gp140, a C3b-binding membrane component of lymphocytes, is the B cell C3dg/C3d receptor (CR2) and is distinct from the neutrophil C3dg receptor (CR4)

gp140, previously identified as a 140-kDa C3b-binding membrane glycoprotein present on Raji cell surface, was shown to be the C3dg/C3d receptor of B lymphocytes (CR2). Specific polyclonal anti-gp140, prepared by immunizing rabbits with this highly purified C3 receptor, blocked Raji cell rosettes with EC3b, EC3bi, EC3dg and EC3d, and also blocked normal lymphocyte rosettes with EC3dg and EC3d without affecting CR1 or CR3 activity. Moreover, a monoclonal anti-C3 (C3b/#130), described by others as reacting with the d region highly expressed on EC3bi, EC3dg and EC3d and poorly exposed on EC3b, completely inhibited EC3bi, EC3dg and EC3d rosettes with Raji cells, but had no effect on EC3b rosettes. Treatment of Raji cells with rabbit anti-gp140 blocked the uptake of three 125I-labeled monoclonal antibodies anti-B2, HB-5 and OKB7 reported to react with C3d-binding proteins, indicating that each of these monoclonal antibodies recognizes epitopes present on gp140. The neutrophil C3dg receptor was examined to determine its relationship to lymphocyte CR2. While neutrophil rosettes with EC3d were undetectable, a specificity for C3d was suggested by the inhibition of EC3dg rosettes by fluid phase C3d-complexes bearing no detectable C3dg. However, such neutrophil EC3dg and EC3bi rosettes were not inhibited by rabbit anti-gp140 nor an excess of anti-CR1, anti-CR2, and anti-CR3. In addition, neutrophils did not bind 125I-labeled anti-gp140, anti-B2, or HB-5. Thus, the neutrophil C3dg receptor is distinct from gp140, the lymphocyte CR2, and should be designated CR4.     

Nuclear localization of the Epstein-Barr virus/C3d receptor (CR2) in the human Burkitt B lymphoma cell, Raji.

Epstein-Barr virus/C3d receptor (CR2) is a glycoprotein of mol. wt 140,000 expressed on the surface of Raji cells. We previously isolated phosphorylated CR2 from purified Raji cell nuclei. We have analyzed the nuclear localization of CR2 by electron microscope immunochemistry of thin sections of Raji cells and we have compared the binding properties of CR2 expressed on purified plasma membranes or nuclei. Anti-CR2 mAb immunogold labeling of thin sections of Raji cells identified CR2 at the nuclear surface and also within the nucleus. Nuclear envelope associated CR2 was localized mainly at nuclear pores. Within the nucleus, CR2 was associated with ribonucleoprotein (RNP) interchromatin fibrils. This labeling was preserved in nuclear matrix preparations. CR2 expressed on the surfaces of purified nuclei or on the cell surface interacted with soluble and particle-bound C3bi/C3d. Monoclonal anti-CR2 antibodies, which recognized extracellular domains of CR2, reacted differently with CR2 depending on its subcellular localization. The presence of CR2 in nuclei may be due to translocation of the cell surface CR2 and/or the presence of two distinct intracellular pathways for mature CR2.     

Different human B cell subsets respond to interleukin 2 and to a high molecular weight B cell growth factor (BCGF)

After activation by anti-μ antibody human B cells acquire the ability to proliferate in the presence of recombinant interleukin 2 (IL2), a 20-kDa mol. mass B cell growth factor (BCGF) and a high mol. mass BCGF (50-kDa BCGF). An anti-IL2 receptor (IL 2R) monoclonal antibody inhibits the IL2-dependent proliferation without affecting that induced by BCGF. B cells expressing the IL2R after anti-μ antibody activation (IL2R⁺ cells) were separated from those not expressing IL2R (IL2R^? cells). IL2 stimulated the proliferation of only IL2R⁺ cells whereas the 20-kDa BCGF acted on both IL2R⁺ and IL2R^? cells. Importantly, the 50-kDa BCGF supported the proliferation of IL2R^? cells whereas it was inactive on IL2R⁺ cells. Thus, the B cell subset responding to the 50-kDa BCGF after anti-μ antibody activation is distinct from that responding to IL2.     

Structure and functions of gp 140, the C3d/EBV receptor (CR2) of human B lymphocytes

Analysis of the interaction of human C3 fragments with human B lymphoma cell line, led us to isolate gp 140, the C3 receptor of Raji cells. Rabbit anti-gp 140 was prepared against this highly purified receptor. Using these polyclonal antibodies, it was found that: gp 140 is the C3d receptor (CR2) which reacts with the C3d site expressed on C3d, C3dg, C3bi and at a less extent on C3b. Gp 140 is a specific marker of human B lymphocytes; gp 140 is also the Epstein-Barr virus receptor (EBVR); CR2 is a membrane site involved in B-cell regulation; and the C3d/C3dg receptor (CR2) of human B lymphocytes is distinct to the C3dg receptor (CR4) of human neutrophils.     

Characterization of neutralizing monoclonal antibodies directed against tetanus toxin fragment C

Abstract

Clostridium tetani causes a life-threatening infectious disease by production of tetanus neurotoxin (TeNT), a 150?kDa molecule composed of light (LC) and heavy chain (HC) polypeptides. The TeNT HC contains an N-terminal domain critical for LC translocation and a C-terminal toxin receptor-binding domain known as fragment C. Despite extensive investigations on epitope specificity of anti-TeNT antibodies, the immunodominant neutralizing epitopes of the toxin are poorly defined. This study describes the generation and characterization of four monoclonal antibodies (MAb) specific for TeNT. The characteristics of each MAb were explored in terms of isotype, specificity, affinity, and immuno-globulin heavy chain variable region (IGHV) gene usage using ELISA, Western blotting, and sequencing techniques. The toxin neutralizing activity of the MAbs was also investigated using the in vitro GT1b neutralizing assay. The data demonstrated that all MAbs bind to tetanus toxin and toxoid. Sub-fragments binding analysis showed that two MAbs react with fragment C, one with both fragment C and LC, and one with LC. Only the two fragment C-specific MAbs were able to neutralize the toxin. Sequencing of the expressed VH and VL genes revealed rearrangements of various VH and VL gene segments in all hybridoma clones. Clonality of the hybridomas was also confirmed by a competition assay that showed recognition of distinct epitopes by these MAbs. The results suggest the importance of TeNT fragment C in terms of immunogenicity and toxin neutralization activity.     

抗bFGF单克隆抗体的鉴定及其意义

用重组牛ｂＦＧＦ免疫Ｂａｌｂ／ｃ小鼠，通过细胞融合，以反向间接血凝和间接ＥＬＩＳＡ筛选，以及有限稀释法克隆化，建立了９株稳定分泌抗ｂＦＧＦ单克隆抗体（ｍＡｂ）杂交瘤细胞。对其中３株用Ｗｅｓｔｅｒｎｂｌｏｔ和生物活性抗体中和实验进行了鉴定。结果显示，ｍＡｂ可结合重组牛或人ｂＦＧＦ，并能抑制ｂＦＧＦ刺激Ｂａｌｂ／ｃ３Ｔ３细胞的生长；腹水的ＥＬＩＳＡ滴度为１∶３０００；用其中２株ｍＡｂ建立的双抗体夹心ＥＬＩＳＡ法灵敏度可达５０ｐｇ／孔。本文还讨论了抗ｂＦＧＦｍＡｂ的应用价值。     

Activation of human platelets through gp140, the C3d/EBV receptor (CR2)

gp140, the C3d/EBV receptor (CR2), previously isolated and characterized from human B lymphocytes, was identified on human platelets: by measuring the specific binding of either polyclonal anti-gp140 IgG and monoclonal anti-C3d/EBVR antibodies, as OKB-7 and HB-5, or human C3d; by isolating gp140 from solubilized platelet components with polyclonal anti-gp140 IgG or monoclonal OKB-7, using immunoprecipitation and electro-immunoblotting assays; by inducing specific activation of human platelets. Cross-linking of this receptor by polyclonal anti-gp140 IgG induced aggregation of human platelets and stimulated ATP release. Absence of lactate dehydrogenase release and inhibition by EDTA and prostacyclin of anti-gp140-induced aggregation, support strongly active aggregation and absence of lysis. Platelet aggregation by anti-gp140 required metabolic activities and was modulated by fibrinogen, paf-acether or thrombin. OKB-7 triggered human platelet aggregation when cross-linked by anti-mouse second-step antibodies. In the same way, platelet activation by C3d fragment was detected, in presence of fibrinogen, only when C3d was cross-linked on the cell surface by anti-C3d F(ab')2 fragments.     

Production of monoclonal antibodies against hepatitis B surface antigen (HBsAg) by somatic cell hybrids

Hybridomas secreting anti-HBs were produced by fusion of either adw or ayw HBsAg primed mouse spleen cells with either P3 X63 Ag8 or P3 NSI 1 Ag4 1 mouse myeloma cell lines. Individual anti-HBs secreting clones were isolated by limiting dilution procedures, and six cell lines have been established, namely, BX182, BX259, BX248, CN324, DN283, and DN296. Progenies of each cell line were derived from a single clone obtained from three subclonings of six anti-HBs positive initial fusion colonies. Clones were passaged in tissue culture and as tumors in syngeneic mice for upwards of six months. Anti-HBs of each line showed characteristic reactivity (detection) patterns in radio-immunoassay using different antigen subtype solid phases followed by either ¹²⁵I-HBsAg or ¹²⁵I-goat anti-mouse IgG probe. The specificity of the anti-HBs from each clone for the subdeterminants of HBsAg was identified by their reaction with ¹²⁵I-HBsAg ligands of several subtypes in a radioimmunoprecipitation assay. Four types of reaction were identified and correlated to the conventional serological subtyping definitions; they were anti-HBs/a (BX259 and CN324), anti-HBs/d (BX182), and possibly anti-HBs/w (BX248 and DN296) and anti-HBs/y (DN283). These monoclonal antibodies will be important for the elucidation of the antigenic structure of native HBsAg and will provide valuable reagents for both antigen detection and subtyping.     

Rapid purification of the human C3b/C4b receptor (CR1) by monoclonal antibody affinity chromatography

The human C3b/C4b receptor (CR1) is a polymorphic glycoprotein that is expressed on erythrocytes, leukocytes and glomerular podocytes. Further structural analysis and molecular genetic studies would be facilitated by the availability of relatively larger amounts of purified CR1. Milligram quantities of CR1 were purified from erythrocyte membranes 10,000-fold with an average yield of 30-40% by a rapid procedure which utilized sequential chromatography on Matrex Red A and a monoclonal anti-CR1 antibody affinity column. The purified receptor was homogeneous by SDS-PAGE and consisted of the 2 most common alleles of CR1. Purified CR1 also retained its function of serving as a cofactor for the cleavage of C3b to iC3b, C3dg and C3c. The amino acid composition was typical of that of a globular protein and sequence analysis of the N-terminus of the purified CR1 revealed that it was blocked.     

Inhibition of killer-target cell interaction by monoclonal anti-H-2 antibodies.

The sites on target cells with which cytotoxic T lymphocytes interact were characterized using three different monoclonal BALB/c anti-CBA antibodies derived from plasma cell hybrids. The antibodies all reacted with H-2K^k and appeared to recognize public specificities H-2.5,11 and 25, respectively. Allogeneic killing directed at products of H-2K^k was inhibited by all three antibodies, irrespective of the H-2 haplotype of the responder; cytotoxicity directed at products of another allele, H-2K^d, or of the H-2 D region on the same target cell was not affected. The antibodies did not inhibit killer cells carrying H-2K^k. Cytotoxic reactions against minor histocompatibility antigens, including the male-specific antigen H-Y, were also blocked by all three monoclonal antibodies when restricted by H-2K^k, but not when restricted by H-2D on the same target cell. Cytotoxic T lymphocytes thus appear to interact with their target via the same, serologically defined H-2K^k molecule which carries public specificities, whether they recognize it as an alloantigen or as self. This argues against the existence of separate H-2 K-encoded molecules recognized by killer cells only and against H-2 specific modifications of minor histocompatibility antigens as the basis of H-2 restriction. One of the antibodies, 27 R9, which reacted with H-2K^k and H-2′ and was thought to recognize specificity H-2.25, showed a weak cytotoxic reaction but bound with a high titer to H-2 D^k, a reaction that has not previously been described. This antibody selectively and with a very high titer inhibited male-specific cytotoxicity restricted by H-2D^k, but did not significantly interfere with allogeneic killing against products of the H-2 D^k region nor apparently with H-2D^k- restricted cytotoxicity specific for other minor antigens. The results suggest the existence of at least two different restriction elements controlled by the H-2D^k region.     

Preparation and characterization of monoclonal antibodies directed against antigenic determinants of recombinant human tumour necrosis factor (rTNF)

A large number of monoclonal antibodies (McAb) binding to antigenic determinants of human tumour necrosis factor (TNF) were prepared from two fusions of mouse myeloma NSO cells with spleen cells from Balb/c mice immunized with highly purified recombinant (r)TNF. Several of these McAbs were highly neutralizing with respect to the biological activity (cytotoxicity) of TNF manifested in L-929 C1.10 cells. Antibody competition experiments suggested the presence of at least two antigenic determinants on the rTNF molecule through which binding of McAb effects neutralization of biological activity. Some of these McAbs were shown to be suitable for the development of immunoassays to quantify rTNF.     

B cell growth factor (BCGF) secreting human T cell clones reactive to a soluble glycoprotein antigen, streptococcal antigen (SA)

A panel of human T cell clones bearing exclusively the helper (T4) phenotype and showing reactivities to a soluble glycoprotein antigen (185,000 dalton Mol. Wt. Streptococcal antigen, SA) is described. Two of these clones namely, SA 1.53 and SA 1.82, are found to co-produce B cell growth factor (BCGF) and interferon-gamma (IFN-gamma) in the absence of interleukin 2 (IL2) upon stimulation with phytohaemagglutinin (PHA) or the specific antigen in the presence of irradiated autologous antigen-presenting cells (APC). Secretion of the lymphokines is genetically restricted in part by DR molecules that are expressed on the cloned cells and APC. Produced BCGF is differentiated from the BCGF-promoting property of IFN-gamma in that only IFN-gamma activity, but not BCGF activity is removed and inhibited by anti-IFN-gamma antibodies. Exogenous IL2 induces secretion of BCGF and IFN-gamma of the cloned cells, an observation which involves interaction of IL2 with IL2 receptors. An analysis of the proliferative responses to antigen of the T cell clones shows that BCGF-producing clones, unlike those that secrete IL2, fail to proliferate significantly to specific antigen restimulation.     

抗KDR单克隆抗体的制备

目的：研制针对肿瘤新生血管内皮细胞生长因子受体（KDR）的特异性单克隆抗体（mAb）。方法：以谷胱甘肽转硫酶耦联的KDR（GST-KDR）可溶性融合蛋白免疫BALB／c小鼠,采用杂交瘤技术制备分泌抗KDRmAb的杂交瘤细胞。结果：获得1株稳定分泌抗KDRmAb的杂交瘤细胞株,命名为3E9B2G11。结论：成功获得能分泌抗KDRmAb杂交瘤细胞,为以后将该mAb改造成小分子酪氨酸激酶抑制剂,使其能进入细胞内与受体KDR特异性结合并阻断血管内皮细胞生长因子的强大生物学功能奠定了重要基础。     

Production of anti-fibroblast growth factor receptor monoclonal antibodies by in vitro immunization.

Monoclonal antibodies against the high affinity tyrosine kinase Fibroblast Growth Factor (FGF) receptor may help define receptor epitopes involved in FGF binding and signal transduction which mediate coronary and tumor angiogenesis (the development of blood vessels). Monoclonal antibodies against the FGF receptor were made by in vitro immunization. Receptor was PAGE-purified and transblotted to nitrocellulose. The nitrocellulose was dissolved with acetonitrile, and the receptor used as antigen in an in vitro immunization system. Screening for FGF receptor positive clones was done using membrane preparations from Coronary Venular Endothelial Cells (CVEC) expressing the FGF receptor, both by Enzyme Linked Immunosorbent Assay (ELISA) and Western blot. Culture supernatants of several clones tested positive for IgM or IgG monoclonal antibodies against the FGF receptor. Antibodies were affinity purified. Ascites fluid was produced in Balb/c mice primed with Incomplete Freund's adjuvant (IFA). The monoclonal antibodies were also found to be suitable for receptor immunoprecipitation. Immunocytochemistry was done on sections from a variety of species. Further characterization of these antibodies, as well as the production of the remainder of a panel of anti-FGF receptor monoclonal antibodies, is underway.     

Mouse monoclonal antibodies to the human C3b receptor

Mouse monoclonal antibodies were raised against the human C3b receptor (CR1) molecule that had been purified from solubilized erythrocytes membranes. Four hybridomas were selected, cloned and expanded because their supernatants reacted strongly with insolubilized CR1 by ELISA and intensely stained B-dependent areas of the spleen and glomerular podocytes by indirect immunofluorescence. The four monoclonal antibodies, named J3D3, J8B10, J3B11 and J7C2, were IgG1 immunoglobulins. J3D3 immunoprecipitated two protein bands of apparent mol. wts 200,000 and 220,000 from 125I-surface-labeled human erythrocytes, which correspond to the two major allotypic forms of CR1. By indirect immunofluorescence, monoclonal antibodies stained polymorphonuclear leucocytes (PMN), most peripheral blood B-cells and a small subset of peripheral blood T-cells. J3D3 bound to CR1 on erythrocytes, PMN and lymphocytes with an affinity of 1-3 X 10(9) M-1 and recognized 170-1330 antigenic CR1 sites with an average of 740 sites/erythrocyte in 100 healthy individuals, approx. 50,000 sites/PMN and 15,000 sites/lymphocyte. There was a bimodal distribution of CR1 numbers on erythrocyte in the normal population. The four monoclonal antibodies similarly inhibited CR1-mediated decay of preformed cell-bound alternative- and classical-pathway C3 convertase sites. Two antibodies, J3D3 and J3B11, inhibited C3b-dependent rosette formation with lymphocytes, although much less efficiently than F(ab')2 polyclonal anti-CR1 antibody. Differences that were observed in the relative capacity of the antibodies to inhibit some of the functions of CR1 and in their ability to compete for binding of 125I-J3D3 to CR1 on erythrocytes, suggested that they are directed against different epitopes on CR1. Monoclonal antibodies provide useful means to assess and analyze the biological and immunoregulatory functions of the C3b receptor.     

Segmental staining of the murine nephron by monoclonal antibodies directed against the GP-2 subunit of laminin

Monoclonal antibodies to GP-2, the 220,000-dalton subunit of laminin, were used in an immunohistologic study to investigate structural variations in basement membranes. Mouse kidney was used because of the wide range of basement membranes represented. Two rat/mouse monoclonal antibodies, designated LAM-I and LAM-II were compared with rabbit polyclonal anti-GP-2 in a light and electron microscopic study that identified nephron segments by morphology, by topography, and by the use of markers specific for individual segments. LAM-I staining is demonstrable on all tubular and glomerular basement membranes but not on those of blood vessels or smooth muscle, differing in this respect from anti-GP-2. LAM-II staining is confined to the basement membranes of the convoluted portion of proximal tubule segments (S1 and S2), not the straight (S3) portion; to the thin limb and the thick ascending limb of Henle's loop, but not distal convoluted tubules or collecting ducts. The heterogeneity of GP-2 localization may be due to differing conformations of the molecule at these sites.     

Identification of viral antigenic determinants by monoclonal antibodies directed against Chilo Iridescent Virus (Iridovirus type 6)

Summary Monoclonal antibodies (McAbs) obtained against Iridovirus type 6 (CIV) were characterized by Western blotting and/or immunoprecipitation. Seven McAbs were found to be strongly reactive with viral polypeptides of molecular weights 16K and 18K by Western blotting. Two McAbs were directed against a complex composed of 30K, 50K, and 100K polypeptides, but failed to react with either of these free polypeptides. This finding could explain the faint reactivity of these McAbs in Western blotting and immunoprecipitation. The reactivity of the other McAbs with their antigenic determinants is also discussed.