Colorectal carcinoma-specific antigen: detection by means of monoclonal antibodies.

Fusion of P3 X 63 Ag8 mouse myeloma cells with splenocytes obtained from mice immunized with cells derived from human colorectal carcinomas resulted in the production of antibody-secreting hybridomas. Two hybridomas (1083-17 and 1116-56) and their clones secreted antibodies binding specifically to human colorectal carcinoma cells either grown in culture or obtained from patients, but did not bind to normal colonic mucosa or other normal and malignant human cells. The binding specificity was consistent in three assays: radioimmunoassay, mixed hemadsorption, and immunofluorescence. Adsorption of these antibodies to colorectal carcinoma cell lines totally eliminated their specific binding.     

The antigenic epitopes of human grown hormone as mapped by monoclonal antibodies

The antigenic epitopes of human GH (hGH) have been investigated using monoclonal antibodies (Mabs) to hGH. A panel of 12 Mabs was incorporated into two-site binding assays in order to investigate the antigenic surface of hGH. Nine reaction patterns were observed. The Mabs were further characterized in terms of their cross-reactivity with human placental lactogen, human PRL, with recombinant hGH molecules (MetLeu8hGH, Met14hGH), and with fragments derived from enzymatic or chemical digestion of hGH. These studies provided information on the antigenic sites that are shared with human placental lactogen and on the N-terminal and C-terminal regions of the hGH molecule. Based upon these findings, we tentatively suggest the epitope model for hGH comprising at least 10 antigenic sites (epitopes) which may be grouped into five antigenic regions.     

Human parathyroid antigen: characterization and localization with monoclonal antibodies.

A cell surface antigen on human parathyroid cells was identified by monoclonal antibodies. The antigen, called parathyroid antigen (PTA), is found on both of two major polypeptides (190 kDa and 160 kDa) apparently associated exclusively with parathyroid cells. To determine whether PTA was a suitable target for in vivo imaging, 125I-labeled anti-PTA was injected into nude mice bearing human parathyroid xenografts. IgG1 anti-PTA antibody showed excellent radiolocalization of the grafts, whereas IgM anti-PTA, containing the same variable domains as the IgG1 antibody, showed little specific binding. These results suggest that antibody isotype is an important parameter for the in vivo immunoscintigraphy of specific cellular antigens.     

Blood group A cross-reacting epitope defined by monoclonal antibodies NCC-LU-35 and -81 expressed in cancer of blood group O or B individuals: its identification as Tn antigen.

Two monoclonal antibodies, NCC-LU-35 and NCC-LU-81, have been established after immunization of mice with membrane preparations of human lung cancer Lu65 tumor xenograft cells grown in vivo and intact cells cultured in vitro, respectively. These two antibodies react specifically with a majority of human adenocarcinomas, irrespective of the host's blood group ABO status, as well as with normal tissues and erythrocytes of blood group A individuals. The antigenicity is associated with a high molecular weight mucin-like glycoprotein separated by gel filtration of Lu65 tumor extracts. The epitope of the mucin-like glycoprotein has been identified as alpha-N-acetylgalactosaminyl residue directly linked O-glycosidically to serine or threonine residues of polypeptides. This epitope was serologically detected several years ago and given the name Tn. Our identification of the epitope is based on the following results: The antigen is sensitive to alpha-N-acetylgalactosaminidase, but not to sialidase or alpha-fucosidase. Various mono- and difucosyl A determinants, either type 1 or type 2 chain, cross-react with both antibodies. The reactivity with both antibodies can be created by treatment of glycophorin A of normal erythrocytes with sialidase followed by beta-galactosidase. N-[3H]acetylgalactosamine can be released by galactose oxidase/NaB3H4 treatment from the Lu65 mucin-like glycoprotein but not from the mucin-like glycoprotein of normal colonic mucosa upon reductive beta-elimination (alkaline borohydride treatment). The antigen may be one of the tumor-associated A cross-reacting antigens occurring in a wide variety of human adenocarcinomas of hosts belonging to all ABO blood groups.     

A human granulocyte-specific antigen characterized by use of monoclonal antibodies

Hybridoma cell lines secreting monoclonal antibodies that bind to a surface antigen of human neutrophils have been prepared by fusion of mouse myeloma cells with spleen cells from mice immunized with human neutrophils. Several of the monoclonal antibodies (AHN 1-6) were specific for a neutrophil surface antigen and did not bind lymphocytes, monocytes, red blood cells, platelets, or basophils. All of the granulocyte-specific antibodies immunoprecipitated a polypeptide of 145,000 daltons and an isoelectric point of about 4.5 and other heterogeneous polypeptides of 105,000 daltons. These same components were the major lactoperoxidase-labeled proteins precipitated by hyperimmune mouse serum. The antibodies were further characterized for binding to several human myeloid leukemia cell lines and cells from patients with myeloid or lymphoid leukemia. All antibodies bound the HL- 60, ML1, ML2, ML3, K562, and U937 myeloid leukemia cell lines. None of the antibodies bound the RPMI 6410 Raji, RPMI 8226, MOLT 4, or Daudi lymphoid cell lines. All of the hybridoma cell lines (AHN 1-6) produced IgM antibodies that were cytotoxic.     

The antigenic surface of human chorionic gonadotropin as mapped by murine monoclonal antibodies

We present a map describing the antigenic determinants (epitopes) of the human CG (hCG) molecule. A panel of monoclonal antibodies to hCG, previously characterized by us was incorporated into two-site binding assays to probe hCG by a 21 X 11 matrix of 231 pairs of MCA. Nine different reaction patterns of reciprocal homology were distinguished and interpreted as representing nine separate and distinct epitopes. Three of them localize on the alpha-subunit, 4 on the beta-subunit and 2 were expressed by the intact hCG (holo-hCG) molecule only, hence designated conformational (c) epitopes. Epitope-contingency analysis revealed that each of the 2 subunits of hCG possesses 1 epitope that is neither adjacent to nor overlapping with any of the other 8 epitopes. The latter were arranged in a complex cluster from which the c-epitopes protrude. Whereas the alpha- and c-epitopes are specific for glycoprotein hormones of human origin and for hCG, respectively, the beta-epitopes appeared to be evolutionarily highly conserved structures: with 1 exception, they were also present on the beta-chain of LH from many different mammalian species.     

Identification of mutant monoclonal antibodies with increased antigen binding.

Sib selection and an ELISA have been used to isolate hybridoma subclones producing mutant antibodies that bind antigen better than the parental monoclonal antibody. Such mutants arise spontaneously in culture at frequencies of 2.5-5 X 10(-5). The sequences of the heavy and light chain variable regions of the mutant antibodies are identical to that of the parent and the Ka values of the mutants and the parent are the same. The increase in binding is associated with abnormalities of the constant region polypeptide and probably reflect changes in avidity of these antibodies.     

Specific modulation of sodium channels in mammalian nerve by monoclonal antibodies.

Monoclonal antibodies (mAbs) were generated against the sodium channels in the intact membrane of the eel electroplax. These antibodies bind to nodes of Ranvier, as indicated by immunofluorescence. When externally applied to rat nerve fibers one of these mAbs blocks impulse conduction. In voltage-clamp experiments, this mAb was found to attenuate sodium current amplitude without affecting the time course. The dose-response curve was very steep and had an ED50 of 133 nM. About half of the mAb effect was shown to be due to a shift, in the hyperpolarizing direction, of the steady-state sodium inactivation versus membrane potential curve. The remaining effect was voltage- and time-independent. This mAb had no effect on the potassium or leakage currents. The results indicate that on the external surface of the sodium channel, there are a number of antigenically similar determinants, which are functionally linked to specific elements of the sodium conductance system. These functionally related determinants were preserved through the course of evolution.     

Human hepatoma-associated cell surface antigen: identification and characterization by means of monoclonal antibodies

A library of murine monoclonal antibodies reactive with human hepatoma cells was generated following immunization of Balb/c mice with an intact cloned human hepatoma cell line, designated PLC/PRF/5-NR. We report the characterization of one such IgG2a antibody, designated anti-PLC1. This antibody specifically stains parental PLC/PRF/5 cell membranes and membranes of SK-Hep 1 and Mahlavu human hepatoma cells grown in culture, using indirect immunofluorescence and horseradish immunoperoxidase techniques. A similar pattern of membranous staining was observed in solid tumors derived from the three hepatoma cell lines which were injected subcutaneously into athymic nude rats and mice. Spontaneous capping on the cell surface was observed in 7 to 30% of the three human hepatocellular carcinoma cell types when incubated in suspension with monoclonal anti-PLC1 at 37 degrees C. Treatment of cells with trypsin or sustained growth in culture did not affect the intensity of membranous staining. Monoclonal anti-PLC1 appeared specific, and antibodies did not stain a variety of human carcinoma cell lines and primary tumors of nonhepatic origin, or several normal human and murine tissues. Purified 125I-labeled monoclonal anti-PLC1 bound specifically to the three hepatoma cell lines in culture. Specificity of the antigen-antibody reaction was demonstrated by competitive binding inhibition in experiments using unlabeled homologous antibody. Binding of 125I-anti-PLC1 was not inhibited by unlabeled monoclonal antibodies to HBsAg or to alpha-fetoprotein. Two hepatoma cell lines secrete a protein that specifically blocks binding of 125I-anti-PLC1 antibodies to cell surface antigenic determinants. This "hepatoma-associated" protein was subsequently purified by affinity chromatography from supernates derived from the three hepatoma cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of antigenic peptide recognized by the anti-JL1 leukemia-specific monoclonal antibody from combinatorial peptide phage display libraries

PURPOSE: In the present study an antigen-mimetic peptide of the anti-JL1 leukemia-specific monoclonal antibody (mAb) was identified and characterized. METHODS: From combinatorial peptide phage display libraries displaying the random linear heptapeptides and dodecapeptides, we selected clones with affinity to anti-JL1 mAb through repeated rounds of panning on a mAb-coated ELISA plate. The antigenicity and immunogenicity of the peptide epitopes were then studied using chemically synthesized peptides. RESULTS: The selected clones had the LXPSIP consensus sequence. Two synthetic peptides LPPSIPFGLTVGGGGS and LLPSIPNQAYLGGGGS specifically reacted with anti-JL1 mAb in ELISA. These two peptides were found to inhibit the interaction between anti-JL1 mAb and JL1 antigen-positive Molt-4 cells. Although the immune sera raised against the keyhole limpet hemocyanin-conjugated peptides failed to react with Molt-4 cells, it showed strong reactivity to the peptide epitope. However, one mAb raised by peptide immunization successfully bound to Molt-4 cells. CONCLUSION: An epitope-mimetic peptide of anti-JL1 mAb was found using combinatorial peptide phage display libraries. It induced strong humoral response against itself, but only a limited fraction of this humoral response was cross-reactive with the original JL1 antigen.     

Localization of factor VIII-procoagulant antigen: an immunohistological survey of the human body using monoclonal antibodies

Various organs, including liver, spleen, heart, lung, kidney, intestines, lymph nodes, pancreas, bone marrow, and thymus, were investigated for the presence of factor VIII-procoagulant antigen (VIIICAg) and factor VIII-related antigen (VIIIRAg), using a panel of monoclonal antibodies directed to factor VIII-von Willebrand factor in combination with a sensitive immunoperoxidase staining technique. In addition to hepatic sinusoidal endothelial cells, the presence of VIIICAg was demonstrated in mononuclear cells sporadically present in lymph nodes, in the alveolar septa of lung, and in the red pulp of spleen. The identity of these mononuclear cells could not be unequivocally determined. Based on morphological criteria, however, it is tentatively concluded that these cells are nonlymphoid and belong to the mononuclear phagocyte system. The presence of VIII-RAg was confined to vascular endothelial cells, hepatic sinusoidal endothelial cells, cells lining the venous sinuses of the red pulp of the spleen, cells lining renal glomeruli and lung capillaries, platelets, and megakaryocytes.     

Efficient neutralization of anthrax toxin by chimpanzee monoclonal antibodies against protective antigen

Four single-chain variable fragments (scFvs) against protective antigen (PA) and 2 scFvs against lethal factor (LF) of anthrax were isolated from a phage display library generated from immunized chimpanzees. Only 2 scFvs recognizing PA (W1 and W2) neutralized the cytotoxicity of lethal toxin in a macrophage lysis assay. Full-length immunoglobulin G (IgG) of W1 and W2 efficiently protected rats from anthrax toxin challenge. The epitope recognized by W1 and W2 was conformational and was formed by C-terminal amino acids 614-735 of PA. W1 and W2 each bound to PA with an equilibrium dissociation constant of 4x10-11 mol/L to 5x10(-11) mol/L, which is an affinity that is 20-100-fold higher than that for the interaction of the receptor and PA. W1 and W2 inhibited the binding of PA to the receptor, suggesting that this was the mechanism of protection. These data suggest that W1 and W2 chimpanzee monoclonal antibodies may serve as PA entry inhibitors for use in the emergency prophylaxis against and treatment of anthrax.     

Trichuris muris: antigen recognition and transfer of immunity in mice by IgA monoclonal antibodies

Mesenteric node lymphocytes from mice that had been infected with the nematode Trichuris muris, and then boosted with adult worm excretory-secretory antigens were fused with myeloma cells to produce a panel of 9 monoclonal antibodies (MoAbs). Five of the MoAbs were of the IgA isotype. The antigen recognition profiles of these MoAbs were studied using SDS-PAGE and immunoblotting; three major profile patterns were identified. Five MoAbs recognized a major band in the MW range 43-48 kD; all recognized a range of antigens. Three MoAbs were used to localize antigens in the bodies of adult worms. Granules within the anterior stichocytes were recognized strongly, as was material within the eggs and pseudocoelom. Two MoAbs stained the cuticle. Although the phosphorylcholine (PC) determinant was widely distributed within worm tissues none of the MoAbs tested recognized PC. Passive transfer of immunity was achieved using two of the IgA monoclonals; no immunity was transferred by the IgM and IgG MoAbs used. The limited recognition profiles of these IgA MoAbs, and the ability to stain stichocyte granules, suggest that their protective activity results from an interaction with ES antigens.     

Surface antigenic change during differentiation of a parasitic protozoan, Leishmania mexicana: Identification by monoclonal antibodies.

The fusion of SP2/0 myeloma cells with spleen cells from mice immunized with Leishmania mexicana amazonensis promastigotes produced hybridoma clones. Indirect immunofluorescent antibody assay with live leishmanias showed that the monoclonal antibody 6H12 recognized only the antigens bound to the surface of L. mexicana amazonensis promastigotes. It also showed that the antibody bound to neither amastigotes of this species nor to other Leishmania species--i.e., L. braziliensis braziliensis, L. tropica, and L. donovani. Monoclonal antibodies from three other clones (4D11, 4H9, and 6A11) were found to compete with 6H12 for binding to L. mexicana promastigotes. With lysates of [35S]methionine-labeled promastigotes, all four monoclonal antibodies precipitated the same triplet set of protein bands at the approximately equal to 68,000-dalton region, whereas another monoclonal antibody (6G5) precipitated a different band at approximately equal to 90,000 daltons. During differentiation of L. mexicana amazonensis from amastigotes to promastigotes, there was a 4- to 8-fold increase above the initial level in the binding of 6H12 monoclonal antibody to leishmanias, as detected by enzyme-linked immunosorbent assay and quantitative fluorometric assay, respectively. Thus, we have demonstrated the use of monoclonal antibodies as probes for antigens that change during leishmanial differentiation.     

组合单克隆抗体多表位检测日本血吸虫循环抗原的研究

目的探讨抗肠相关抗原单克隆抗体（单抗）JPG5和抗虫卵可溶性抗原单抗JPS6、JPG5＋JPG1组合和JPS6＋JPG1组合对血吸虫循环抗原（CAg）的诊断效能。方法采用双抗体夹心酶联免疫吸附试验（ELISA）对慢性血吸虫病、急性血吸虫病、晚期血吸虫病、卫氏并殖吸虫病、华支睾吸虫病、乙型肝炎患者血清,以及治疗前、治疗后半年、治疗后1年的慢性血吸虫病疗效考核血清进行CAg单位点和多位点检测。结果JPG5检测慢性血吸虫病患者的敏感性和特异性分别为74.3%和96.8%,JPS6分别为78.7%和95.3%;两株单抗与卫氏并殖吸虫病、华支睾吸虫病及乙型肝炎患者血清的交叉反应率分别为15.2%、15.6%、3.2%和21.5%、15.6%、16.0%。JPG5检测治疗后1年的慢性血吸虫病患者血清CAg阴转率高于JPS6。JPG5＋JPG1组合检测慢性血吸虫病患者的敏感性和特异性分别为80.1%和93.7%,JPS6＋JPG1组合分别为73.5%和96.1%;两种组合与卫氏并殖吸虫病、华支睾吸虫病及乙型肝炎患者血清交叉反应率分别为21.5%、15.6%、18.0%和22.8%、15.6%、16.0%。两种组合多位点检测治疗后1年的慢性血吸虫病患者血清CAg阴转率分别为66．7％和45．5％。结论JPG5、JPS6可用作CAg检测的备选探针。JPG5＋JPG1组合与JPS6＋JPG1组合对CAg的诊断效能相近;较之单位点检测,多位点检测敏感性无明显提高。     

Specificity variants in monoclonal antibodies reactive with peptide epitopes of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum

It has been suggested that repeat sequence antigens of Plasmodium falciparum may serve the parasite in immune evasion by modifying the host antibody response and impairing the development of protective immunity. According to this proposal networks of cross-reactive, repeat sequence malarial antigens have the ability to stimulate a high proportion of all somatically mutated B cells with altered antibody specificity, and thus to hinder the normal process of antibody affinity maturation. To determine the rate at which immunoglobulin mutations produce new reactivities with repeat sequence antigens, hybridoma cell lines specific for the ring-infected erythrocyte surface antigen (RESA) were examined for the incidence of specificity variants that arose naturally or as a result of treatment with the chemical mutagen ethylmethane sulphonate (EMS). From one of the cell lines variants were readily isolated having reactivity towards a very closely related repeat sequence epitope within the same RESA antigen. However, the other hybridoma/antigen combinations revealed no variants. In general, mutations giving rise to antibodies with altered specificity for related repetitive antigens were not readily induced and only limited support of the hypothesis was obtained.     

Cell surface antigens of human malignant melanoma: definition of six antigenic systems with mouse monoclonal antibodies.

Eighteen mouse monoclonal antibodies were selected for reactivity with cell surface antigens of the immunizing human melanoma cell line SK-MEL-28. Six distinct antigenic systems were defined by direct serological assays and absorption tests with a panel of 41 cell lines derived from normal and malignant human tissues. Biochemical analysis indicated that two of the antigens are glycoproteins with molecular sizes of 95,000 and 150,000 daltons (gp95 and gp150). Two other antigenic systems (O5 and the R24 group) are associated with heat-stable molecules having the characteristics of glycolipids. The remaining two antigens (M19 and R8) are heat labile, but molecular characterization has not been possible. Each of the antigenic systems has a distinctive pattern of distribution on various cell types, varying from a broad representation to a more restricted occurrence. O5 appears to be a species antigen, being present on virtually every human cell type tested. gp95, gp150, M19, and R8 are found on a characteristic proportion of melanomas, astrocytomas, and epithelial cancers and on normal kidney cells. The antigen defined by the R24 antibody has the most restricted distribution of all. Reactivity is found with melanomas and astrocytomas, whereas epithelial cell types, fibroblasts, and cells of hematopoietic origin lack R24. Although occurrence of gp95, gp150, M19, and R8 distinguishes a small subset of melanomas not expressing these antigens, R24 is found on all melanoma cells.     

Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system.

A convenient and versatile approach to the direct synthesis of a peptide-antigen matrix by the solid-phase method is described. The approach is called the multiple antigen peptide system (MAP) and it utilizes a simple scaffolding of a low number of sequential levels (n) of a trifunctional amino acid as the core matrix and 2n peptide antigens to form a macromolecule with a high density of peptide antigens of final Mr 10,000. The MAP model chosen for study was an octa-branching MAP consisting of a core matrix made up of three levels of lysine and eight amino terminals for anchoring peptide antigens. The MAP, containing both the core matrix and peptides of 9-16 amino acids, was prepared in a single synthesis by the solid-phase method. Six different MAPs elicited specific antibodies in rabbits and mice, of which five produced antibodies that reacted with their corresponding native proteins. In rabbits, the sera had a considerably higher titer of antibodies than sera prepared from the same peptides anchored covalently to keyhole limpet hemocyanin as carrier. Thus, the MAP provided a general, but chemically unambiguous, approach for the preparation of carrier-bound antigens of predetermined and reproducible structure and might be suitable for generating vaccines.