Monoclonal antibody to a human leukocyte-specific membrane glycoprotein probably homologous to the leukocyte-common (L-C) antigen of the rat

The monoclonal antibody (F10–89–4) described in this study recognizes an antigen which by quantitative absorption analysis is absent from human brain, kidney, liver, heart, erythrocytes, platelets and normal serum, but is present on spleen, lymph node, chronic lymphatic leukemia cells, bone marrow, thymus and granulocytes at a ratio of 1 : 1 : 0.8 : 0.3 : 0.3 : 0.1, respectively. Analysis with the fluorescence-activated cell sorter showed that 100% of thymocytes, lymph node lymphocytes, blood mononuclear cells and granulocytes carry the antigen, while 83% of bone marrow cells are positive. There was marked heterogeneity in the amount of labeling of thymocytes, with 3 major peaks. There was also heterogeneity of labeling of blood mononuclear cells and lymph node lymphocytes, with a weakly staining hump containing approximately 20% of the cells in the case of lymph node lymphocytes. Double labeling experiments demonstrated that the weakly staining cells of blood and lymph node were B lymphocytes, while frozen sections of thymus showed that the antigen was expressed most weakly in subcapsular cortical thymocytes, and most strongly on medullary thymocytes. Biochemical studies established that the antigen bound to lentil lectin columns, and sodium dodecyl sulfate polyacrylamide gel electrophoresis studies using NaB³ H₄-labeled blood mononuclear cells established that the antigen was a major glycoprotein of lymphocytes, and that its molecular weight was in the region of 190 000 to 215 000.     

抗人全T细胞单克隆抗体大鼠杂交瘤细胞株的建立

通过融合大鼠免疫细胞株IR983F和经T细胞株HPB—ALL免疫大鼠脾细胞,获得分泌单克隆抗体LO—CD6a的杂交瘤细胞株。实验证实,LO—CD6a是人全T细胞特异性的。     

Monoclonal antibody (F5) to human prostate antigen

Hybridoma culture F5 has been developed which secretes monoclonal antibody (McAb) directed to an epitope of a prostatic glycoprotein of Mr 34 kD (Prostate Antigen, PA). Tissue levels of PA have been evaluated using a competitive-binding enzyme-immunoassay based upon the inhibition of McAb binding activity to purified antigen. Results indicated the specific occurrence of high antigen concentrations in extracts prepared from prostatic tissues. The antigenicity of epitope F5 is resistant to tissue fixation and embedding protocols, and has been demonstrated upon immunoperoxidase staining procedures. Immunoperoxidase data strongly indicate that McAb F5 possesses a singular specificity towards prostatic epithelial cells. Other tissues, whether normal or cancerous, fail to express this determinant. Specimens examined included epithelial and nonepithelial tissues along with a panel of carcinomas and sarcomas. The antibody was able to detect tumor cells at extra-prostatic sites and represents a powerful probe for the detection and differential diagnosis of metastatic cancer of the prostate.     

A unique human B lymphocyte antigen defined by a monoclonal antibody

We produced a hybridoma designated 4G7 from a mouse immunized with chronic lymphocytic leukemia cells. The 4G7 hybridoma secretes an IgG1 antibody that is specific for normal and malignant B lymphocytes. Using dual color immunofluorescence staining, this antibody reacted with all immunoglobulin-positive cells but no T cells in normal peripheral blood. There was no detectable 4G7 antigen on monocytes, platelets, red cells, granulocytes, or phytohemagglutinin-activated T cells. When PBL were depleted of 4G7 positive cells and stimulated with pokeweed mitogen, secreted immunoglobulin levels fell to less than 10% of control values on Day 5 and less than 1% of control on Day 7. This antibody was reactive with 155 of 176 B lineage neoplasms on which it was screened. Thirty-five cases of myeloid or T-lymphoid malignancy were negative. Our studies show that the 4G7 antigen modulates in the presence of excess antibody. Free 4G7 antigen was not found circulating in human serum. The cell surface antigen identified by 4G7 was sensitive to pronase proteolysis but resistant to trypsin and chymotrypsin digestion. A comparison of 4G7 with other known B-cell antibodies indicates that the 4G7 antigen has not been previously identified. This antibody is of use for the identification of normal B lymphocytes, the study of B-cell differentiation, and the characterization of lymphoid malignancies.     

Development and characterization of a human monoclonal antibody probably detecting the leukocyte differentiation antigen CD39

A human monoclonal IgM antibody, referred to as TU223, has been produced. The reactivity of TU223 was tested in various cells and cell lines by complement-dependent microcytotoxicity test and fluorescence-activated cell sorter analysis. The antigen defined by TU223 was expressed on Epstein-Barr virus-transformed B-cell lines and on some Burkitt's lymphoma cell lines, but was not expressed on normal T cells, B cells or erythrocytes. In addition, expression of the antigen defined by TU223 was also induced on B cells activated by Epstein-Barr virus or pokeweed mitogen, and on T cells activated by phytohemagglutinin, concanavalin A, pokeweed mitogen or recombinant interleukin-2. However, no expression of the antigen detected by TU223 was induced at all on recombinant interleukin-4-treated B cells or macrophage-like cell line U937. When the ability of TU223 and various mouse monoclonal antibodies to bind to human differentiation antigens was compared, interestingly, the reactivity of TU223 was found to be very similar to that of mouse monoclonal antibody CD23 (H107), which reacts with Fc epsilon receptor II. Two-color analysis revealed that the antigen defined by TU223 is expressed on the cell surface of certain lymphoid cells expressing CD23 antigen. However, it can be concluded that the antigen defined by TU223 is clearly distinct from Fc epsilon receptor II, based on assay of cross-blocking between H107 and TU223. The surface antigen on B85 cells recognized by TU2232 had the molecular size of 80-82 kiloDaltons as determined by immunoblotting analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibody to HLA-A3

The monoclonal antibody GAP A3 detects the HLA allospecificity A3. Reactivity of the monoclonal was in exact concordance with the presence of the A3 antigen as defined by conventional alloantisera on a panel of 59 cells from individuals with well-characterized HLA antigens. Reactivity with GAP A3 segregated with HLA-A3 in a family where three of eight siblings inherited the paternal A3 antigen. GAP A3 precipitated appropriate 44,000- and 12,000-dalton bands on SDS-polyacrylamide gels under reducing conditions from an HLA-A3-positive, but not an HLA-A3-negative B lymphoblastoid cell line. Thus, by serological, familial, and biochemical criteria, GAP A3 defines the allospecificity HLA-A3.     

Monoclonal antibody to human cross-linked fibrin

Cross-linked Fibrin II was prepared using Kabi grade (L) fibrinogen. Fibrin plasmic digest was separated on Sepharose CL-6B. Fragments Mr 135-300 kDa were used to immunize 6-9 weeks old female BALB-c mice. A stable hybridoma secreting monoclonal antibody (MAb) TD-1 (IgG 2a, Kappa) was prepared by fusion using myeloma cells (P3-NS1/1-Ag4-1) and immunized cells. Fibrinogen and plasmin digest of fibrinogen in serial dilutions did not compete with the immunizing antigen. To prove that TD-1 binds specifically to cross-linked fibrin, immunoprecipitation with S. aureus and affinity chromatography were performed. In both experiments, we demonstrated that TD-1 binds specifically to a protein Mr greater than 200 kDa which is found in XL-fibrin and not fibrinogen. Reduced samples showed the antibody bands (heavy and light chains) and three protein bands, Mr greater than 80 kDa (gamma-gamma dimer), Mr greater than 45 kDa (beta chain of fragment D) and Mr greater than 16 kDa (alpha chain from fragment D) were present. TD-1 reacted strongly with HPLC fraction of the immunizing antigen Mr 220 kDa (probably DD/E complex). Affinity binding constants (Scatchard Plot Analysis) were determined. The highest affinity was obtained with XL-fibrin fraction Mr 220 kDa, KD = 1.39 X 10(-8) and high molecular weight XL-fibrin fragments, KD = 1.6 X 10(-7). Fragment DD had KD of 2.8 X 10(-6). These results suggest that TD-1 is specific for the DD region of human cross-linked Fibrin II.     

Monoclonal antibody to human epidermal growth factor

A monoclonal antibody directed to a species-specific determinant of human epidermal growth factor (h-EGF) was obtained by fusing murine myeloma cells with BALB/c mouse splenocytes sensitized to h-EGF. This antibody, referred to as 863.D4, did not react with either rat or mouse epidermal growth factor or with 11 other polypeptide hormones tested as shown by solid-phase radioimmunoassay (SPRIA), and immunoprecipitation followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Scatchard analysis of the antibody binding to purified h-EGF revealed an apparent equilibrium dissociation constant of 1 X 10(-8) M. The antibody blocked both the binding of h-EGF and h-EGF stimulation of 3H-thymidine incorporation into DNA by greater than 90% in confluent cultures of human foreskin fibroblasts.     

Monoclonal antibody analysis of responder and stimulator cells in the human autologous mixed lymphocyte reaction

Responder and stimulator cell subpopulations in the autologous mixed lymphocyte reaction (AMLR) were determined with the OK series of monoclonal antibodies. Mitomycin-C-treated, monocyte-enriched cell populations were used as stimulator cells in the AMLR. Treatment of these monocytes with either OKM and/or OKI monoclonal antibodies and complement resulted in a marked loss of ability of these cells to act as stimulators in the AMLR. Removal of OKT3+ and OKT4+ cells diminished the proliferative responses of AMLR cultures. Interaction of T cells with autologous monocytes resulted in generation of cells capable of suppressing both MLR and AMLR cultures. The suppressor activity of these cells was diminished by treatment with OKI , OKT4 or OKT8 monoclonal antibodies. No cytotoxic activity to autologous or allogeneic monocytes was observed. Additional studies showed an increased number of OKT9 + and OKI + as well as OKT8+ T cells in the AMLR responder cell population. This study indicates that cultures of T lymphocytes with autologous monocytes yield T cell subset(s) which suppress MLR and AMLR reactivity.     

Monoclonal antibody CI-panHu defines a pan-human cell-surface antigen unique to higher primates.

The murine monoclonal antibody CI-panHu reacts strongly with the cell surface of all human cells, including erythrocytes, tumour cells and HLA-A,B,C-negative cell lines. As such, this antibody defines the first pan-human cell-surface antigen reported. The antigenic determinant detected is associated with a protein doublet of 16,000 MW whose expression is restricted to cells from humans, apes and some species of Old World monkeys. Antibody reactivity is not diminished by routine fixation procedures, nor by paraffin-embedding, and the antigenic determinant is relatively protease-resistant. The use of this antibody as a positive control in immunoassays of human cells is discussed.     

Monoclonal antibody against human macrophages/monocytes and granulocytes

A monoclonal antibody of the IgG2a isotype directed against lymphokine-activated human macrophage cell line (U937) was produced and characterized. By indirect immunofluorescence microscopy or binding assay, this antibody (MaG-1) reacted with human peripheral monocytes, peritoneal macrophages, alveolar macrophages, and peripheral granulocytes but not with lymphocytes, erythrocytes, and platelets. MaG-1 antigen was also expressed by 34% of bone-marrow cells which includes monocytic and granulocytic precursors, whereas lymphoid and erythroid precursor cells were found on MaG-1 negative population. Immunoprecipitation of 125I-labeled U937 cell extract with the Mag-1 antibody under nonreducing and reducing conditions yielded a specific band of 66 kD. Thus, this antibody defines a new human macrophage/monocyte and granulocyte-specific antigen and may be useful for studying differentiation of human macrophages/monocytes.     

A monoclonal antibody to a rat hepato-renal membrane antigen.

A monoclonal antibody against a membrane glycoprotein of rat hepatocytes has been produced. The nature of this antibody designated as HAM.4 was analysed by cellular radioimmunoassay, flow cytofluorography and indirect immunoperoxidase procedures. The following characteristics of HAM.4 were elucidated. First, an immunohistochemical study revealed that this antibody stained preferentially the bile canalicular face of hepatocyte membrane. Secondly, HAM.4 cross-reacted with kidney, spleen and thymus as well as liver. The kidney expressed much more the antigen molecules detected by this antibody than the liver did. The antigen was located predominantly on the brush border of proximal tubules in kidney. Thus, HAM.4 would be useful for analysing one of the brush border antigens of renal tubules which has been thought to be a pathogenic antigen for inducing experimental membranous glomerulonephritis. Finally, HAM.4 failed to label the cell membrane of rat hepatoma cell lines examined, indicating that the antigen detected by HAM.4 may disappear from cell surface during the course of hepatocarcinogenesis.     

Ber-ACT8: new monoclonal antibody to the mucosa lymphocyte antigen.

Using a newly established HTLV-1 positive T cell line as an immunogen, a new monoclonal antibody, Ber-ACT8, was produced. It reacts with in vitro activated T cells and a small subset of normal resting T cells, but not with resting B cells or any of the 29 established human permanent cell lines tested. Immunohistological analysis of a wide spectrum of human tissues showed that Ber-ACT8 reactivity is restricted to a few T cells in the peripheral blood, the extrafollicular areas of lymph nodes and tonsils, and splenic red pulp. In the gut Ber-ACT8 labelled most intraepithelial T cells and up to 50% of lamina propria T cells. The antibody also immunostained T cells present in the oral and bronchial mucosa. Double labelling on splenic cells, fresh blood lymphocytes, and in vitro activated T cells showed that most Ber-ACT8 positive cells coexpressed CD8. Ber-ACT8 did not react with any of the 14 Hodgkin's lymphomas nor any of the 172 non-Hodgkin's lymphomas tested, with the exception of 10 cases of T cell lymphomas, five of which were located in the jejunum and associated with coeliac disease, and one B cell lymphoma, and most cases of hairy cell leukaemia tested. Parallel immunostainings with Ber-ACT8, anti-TCR-beta (beta F1), and anti-TCR-delta showed that most Ber-ACT8 positive T cells carry the TCR of alpha beta type. Comparison of Ber-ACT8 with HML-1, B-ly7, and LF61 showed essentially the same reactivity and an identical molecular target. The molecular structure recognised seems to be a trimeric molecule with components of 150, 125 and 105 kilodaltons, with the Ber-ACT8 epitope localised on the 150 kilodalton chain. The 150 kilodalton molecule contains an 0-linked carbohydrate moiety of about 10 kilodaltons. Because of its very selective distribution, the trimeric antigen is a powerful reagent for the diagnosis of gut T cell-derived T cell lymphomas and other extranodal T cell lymphomas, as well as hairy cell leukaemia.     

Monoclonal anti-galactan IgA J 539 binds intercatenarily to its polysaccharide antigen. Observations on the binding of antibody to a macromolecular antigen

Highly purified P. Zopfii galactan of mol. wt 2 X 10(5) binds monoclonal IgA J 539 with a Ka of 5.80 X 10(5) M-1 if the polysaccharide concn is expressed in blocks of 30 galactosyl residues. This is the same Ka as found for the antibody and methyl beta (1,6)-beta-D-galactopyranosyltetraoside, the ligand capable of filling the entire combining area of the immunoglobulin. This same polysaccharide precipitates monomeric IgA J 539 on agar-double diffusion. It is concluded that the antibody binds to intercatenary galactosyl residues of the antigen.     

Monoclonal antibody to Streptococcus mutans type e cell wall polysaccharide antigen.

A monoclonal antibody against the polysaccharide antigen of Streptococcus mutans serotype e was prepared. It was found that beta-methyl-D-glucopyranoside and cellobiose markedly inhibited the precipitin reaction, whereas maltose showed no inhibition. The beta-glucosyl moiety of the type e polysaccharide seems to be the predominant antigenic determinant of the antigen.     

Monoclonal antibody to human basement membrane collagen type IV

We have developed a hybridoma cell line which secretes monoclonal antibody to human basement membrane type IV collagen. The monoclonal antibody secreted by this hybridoma has been obtained in large amounts by either concentrating it from culture supernatants or from the ascites fluid of mice bearing the hybridoma tumour. This monoclonal antibody to type IV collagen does not cross-react with other types of collagens, including types I, III and V, as determined by an enzyme-linked immunosorbent assay (ELISA) and by immunohistochemical staining of corneal and lens sections. Descemet's membrane of mouse, rabbit and human corneal endothelium and lens capsule, both rich in type IV collagen, bind the antibody when stained immunohistochemically. By the indirect precipitation technique, the antibody is found to bind more than three peptides in the basement membrane collagen-rich fraction of human placenta. Based on the observations of other investigators, these peptides are probably derived by proteolysis of the larger polypeptides, since the purification steps in involve extensive pepsin digestion.