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A H Ross

Publications and source records attributed to A H Ross.

At least 91 records · Page 5Linked to original sources

Purification of monoclonal antibodies from ascites using ABx liquid chromatography column.

Murine and rat monoclonal antibodies (MAbs) were purified to 90-95% homogeneity from ascites in a single step using an ABx liquid chromatography column which utilizes a silica-based mixed-mode ion-exchange matrix. The resulting MAbs were highly immunoreactive reflecting the mild conditions used for this procedure. The column was also useful for detecting and separating multiple immunoglobulins present in the same ascites. For one ascites sample, three immunoglobulin species, which apparently differed only in their light chains, were resolved using this column and differed greatly in their immunoreactivity.

Animals↗

Expression of melanoma-associated antigens in rapidly dividing human melanocytes in culture.

Conditions were established to induce rapid clonal growth of melanocytes from newborn foreskin. Surface antigen expression was analyzed using monoclonal antibodies derived by immunization of mice with melanoma cell, melanocyte, and placental membrane preparations. Unlike resting melanocytes in normal skin, cultured melanocytes expressed most major melanoma-associated antigens tested, e.g., nerve growth factor receptor, proteoglycan, transferrin-related Mr 97,000 protein antigen, Mr 120,000 protein, and gangliosides 9-O-acetyl GD3 and GD3. HLA-DR antigen and ganglioside GD2 were expressed at very low levels or not expressed. After several subpassages, most melanocyte cultures, including clones and melanocytes, initially sorted by rosetting with monoclonal antibody to nerve growth factor receptor, lost their characteristic bipolar morphology and expression of nerve growth factor receptor and Mr 97,000 antigen but continued to express high molecular weight proteins such as proteoglycan, Mr 130,000/105,000 and 120,000 antigen. The few melanocyte cultures that did maintain their characteristic bipolar to spindle morphology continued to express all melanoma-associated antigens and even began to express HLA-DR antigens. Melanocytes cultured in the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate also maintained their bipolar morphology, were often pigmented, and continued to express melanoma-associated antigens for several passages; they did not express HLA-DR antigen. Our studies indicate that rapidly proliferating melanocytes in culture undergo antigenic changes associated with malignancy.

Antibodies, Monoclonal↗

Binding of an antagonistic monoclonal antibody to an intact and fragmented EGF-receptor polypeptide.

A murine monoclonal antibody (No. 425) raised against human A431 carcinoma cells specifically immunoprecipitates the 170,000 molecular weight epidermal growth factor (EGF)-receptor from extracts of A431 cells as well as from extracts of human placenta and cultured fibroblasts, but does not recognize the murine receptor. Binding to the external domain of the human EGF-receptor was indicated by indirect immunofluorescent staining of fixed nonpermeable cells. The antibody binds to both glyco- and aglycoreceptor forms, indicating that the epitope is a part of the polypeptide chain. Binding of the antibody to the receptor is conformation dependent; i.e., denatured receptors lacking EGF-binding activity are not recognized by the antibody. The results of antibody binding studies indicate that the epitope is closely linked to the EGF binding active site, and is common to both high- and low-affinity EGF-receptors. Interaction of this epitope with the antibody inhibits EGF binding and bioactivity, and triggers receptor down-regulation, but does not generate EGFlike kinase-stimulatory or mitogenic responses either in vitro or in vivo. The antibody was tested for its ability to bind to domain-sized fragments of the 170-kDa EGF-receptor. It can recognize both the proteolytically generated 110-kDa EGF binding peptide, and a soluble 100-kDa EGF-receptor secreted by A431 cells. This indicates that the epitope recognized this antibody retains its conformation after proteolytic separation of the EGF binding domain from the rest of the receptor molecule.

Antibodies, Monoclonal↗

Induction of specific immunity to human colon carcinoma by anti-idiotypic antibodies to monoclonal antibody CO17-1A.

Anti-idiotypic antibodies (Ab2) to murine monoclonal antibody (mAb) CO17-1A (Ab1), which defines an antigen associated with human colon carcinoma, were produced in goats. The Ab2 inhibited binding of Ab1 to colon carcinoma cells, and purified tumor antigen inhibited binding of Ab1 to Ab2. The Ab2 elicited in rabbits anti-anti-idiotypic antibodies (Ab3) that bound to cultured human cells of various tissue origins with a binding pattern identical to that of Ab1. Moreover, both Ab1 and Ab3 bound to the isolated tumor antigen, and the Ab3 lysed human colon carcinoma cells in culture in the presence of rabbit complement. Thus, Ab2 raised to mAb CO17-1A are candidates for use in immunotherapy trials in cancer patients.

Animals↗

PDGF induces c-myc mRNA expression in MG-63 human osteosarcoma cells but does not stimulate cell replication.

The platelet-derived growth factor (PDGF) modulated growth response of the MG-63 human osteosarcoma cell line, which neither expresses c-sis mRNA nor secretes a PDGF analogue, was characterized. Scatchard analysis demonstrated that the MG-63 cells have 23,000 receptors per cell with a Kd of 5 X 10(-11) M. The receptor became phosphorylated, in a PDGF concentration-dependent manner, when 32P-orthophosphate-labeled cells were treated with PDGF for 3 h at 4 degrees C. The phosphorylated receptor was identified by autoradiography and gel electrophoresis after isolation of the 32P-labeled receptor using a solid-phase monoclonal antibody directed against phosphotyrosine. Binding of the receptor to the antibody was inhibited by 5 mM phenyl phosphate, further suggesting that PDGF stimulated tyrosine-specific receptor autophosphorylation. In addition, treatment of MG-63 cells with PDGF for 3 h at 37 degrees C induced a 7.5-fold increase in c-myc mRNA accumulation as analyzed on Northern gels. However, MG-63 cells grew equally well in either serum-(which contains PDGF) or plasma-(which does not) supplemented medium. Furthermore, PDGF did not stimulate DNA synthesis in growth arrested MG-63 cells, nor did it potentiate DNA synthesis modulated by somatomedin C. Thus MG-63 cells are a naturally occurring cell variant in which PDGF stimulates c-myc expression but does not modulate mitogenesis.

Animals↗

The significance of controls in Lyme arthritis of children.

In order to better define the specificity of Lyme disease (LD) seropositivity and seronegativity during a study of Lyme arthritis in a pediatric rheumatology population, three groups of controls are in the process of being developed. Children with other rheumatic disorders. Siblings and parents of children with Lyme arthritis. Individuals with intense environmental exposure to LD. Sera from each have been tested for antibody to Borrelia burgdorferi (Bb), by indirect immunofluorescence assay (IFA), enzyme linked immunosorbent assay (ELISA), or both. Of the 57 children with rheumatic disorders of whom 25 had JRA, 4 (7%) reached a border zone, non-specific level of 1:64 by IFA, and 53 sera were non-reactive. 7 household relatives of LD cases had non-reactive sera. The 42 intensely Bb-exposed individuals all had non-reactive sera. The fact that none of the 106 controls attained a clinically significant level of antibody is a general indication of the specificity of the serologic tests. The use of selected control groups such as these might potentially solve the ambiguities in clinical-laboratory correlations in Lyme arthritis.

Adolescent↗

Anti-idiotype immunization of cancer patients: modulation of the immune response.

Thirty patients with advanced colorectal carcinoma (CRC) were treated with alum-precipitated polyclonal goat anti-idiotypic antibodies (Ab2) to monoclonal anti-CRC antibody CO17-1A (Ab1) in doses between 0.5 and 4 mg per injection. All patients developed anti-anti-idiotypic antibodies (Ab3) with binding specificities on the surfaces of cultured tumor cells similar to the specificity of Ab1. Furthermore, the Ab3 competed with Ab1 for binding to CRC cells. Fractions of Ab3-containing sera obtained after elution of the serum immunoglobulins from CRC cells bound to purified tumor antigen and inhibited binding of Ab2 to Ab1. The Ab3, therefore, may share idiotopes with Ab1. Six patients showed partial clinical remission and seven patients showed arrest of metastases following immunotherapy. Four of the thirteen patients with measurable clinical responses had received Ab2 alone, whereas 9 patients had also received chemotherapy.

Antibodies, Anti-Idiotypic↗

Purification and amino terminal sequencing of human melanoma nerve growth factor receptor.

The nerve growth factor (NGF) receptor, solubilized with Triton X-100 detergent, has been purified from human melanoma cell line A875. Purification to near-homogeneity was achieved by chromatography on wheat germ agglutinin-agarose, followed by immunoaffinity chromatography on Sepharose columns coupled with anti-NGF receptor monoclonal antibody (MAb). The purified receptor, a 75,000-dalton protein, retains the capacity to bind NGF as well as anti-receptor MAbs. Final purification was achieved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of amino acid residues at the amino terminus has been determined. Possible sequence homology between the NGF receptor and several other proteins is discussed. Using the purified receptor as immunogen, new MAbs to the NGF receptor have been produced. The NGF receptor was visualized by immunoperoxidase staining in tissue sections of dorsal root ganglia from monkeys.

Amino Acid Sequence↗

Non-specific binding of insulin in an equilibrium binding assay of insulin antibodies.

In liquid phase assays for insulin binding antibodies (IBA), total binding of insulin is composed of specific and non-specific binding (NSB). Sometimes NSB is determined in serum of healthy individuals and then subtracted from total binding of IBA positive serum to obtain specific binding. This method does not take into account that NSB might vary from plasma to plasma. This possibility was investigated by means of a computerised non-linear curve fitting routine for the evaluation of measurement results of an (equilibrium) binding assay for IBA, which yields estimates of NSB for each plasma individually. From each of 19 insulin treated diabetic patients, 4 blood samples, taken at different points in time, were available for IBA and NSB measurement. It was found that inter-patient variance of NSB exceeded within-patient variance (p less than 0.01) and, in a number of instances, within-patient variance was greater than experimental variance. Our results indicate that it is advisable to use methods of IBA evaluation that take these NSB variations into account.

Antibodies↗

Gene transfer and molecular cloning of the human NGF receptor.

Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

Animals↗

Anti-idiotypic antibodies bear the internal image of a human tumor antigen.

Goat antibodies to idiotypes (anti-idiotypic antibodies; Ab2) that recognize an idiotype associated with the combining site of a BALB/c mouse IgG2a monoclonal antibody (Ab1) to human gastric carcinoma were used to immunize BALB/c mice and rabbits. A monoclonal anti-anti-idiotypic antibody (Ab3) of IgG1 isotype was obtained after immunization of mice. The Ab3 and the Ab1 showed identical binding specificities and bound with similar avidities to the same tumor antigen. The induction of Ab1-like Ab3 by Ab2 was not restricted to mice, since Ab3 could also be induced in rabbits. Both the mouse- and the rabbit-derived Ab3 bound the same gastrointestinal cancer-associated antigen as Ab1. These findings indicate that Ab2 induced the formation of antigen-specific Ab3, probably because it bears the internal image of the tumor-associated antigen. This Ab2 may therefore have potential for modulating the immune response of cancer patients to their tumors.

Animals↗

Isolation and characterization of a carcinoma-associated antigen.

GA733 is a murine IgG2a monoclonal antibody (MAb) against human gastric carcinoma and is highly tumoricidal in nude mice. The GA733 antigen is a cell surface protein with two subunits of 30,000 and 40,000 daltons. The antigen isolated by immunoaffinity chromatography consists mainly of the 30,000-dalton subunit which bears the GA733 epitope. This subunit displayed several isoelectric points between 6.9 and 7.7. Anti-colon carcinoma MAb 17-1A also detects this antigen, but probably binds to a different epitope.

Antibodies, Monoclonal↗

Childhood Lyme arthritis: experience in an endemic area.

We report 25 children with oligoarticular arthritis associated with Lyme disease. There were 16 boys (male/female ratio 1.8:1); ages ranged from 2 to 15 years. Thirteen (52%) children had no history of erythema chronicum migrans or other rash. Thirteen had temperatures as high as 41 degrees C for up to 2 months before the onset of arthritis. Twelve recalled definite tick bites. Ten (40%) children, of whom seven had no history of rash, were hospitalized for presumed septic arthritis. Another four had diagnoses of pauciarticular juvenile rheumatoid arthritis for as long as 3 years. Seven patients had less acute, recurrent episodes of synovitis. Two children had seventh nerve palsies 2 months before onset of arthritis. All patients had antibodies to the Lyme spirochete. In 14 patients, synovial fluid white blood cell counts ranged from 180 to 97,700/mm3 (greater than or equal to 76% polymorphonuclear leukocytes). Antibiotic therapy was effective in all patients; in 13, orally administered therapy alone resulted in elimination of synovitis and recurrent attacks. Lyme arthritis may be confused with acute bacterial septic arthritis or recurrent "pauciarticular juvenile rheumatoid arthritis," particularly when there is no history of erythema chronicum migrans.

Adolescent↗

Purified human platelet-derived growth factor receptor has ligand-stimulated tyrosine kinase activity.

The platelet-derived growth factor receptor (PDGF-R), a 180-kDa single-chain polypeptide, was purified from membranes of the human osteogenic sarcoma cell line MG-63. Purification was achieved by treatment of membranes with PDGF and ATP, followed by solubilization with nonionic detergent and successive chromatography on solid-phase anti-phosphotyrosine monoclonal antibody and DEAE-cellulose. The PDGF-R, which was estimated to be 50-80% pure by NaDodSO4/polyacrylamide gel electrophoresis of 32P-labeled preparations, was free of contaminating epidermal growth factor receptor and had no detectable phosphatase activity. It specifically bound 125I-labeled PDGF, a reaction quantified by binding of the ligand-PDGF-R complex to the anti-phosphotyrosine antibody. The purified receptor displayed PDGF-stimulatable tyrosine kinase activity, assayed by autophosphorylation of PDGF-R at tyrosine residues and by phosphorylation of angiotensin II. The Km for ATP in the autophosphorylation reaction was 7.5 microM. Addition of PDGF did not change the Km but increased the Vmax 1.7-fold.

Adenosine Triphosphate↗

The nerve growth factor receptor gene is at human chromosome region 17q12-17q22, distal to the chromosome 17 breakpoint in acute leukemias.

Genomic and cDNA clones for the human nerve growth factor receptor have been used in conjunction with somatic cell hybrid analysis and in situ hybridization to localize the nerve growth factor receptor locus to human chromosome region 17q12-q22. Additionally, part, if not all, of the nerve growth factor receptor locus is present on the translocated portion of 17q (17q21-qter) from a poorly differentiated acute leukemia in which the chromosome 17 breakpoint was indistinguishable cytogenetically from the 17 breakpoint observed in the t(15;17)(q22;q21) translocation associated with acute promyelocytic leukemia. Thus the nerve growth factor receptor locus may be closely distal to the acute promyelocytic leukemia-associated chromosome 17 breakpoint at 17q21.

Chromosome Mapping↗