino acid sequence identical to that of the segment spanning residue 63-84 of the major HLA-B27.1 subtype antigen has been obtained. Specific antibodies were raised in rabbits against this peptide, coupled to keyhole limpet hemocyanin carrier. These antibodies lysed lymphoblastoid cell lines expressing HLA-B27.1 in a complement-mediated cytotoxicity assay. They lysed neither B27-negative target cells, nor B27-positive cells expressing other B27 subtype antigens. Complement-mediated lysis of B27.1-positive targets was inhibited by free peptide and by peptide coupled to an unrelated carrier. In addition, the lytic action of the rabbit antiserum was blocked by a monoclonal antibody with no complement-activating capacity that under the conditions of the assay, was specific for HLA-B27. These results indicate that rabbit antibodies against the 63-84 peptide recognize the native HLA-B27.1 antigen; this antiserum is allospecific in character; and it discriminates among B27 subtypes. Thus the data provide direct evidence on the contribution of the hypervariable region spanning residues 63-84 to the alloantigenic specificity of HLA-B27.     

The peptide binding specificity of HLA-B27 subtypes

Five HLA-B27 subtypes, B^*2701, B^*2703, B^*2704, B^*2705, and B^*2706, were tested for direct binding with twenty-six synthetic nonapeptides carrying the primary anchor residue motifs (combination of amino residues at positions 2 and 9) relevant to B^*2705. The peptide sequences were derived from human HSP89, P53 and MBP. The alpha chains were immunospecifically isolated from LH (B ^* 2701), CH (B ^* 2703), WE1 (B ^* 2704), BTB (B ^* 2705), and LIE (B ^* 2706) cells and their peptide binding was measured by the HLA class I alpha chain refolding assay. The data obtained indicated that the B27 subtypes tested can bind a common set of peptides carrying several different anchor residue motifs. The motifs, R-K and R-R, reported for B^*2705 and a new motif H-R were accepted by B^*2703, B^*2704, and B^*2706, but not by B^*2701. However, other motifs, including known B^*2702 and/or B^*2705 motifs, R-H, R-L, R-A, and R-F, and a new motif found here, R-G, were apparently accepted by all B27 subtypes tested. The observed cross-peptide binding in the B27 subgroup is compatible with the so-called arthritogenic peptide hypothesis in the pathogenesis of ankylosing spondylitis.     

Serological analysis of species specificity in the high mobility group chromosomal proteins.

The non-histone chromosomal protein of the high mobility group (HMG-1) present in mouse liver was purified to homogeneity. Antibodies against this protein as well as pure HMG-1 derived from calf thymus and HMG-E purified from duck erythrocytes were elicited in rabbits. The interaction between the antibodies and the immunogens was measured by passive hemoagglutination and by quantitative microcomplement fixation. Quantitative microcomplement fixation assays revealed that the immunological distance between HMG-1 from calf thymus and HMG-1 from mouse liver and duck erythrocytes was 15. This corresponds to 3% sequence differences. It was estimated that amino acid substitution occurred at about seven positions in the polypeptide chain. Thus, HMG-1 proteins display remarkable evolutionary conservation in their primary sequence, similar to that displayed by histones H4 and H3, suggesting that their biological function is dependent on stringent structural requirements. HMG-E protein is significantly different from both HMG-1 and HMG-2 derived from calf thymus. As such, it is a protein unique to avian erythrocytes.     

Antigenic specificity of antibody-dependent cell-mediated cytotoxicity directed against human immunodeficiency virus in antibody-positive sera.

Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for human immunodeficiency virus (HIV) has been described for HIV-infected individuals. To determine the antigenic specificity of this immune response and to define its relationship to the disease state, an ADCC assay was developed using Epstein-Barr virus-transformed lymphoblastoid cell line targets infected with vaccinia virus vectors expressing HIV proteins. The vaccinia virus vectors induced appropriate HIV proteins (envelope glycoproteins gp160, gp120, and gp41 or gag proteins p55, p40, p24, and p17) in infected lymphoblastoid cell lines as demonstrated by radioimmunoprecipitation and syncytia formation with c8166 cells. Killer cell-mediated, HIV-specific ADCC was found in sera from HIV-seropositive but not HIV-seronegative hemophiliacs. This HIV-specific response was directed against envelope glycoprotein but was completely absent against target cells expressing the HIV gag proteins. The ADCC directed against gp160 was present at serum dilutions up to 1/316,000. There was no correlation between serum ADCC titer and the stage of HIV-related illness as determined by T-helper-cell numbers. These experiments clearly implicated gp160 as the target antigen of HIV-specific ADCC activity following natural infection. Vaccines which stimulate antibodies directed against gp160, which are capable of mediating ADCC against infected cells, could be important for protection against infection by cell-associated virus.     

Avidity dictates the lytic capacity of human cytolytic T lymphocyte clones with similar fine specificity against murine cells expressing HLA-B27 antigen

The role of the avidity of human CTL in the recognition and lysis of murine P815 cells expressing HLA-B27.1 Ag has been examined. Seven B27-specific alloreactive CTL clones were tested for their ability to lyse a B27.1+-P815 transfectant clone 1-7E, obtained after cotransfection of P815-HTR cells with HLA-B27.1 and human beta 2-microglobulin genes. The expression level of HLA-B27.1 on 1-7E cells was comparable to that on a human lymphoblastoid cell line, as determined by flow cytometry. Of the seven CTL clones used, CTL 1, 26, and 29 displayed the same fine specificity as established with a panel of target cells expressing six structurally different HLA-B27 variants. However, CTL 1 and 29 were of higher avidity than CTL 26, in that the lysis of human target cells by only this latter clone was inhibited by an anti-CD8 mAb. Based on the same criteria, CTL 2, 15, and 48 possessed the same or very similar fine specificity, but CTL 48 was of higher avidity than CTL 2 or 15. The seventh clone, CTL 40, was of a different fine specificity and its lysis of human target cells was also inhibited by the same anti-CD8 mAb. Only those clones whose lysis of human targets could not be inhibited by anti-CD8 antibody were able to lyse the 1-7E murine transfectants. These results indicate that, for human CTL clones with identical or very similar fine specificity, only those of higher avidity are able to lyse P815 murine cells expressing the HLA-B27 antigen. The lysis of HLA-B27.1+-murine transfectants by relevant clones was inhibited by anti-CD8 antibody. This result strongly suggests that the relative contribution of CD8 in stabilizing the interaction between human CTL and HLA-B27+-murine target cells is more significant than with human target cells.     

Serological analysis of early mouse embryo with rat monoclonal antibodies produced against mouse teratocarcinoma cells

Rat-mouse hybridoma antibodies were produced against mouse teratocarcinoma F9 or PCC4 aza1 cells, and four clones were established. Both the F11 (IgM) and F20 (IgG2c) antibodies showed a similar specificity, reacting only with nullipotential teratocarcinoma cells. They were also found to agglutinate sheep red blood cells. Solid-phase enzyme-linked immunofluorescence assay showed that, among the neutral glycolipids studied, they only reacted with the Forssman antigen. P2 antibody (IgG2b) reacted with the undifferentiated-type and embryonal endodermtype teratocarcinoma cells. During the preimplantation stage, this antibody did not stain mouse embryos, but it reacted very weakly with the inner cell mass of blastocysts cultured in vitro. In the 5th-day embryo, the embryonic ectoderm as well as the visceral and parietal endoderm were positive, but the extraembryonic ectoderm was not. Mesoderm of the 7.5th-day embryo also reacted with this antibody. However, P2 antigen was not observed in the 16th-day embryo or in adult tissues. F2 antibody (IgG2a), which was reactive with all of the cultured cell lines tested, showed an immunoreaction with mouse embryos throughout the preimplantation stage. However, in the 7.5th-day embryo, the presence of F2 was limited to the cells forming the parietal endoderm. This antigen was present in some epithelial tissues of the 16th-day embryo and adult mouse. Of these antigens, P2 and F2 are probably novel differentiation antigens of the early mouse embryo. Together with the Forssman antigen, these will be important markers for analyzing cell-surface antigens of mouse teratocarcinoma cells as well as embryos.     

Substrate specificity of rat sera IgG antibodies with peroxidase and oxidoreductase activities

We have recently shown that intact IgGs from the sera of healthy Wistar rats oxidize 3,3'-diaminobenzidine (DAB) in the presence and in the absence of H(2)O(2) similar to horseradish peroxidase (HRP). Here we demonstrate for the first time that the peroxidase and oxidoreductase activities of IgGs can efficiently oxidize not only DAB but also o-phenylendiamine, phenol, p-dihydroquinone, alpha-naphthol, and NADH but, in contrast to HRP, cannot oxidize adrenalin. In contrast to IgGs, HRP cannot oxidize phenol, p-dihydroquinone, or alpha-naphthol in the absence of H(2)O(2). In contrast to plant and mammalian peroxidases, IgGs were more universal in their metal dependence. The specific wide repertoire of polyclonal peroxidase and oxidoreductase IgGs oxidizing various substances could play an important role in protecting the organism from oxidative stress and serve as an additional natural system destroying different toxic, carcinogenic, and mutagenic compounds.     

截短表达的TTSuV2 ORF1重组蛋白对猪血清的免疫学反应

【目的】本文旨在了解猪细环病毒2(Torque teno sus virus 2,TTSu V2)病毒蛋白对猪血清的免疫学反应,明确TTSu V2 ORF1编码的氨基酸序列上特定片段的免疫原性,为TTSu V2的免疫学检测方法建立提供实验依据。【方法】以本实验室测序的TTSu V2毒株为研究对象,在大肠杆菌表达系统中克隆表达了TTSu V2ORF1基因上两相互重叠的基因片段,对表达产物进行纯化,分析了两重组蛋白(TTSu V2 ORF1a与TTSu V2ORF1ab)对猪血清抗体的免疫学反应。【结果】应用标签抗体进行Western Blotting分析的结果显示,重组TTSu V2 ORF1a与TTSu V2 ORF1ab蛋白成功表达。应用建立的ELISA方法对212份猪血清进行检测的结果表明,含有179个氨基酸的重组TTSu V2 ORF1a与含有416个氨基酸的重组TTSu V2 ORF1ab蛋白之间有显著的相关性,二者对猪血清的反应基本一致。采用猪血清进行Western Blotting分析的结果显示,血清抗体可特异性识别两重组蛋白。【讨论】TTSu V2 ORF1a与TTSu V2 ORF1ab相互重叠的179个氨基酸序列上(168-346)含有TTSu V2 ORF1关键的B细胞表位,重组蛋白TTSu V2 ORF1a适用于TTSu V2血清抗体免疫学检测。