Structural relatedness of distinct determinants recognized by monoclonal antibody TP25.99 on beta 2-microglobulin-associated and beta 2-microglobulin-free HLA class I heavy chains

The association of HLA class I heavy chains with beta2-microglobulin (beta2m) changes their antigenic profile. As a result, Abs react with either beta2m-free or beta2m-associated HLA class I heavy chains. An exception to this rule is the mAb TP25.99, which reacts with both beta2m-associated and beta2m-free HLA class I heavy chains. The reactivity with beta2m-associated HLA class I heavy chains is mediated by a conformational determinant expressed on all HLA-A, -B, and -C Ags. This determinant has been mapped to amino acid residues 194-198 in the alpha3 domain. The reactivity with beta2m-free HLA class I heavy chains is mediated by a linear determinant expressed on all HLA-B Ags except the HLA-B73 allospecificity and on <50% of HLA-A allospecificities. The latter determinant has been mapped to amino acid residues 239-242, 245, and 246 in the alpha3 domain. The conformational and the linear determinants share several structural features, but have no homology in their amino acid sequence. mAb TP25.99 represents the first example of a mAb recognizing two distinct and spatially distant determinants on a protein. The structural homology of a linear and a conformational determinant on an antigenic entity provides a molecular mechanism for the sharing of specificity by B and TCRs.     

Preliminary structural characterization of the leukocyte cell surface molecule recognized by monoclonal antibody TA-1

TA-1 is a monoclonal antibody identifying a cell surface molecule with a broad distribution on normal and malignant human leukocytes. Preliminary structural studies performed by using radioimmunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that TA-1 recognizes a two-chain polypeptide of approximately 170 kilodaltons (KD) and 95 KD under reducing conditions. The same experiment conducted under nonreducing conditions yielded bands of approximately 155 KD and 110 KD, suggesting the existence of intrachain disulfide bonds in both subunits. Both polypeptide chains were labeled with tritiated sodium borohydride after treatment of cells with neuraminidase and galactose oxidase, thereby demonstrating that both were glycosylated. Tryptic peptide mapping indicated that the 170-KD and 95-KD subunits did not have significant homology in peptide composition. We are designating this newly defined human leukocyte bimolecular complex gp 170/95.     

从噬菌体表面展示肽库中筛选衣原体单克隆抗体识别的抗原表位

利用抗体捕获法,经三轮淘洗,从表面展示随机肽序列的噬菌体文库中筛选到与衣原体单克隆抗体C17特异结合的噬菌体克隆,其一致序列为：（L／I）PGGS（P／W）,竞争抑制实验表明含特异序列的克隆能与天然抗原竞争。据此,我们认为此序列为衣原体的B细胞抗原表位。     

NMR study on the interaction between MHC class I protein and its antigen peptide

A major histcompatibility complex (MHC) class I protein H-2K(b) was expressed in a large scale as a fusion protein with thioredoxin and hexahistidine at the N-terminus to analyze the interaction with the antigen peptide SIYRYYGL. NMR spectra of the peptide in the mixture solution with the protein showed very broad signals, indicating the obviously clear existence of the dynamic interaction between the class I protein and the antigen peptide. The interaction of the protein and peptide was discussed as well as the surrounding atmosphere of the peptide in the complex.     

Characterization of the antigen recognized by monoclonal antibody 1D3

Human ovarian mucinous cystadenocarcinoma-associated antigen recognized by murine monoclonal antibody 1D3 (Bhattacharya et al., 1982) was characterized. Gel filtration and sodium dodecylsulfate polyacrylamide gel electrophoresis, followed by Western-blot analysis showed that 1D3 is a high molecular weight glycoprotein. Isoelectric focusing of 1D3 antigen showed 2 overlapping antigenic components with PI 2.5 and 2.6. 1D3 antigen was extremely stable (10 min at 100 degrees C) to heating. The antigenic activity was slightly stimulated by treatment with galactosidases, but neuraminidase treatment enhanced the antigenic activity about 3-fold. Antigen activity was completely stable to periodate oxidation. Pronase and trypsin treatment completely destroyed the antigenic activity. Properties of 1D3 antigen suggest that this is a high molecular weight (approximately 5-20 x 10(6) Dalton), sialomucin. Monoclonal antibody 1D3 recognizes only the protein part of this molecule.     

Relationship between the structural conformation of monoclonal antibody layers and antigen binding capacity

Neutron reflection has been used to determine the pH-dependent structural conformation of monoclonal antibody layers adsorbed at the hydrophilic silicon oxide/solution interface, within the pH range 4-8, over which the silicon oxide surface carried weak negative charges and the net charge on the antibody reversed. The depth resolution achieved, by use of D2O as solvent to enhance the neutron contrast of the adsorbed antibody layer, was around 2-3 A. The results have been correlated with the ellipsometric measurement of antigen binding capacity (AgBC). The antibody was a mouse monoclonal anti-hCG (human chorionic gonadotropin) directed against the beta subunit of hCG, with molecular weight of 150 000 and isoelectric point around pH 6.0. At pH 4, the adsorbed antibody could be described as a single layer 40 A thick, consistent with an almost perfect flat-on orientation with all three fragments (Fc, Fab) lying flat on the surface. With increasing pH, the antibody layer swelled (65 A at pH 6, 75 A at pH 8) and could be described as three sublayers of different protein density, consistent with some twisting of molecules so that some fragments became more loosely attached to the surface. At pH 8, the repulsive interaction between protein and surface was reflected in a significantly decreased total adsorbed amount. The dominant effect acting to increase AgBC was decreased surface packing density. The effect of the conformational changes revealed at different pH was less important. The results have shown that within the flat-on orientation adopted by the adsorbed antibody, steric hindrance is the main constraint on binding, restricting the access of the antigen to active sites within the antibody layer.     

Purification and characterisation of MHC class I antigen from rat liver with monoclonal antibody

We have described three monoclonal antibodies (HAM1, HAM2, and HAM3) to rat liver cell membrane glycoproteins. Recently also we reported another monoclonal antibody (HAM4) to rat hepato-renal membrane antigen. Using these monoclonal antibodies, it is possible to purify membrane antigens. This paper describes the details of the purification and the nature of the antigen purified with one of the monoclonal antibodies (HAM2) to rat liver cell membrane glycoproteins. Antigen was purified with immunoaffinity column. The amino acid composition was determined and compared with those of mice MHC class I antigen (H-2) and with the rat lymphocyte membrane antigens which were purified with monoclonal antibodies and of which amino acids compositions were determined.     

Idiotypic replica of an anti-human tumor-associated antigen monoclonal antibody. Analysis of monoclonal Ab1 and Ab3 fine specificity

CaMBr1 is a tissue-specific and tumor-associated saccharidic epitope, defined by mAb MBr1 (Ab1), expressed on glycoconjugates of the human mammary carcinoma cell line MCF-7 and of normal and neoplastic mammary epithelial cells. An anti-anti-idiotypic monoclonal Ab3, 2G-3, identifying a human breast tumor associated antigen, was raised by using as immunogen a mouse anti-idiotypic monoclonal Ab2, A3B10, which behaves as the internal image of CaMBr1. mAb 2G-3, as well as MBr1, defines a saccharidic epitope on glycoconjugates extracted from MCF-7 cells and shows MBr1-like reactivity on normal and neoplastic-tissues. Experimental evidence, however, suggests that the fine immunoreactivity of the two antibodies is not identical, because MBr1 has a preferential reactivity with glycolipids and 2G-3 with glycoproteins. We suggest that a possible biologic explanation for our findings could reside in the nature of the immunogens used to raise the two mAb (glycolipid vs protein "internal image").     

Isolation and characterization of monoclonal antibody directed against antigenic peptide presented by class I histocompatibility molecule]

We describe the isolation and several characteristics of a monoclonal antibody (X5.3.7) which recognizes a peptide derived from influenza virus nucleoprotein and presented by the murine class I major histocompatibility molecule Kd. X5.3.7 is thus an example of an antibody capable of recognizing an epitope normally recognized by T cells.     

Identification, synthesis and properties of a consensus peptide recognized by a monoclonal antibody directed to various type I cytokine receptors

Previous works demonstrated that the monoclonal antibody (MAb) called R7B4 is directed to an epitope shared by receptors for lactogenic and somatogenic hormones as well as interleukins 2 and 6 (IL-2 and IL-6). The MAb inhibited the biological effects of those hormones and cytokines by impairing their binding to receptors. It is known that the receptors for growth hormones (GH), prolactins (PRL), IL-2, and IL-6 pertain to the type I cytokine receptor family, sharing the common motif WSXWS or the homologous F(Y)GEFS. Thus, a set of 34 decapeptides corresponding to diverse receptors containing those sequences were synthesized by the PEPSCAN method and their reactions with MAb R7B4 were measured by ELISA. The MAb significantly recognized 21 peptides, allowing us to establish the consensus sequence HGYWSEWSPE as a portion of the R7B4 epitope. The consensus peptide was synthesized and purified by conventional methods, and its capacity to bind to MAb R7B4 paratope confirmed. Moreover, polyclonal Ab to the peptide elicited in mice were able to inhibit the hGH binding to lactogenic, somatogenic and human specific liver receptors. This fact suggests that the consensus peptide could be used as an immunogen to produce anti-hGH receptor Ab behaving as hormone or cytokine antagonists in certain pathological conditions.     

Molecular and developmental studies of a sperm acrosome antigen recognized by HS-63 monoclonal antibody.

A conserved mouse sperm antigen (MSA-63) recognized by a monoclonal antibody (HS-63) was isolated from mouse testes by single-step immunoaffinity chromatography. Isolated MSA-63 preparation was shown to be a group of proteins ranging from 24-84 kDa and with isoelectric points (pIs) ranging from 4.0-6.0 when analyzed by two-dimensional (2-D) gel electrophoresis. Microsequencing techniques were employed to determine the relationships of various protein spots on 2-D gels. Partial amino acid sequences of some protein spots in isolated MSA-63 preparation were shown to be homologous to mouse actins, while others revealed homology only to the SP-10 protein. Rabbit antisera raised against isolated MSA-63 antigen preparation were used to immunoscreen a mouse testis cDNA library. Isolated cDNA clones carrying a 1.2-kb insert were used to obtain nucleotide sequences containing open-reading frames and to deduce the corresponding amino acid sequence of MSA-63. A high degree of homology was observed between MSA-63 and a known human sperm antigen, SP-10, at DNA/protein levels. Amino acid sequences of tryptic peptides derived from protein spots of 24-47 kDa and pIs of 4.2-4.4 were found to be identical to those deduced from isolated cDNA clones. The gene expression of MSA-63 during spermatogenesis in mice was studied using a specific cDNA probe as well as HS-63. It was observed that MSA-63 was not expressed until the postmeiotic stages of spermatogenesis.     

Differential regulatory role of monomorphic and polymorphic determinants of histocompatibility leukocyte antigen class I antigens in monoclonal antibody OKT3-induced T cell proliferation

Monoclonal antibodies (mAb) to monomorphic and polymorphic determinants on the heavy chain of histocompatibility leukocyte antigen (HLA) class I antigens inhibit mAb OKT3-induced T cell proliferation, whereas the anti-beta 2-microglobulin mAb NAMB-1 does not affect it. The inhibitory effect of anti-HLA class I mAb is specific, is not an Fc-mediated phenomenon, does not require accessory cells, and does not involve early stages of T cell activation. Distinct determinants of HLA class I antigens regulate T cell proliferation by different mechanisms, because the anti-HLA-A2, A28 mAb CR11-351, and the mAb W6/32 to a framework determinant of HLA class I antigens block interleukin 2 (IL-2) secretion and IL-2 receptor expression, whereas the mAb CR10-215 to a monomorphic determinant blocks only IL-2 receptor expression. The mAb CR10-215 and W6/32 induced a 50% of maximal inhibition of T cell proliferation, when added after 27 and 12 hr, respectively, of incubation of peripheral blood mononuclear cells with mAb OKT3. On the other hand, the mAb CR11-351 inhibited T cell proliferation even when added after 38 hr of incubation of peripheral blood mononuclear cells with mAb OKT3 and was the only one to inhibit proliferation of cycling T lymphocytes. It is suggested that HLA class I antigens regulate T cell proliferation by interacting with cell-surface molecules involved in T cell activation. The differential inhibitory activity of the anti-HLA class I monoclonal antibodies tested may reflect the different ability of the corresponding determinants to interact with activation molecules.     

Induction by immunomodulatory agents of a macrophage antigen recognized by monoclonal antibody 158.2 and correlation with macrophage function

MA158.2, a rat monoclonal antibody with binding specificity for cells of the monocyte-macrophage lineage, reacts with an antigen (158.2) whose expression is enhanced on mononuclear cells activated to the tumoricidal phenotype by treatment with lymphokine supernatant containing macrophage activating factor (MAF). The functional relevance of enhanced expression of this antigen has been examined in mouse peritoneal macrophages treated with a variety of immunomodulatory agents and assayed for augmented macrophage-mediated defense reactions, including O-2 production, microbicidal, and tumoricidal activity. An interferon-gamma (IFN-gamma) preparation produced by recombinant DNA technology induced a dose-dependent increase in expression of the 158.2 antigen in inflammatory macrophages which was accompanied by acquisition of microbicidal activity against Listeria monocytogenes. However, these cells did not express tumoricidal activity and induction of this property required concomitant exposure to lipopolysaccharide (LPS). Similar results were obtained using macrophages elicited with pyran copolymer. Exposure to LPS alone induced enhanced expression of antigen 158.2 but did not elicit microbicidal activity. Macrophages challenged with IFN-alpha, IFN-beta, MDP, and bestatin did not exhibit increased 158.2 and also failed to acquire tumoricidal activity when treated concomitantly with LPS. Collectively, these data indicate that the MA 158.2 antibody recognizes an antigen expressed by macrophage populations displaying the so-called primed phenotype in which microbicidal activity is expressed but in which induction of tumoricidal activity requires the addition of a second signal such as LPS.     

Saturation-transfer difference NMR studies for the epitope mapping of a carbohydrate-mimetic peptide recognized by an anti-carbohydrate antibody

Saturation-transfer difference NMR spectroscopy (STD-NMR) experiments have been performed to analyze the topography or epitope of the octapeptide MDWNMHAA recognized by the anti-carbohydrate antibody SYA/J6 in solution; the antibody is directed against the Shigella flexneri Y O-antigen polysaccharide. The results permit a valuable comparison of solution versus crystal-structure data, and provide insight for the design of the next-generation binding ligands.     

Analysis of structural properties and cellular distribution of avian Ia antigen by using monoclonal antibody to monomorphic determinants

In this report, we describe the analysis of Ia-like antigens in the chicken by using a monoclonal antibody (CIa-1) reactive with monomorphic determinants of the Ia-like (B-L) antigens. This antibody reacts with determinants on B cells in all avian species tested, but does not detect antigens on lymphocytes of representative mammals, reptiles, and amphibians. In addition to B cells, this antibody defines a subpopulation of the monocyte-macrophage series and reacts with mitogen-activated T cells. Immunochemical analysis indicates that the CIa-1 reactive antigen is a 65,000-dalton glycoprotein consisting of an alpha-chain of 32,000 daltons noncovalently bound to a beta-chain of 27,000 daltons. Under nonreducing conditions, the beta-chain migrates with slightly faster mobility. Two-dimensional gel analysis indicates that the beta-chain is the more heterogeneous of the two chains. Thus, the antigen detected by CIa-1 antibody is similar in cell distribution and structure to the murine Ia antigens and human DR antigens. During in ovo development, Ia+Ig- cells were not found in the yolk sac but were detected in the spleen, mesonephros, and bursa of 9-day embryos. Two populations of Ia+Ig- cells were identified in the bursa: 40 to 60% of the bursacytes, mostly larger cells, exhibited brighter immunofluorescence reactivity than the smaller bursacytes.     

Cell type specificity of a neural cell surface antigen recognized by the monoclonal antibody A2B5

Summary The monoclonal antibody A2B5 reacts with the surface membrane of most neurons in monolayer cultures of cerebellum, retina, spinal cord, and dorsal root ganglion of embryonic and early postnatal C57BL/6J mice maintained in vitro for culture periods of 2 to 10 days. A small percentage of astroglial cells also expresses A2B5 antigen in murine, chicken and rabbit cerebellum, in chicken retina, and in murine spinal cord and dorsal root ganglion. Less mature astroglial cells are stained for A2B5 antigen to a greater extent than the more mature astrocytes. Astrocytes from rat cerebellum and mouse retina were not found to express A2B5 antigen under the present culture conditions. Some of the less mature oligodendrocytes recognized by 04 antibodies express A2B5 antigen, while the more mature 01 antigen and galactocerebroside-positive oligodendrocytes were not found to be A2B5 antigen-positive. Fibroblasts or fibroblast-like cells do not express detectable levels of A2B5 antigen. After fixation of the cells with paraformaldehyde and ethanol, all cell types present in culture are labeled by the A2B5 antibody intracellularly.     

Immunogold co-localization of ovine placental lactogen and the antigen recognized by the SBU-3 monoclonal antibody in sheep placental granules

Ovine placental lactogen and the SBU-3 antigen (derived from a trophoblast membrane preparation), two proteins of widely different structure, function and destination, were shown by ultrastructural immunogold techniques to localize in identical structures in the sheep placentome throughout most of pregnancy. Both were restricted to the ultrastructurally similar membrane-bounded granules in the chorionic fetal binucleate cell and the syncytium at the fetomaternal interface. The Golgi body from which the granules form was also doubly labelled but only in the binucleate cell, never the syncytium. This provides further evidence that the binucleate cells migrate and fuse to form the syncytium. The two proteins were homogeneously distributed in the granules and would be released together by exocytosis. Only the lactogen reaches the fetal and maternal circulations so the SBU-3 may have some more local function. In early pregnancy the SBU-3 antigen is found by itself in the granules, indicating that the association with the lactogenic hormone is not obligatory. Neither antigen was found consistently in the otherwise ultrastructurally similar interplacentomal binucleate cell granules, corroborating the presence of two functional populations of binucleate cells.     

Identification of a linear peptide recognized by monoclonal antibody 2D7 capable of generating CCR5-specific antibodies with human immunodeficiency virus-neutralizing activity

CCR5 is the major coreceptor for human immunodeficiency virus (HIV) infection. The murine monoclonal antibody (MAb) 2D7, which recognizes a conformation-dependent epitope in the second extracellular loop of CCR5, is one of the most potent inhibitors of R5 virus cell entry. However, attempts to humanize 2D7 for in vivo human use have been unsuccessful so far. A filamentous phage library expressing random peptides was used to identify a peptide mimitope that is recognized by MAb 2D7. A synthetic peptide containing this sequence (2D7-2SK) bound to MAb 2D7 with high affinity and reversed its HIV type 1 (HIV-1) fusion inhibitory activity. The peptide contains sequence homologies to two distal regions of the second extracellular loop of human CCR5, both of which are required for MAb 2D7 binding. Rabbit anti-2D7-mimitope antibodies competed with MAb 2D7 for binding to the 2D7-2SK peptide in Biacore biosensor testing. Importantly, the rabbit anti-2D7-2SK antibodies bound to CCR5 on cells and specifically inhibited R5 (but not X4) envelope-mediated syncytium formation. These antibodies also neutralized infection of human peripheral blood mononuclear cells with R5 HIV isolates comparably to MAb 2D7. In summary, we have identified a novel peptide that closely mimics the MAb 2D7 epitope on CCR5. This peptide could be included as a potential vaccine candidate or to isolate 2D7-like human antibodies as entry inhibitors for R5 viruses.     

Development of T lymphocytes in Xenopus laevis: appearance of the antigen recognized by an anti-thymocyte mouse monoclonal antibody

Development of T lymphocytes in Xenopus laevis was studied using a mouse monoclonal antibody (mAb), XT-1, that was produced against surface determinants on thymocytes of J strain frog. Ontogenic studies, employing immunofluorescence, showed that cells positive for the determinant recognized by XT-1 mAb (XT-1⁺ cells) were first detected in the thymus of J strain Xenopus by Nieuwkoop and Faber stage 48 (7 days postfertilization) and then in the spleen, liver and kidney by stage 52 (20 days postfertilization). Percentages of XT-1⁺ cells in the thymus increased rapidly by stage 49 (10 days postfertilization) and reached adult levels by stage 52, and those in the spleen, liver, and kidney reached adult levels by stage 56 (40 days postfertilization). Electron microscopic immunohistochemistry revealed that most XT-1⁺ cells in thymuses from stage 56 larvae were typical small lymphocytes (4–7 μm in diameter). In contrast, many XT-1⁺ cells in larval thymuses at stage 49 are large (8–10 μm in diameter) lymphoblastoid cells. Thymectomy at stage 46 (5 days postfertilization) depleted XT-1⁺ cells in larval and adult lymphoid organs to background levels. These results suggest that the XT-1⁺ cells are differentiated from the lymphoid precursor cells in the thymus before the appearance of small lymphocytes and migrate into peripheral lymphoid organs. The cell surface determinant recognized by the XT-1 mAb may provide an important marker for the differentiation of T lymphocytes in Xenopus.     

Mapping and regulation of the tumor-associated epitope recognized by monoclonal antibody RS-11

We have previously described a rat monoclonal antibody, RS-11, which recognizes a tumor-associated antigen common to several species. In the present study, we have cloned and characterized the antigen recognized by RS-11. We screened a phage expression library prepared from HeLa cDNA and identified a clone that reacts with RS-11. DNA sequence analysis revealed that this clone contains sequences of keratin 18 (nucleotides 568-1196). We constructed several glutathione S-transferase fusion proteins and synthetic peptides based on this DNA sequence analysis and examined their reactivity with RS-11 to accurately map the RS-11 epitope. We determined that the epitope resides within a region of seven amino acids on the alpha-helix 2B domain of keratin 18 in which two amino acids (Leu(366) and Lys(370)) are completely conserved among intermediate filaments as well as other keratin members that are immunoreactive with RS-11. These two residues are sequentially discontinuous but spatially adjacent. The RS-11 epitope is constitutively present in human primary cultured hepatocytes; however, its immunoreactivity with RS-11 is up-regulated by malignant transformation or stimulation with either epidermal growth factor or transforming growth factor alpha.