Linda J. Foote

Rat monoclonal antibodies to mouse lung components for analysis of fibrosis

Six rat monoclonal antibodies to mouse lung membrane fraction have been characterized. Each has unique binding properties and can be used to stain particular lung components in paraffin sections. One antibody, 133-13A, recognizes a 100-kDa glycoprotein on lung tumor cells, but stains only macrophage-like cells in normal or fibrotic lung sections as determined by immunogold electron microscopy. The monoclonal antibody 273-34A binds to a 112-kDa protein on the surface of normal mouse lung fibroblasts. Immunogold electron microscopy demonstrates antibody binding to capillary endothelial cells, but not to fibroblasts. Type I, or Type II cells. Staining of fibrotic sections with MoAb 273-34A is dramatically enhanced over staining of normal lung. A third antibody, 370-8A, gives a general staining pattern throughout normal lung that is intensified in fibrotic lung. Another MoAb 328-41A mediates intense nuclear fluorescence of lung tumor cells and cells in lung sections. It binds to 14- and 17-kDa proteins that may be high mobility group (HMG) nuclear proteins. Enzyme inhibition studies and immunofluorescence staining patterns on normal lung indicate that elastin may be the target of MoAb 328-35B. MoAb 327-5B binds to normal mouse lung fibroblasts and red blood cell membranes. These last three MoAbs stain macrophages in fibrotic lung, but give a general pattern of light epithelial cell stain in normal lung sections indicating macrophage engulfment of the normal cell antigen during fibrosis. These antibodies should be useful in identifying cell types and molecular mechanisms involved in early stages of fibrosis induced by different chemical insults.

Mutation analyses of a series of TNT-related compounds using the CHO-hprt assay.

Trinitrotoluene (TNT) and related compounds were tested for induction of mutation in the CHO‐hprt mutation assay. The parent compound, TNT, was consistently found to be mutagenic at concentrations above 40 μg ml−1, whether or not S9 activating enzymes were added. Five TNT metabolites gave statistically significant but small increases in mutation frequency over solvent controls: 4‐amino‐2,6 dinitrotoluene, 2,4′,6,6′‐tetranitro‐2′,4‐azoxytoluene, 2,2′,6,6′‐tetranitro‐4,4′‐azoxytoluene, 2′,4,6,6′‐tetranitro‐2,4′‐azoxytoluene and triaminotoluene. Clear dose–response relationships could not be established for the mutagenic response of these compounds. They are considered as very weak mutagens in this mammalian test system. Five compounds did not produce statistically significant mutation frequencies at the levels tested: 2‐amino‐4,6‐dinitrotoluene, 2,4‐diamino‐6‐nitrotoluene, 1,3,5‐trinitrobenzene, 2,6‐diamino‐4‐nitrotoluene and 4,4′,6,6′‐tetranitro‐2,2′‐azoxytoluene. The results indicate that none of the TNT metabolites tested pose a significant mutational health risk, at least as judged by the CHO‐hprt assay. Copyright

Monoclonal antibodies from rats immunized with fragment D of human fibrinogen

Abstract Fischer rats were immunized with fragment D (Fg-D) of human fibrinogen (Fg) to obtain antibody specific for neoantigens unique to this molecule. Absorption of serum with whole Fg indicated that some of the antibody produced reacted preferentially with Fg-D. Hybridoma cultures were prepared by fusion of immune rat spleen cells with mouse myeloma P3-X63-Ag8. Monoclonal antibodies obtained from these cultures fell into two classes: (a) Those reacting equally well with Fg and Fg-D. (b) Those reacting preferentially but not absolutely with Fg-D. Antibody from hybridoma 104-14, a member of the first group had an affinity for Fg-D of 1.5 × 10 9 M −1 while antibodies from 106-59 and 106-71 (group 2) demonstrated much lower affinities of 1.0 × 10 7 and 4.7 × 10 6 M −1 , respectively. The cross reactivity of antibodies in the second group indicated that they react with protein conformations that are altered during production of Fg-D from Fg.

Phage Display Selection of scFv to Murine Endothelial Cell Membranes

The diversity of endothelial cells is becoming more apparent and more important in defining vessel systems that supply blood to normal organs and to tumors. Reagents that identify expression of cell surface determinants on these cells are crucial for differentiating among different vessel types. As a first step in this process we have selected a panel of 25 scFvs from a phage display library that bind to the endothelial cell line LEII. The scFvs are of high affinity and bind to some tumor cells as well as to the target endothelial cell. The scFvs can be divided into 8 epitope groups by use of competition binding studies. DNA sequencing of the members of these groups generally support the classification. This work shows that phage display is a rapid and efficient method for identification of reagents for cell surface molecules.

Genetic analysis of Pseudomonas aeruginosa by enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR) and arbitrarily primed PCR: gel analysis compared with microchip gel electrophoresis.

OBJECTIVES To assess the applicability of a newly emerging microchip gel electrophoresis for rapid strain differentiation among clinical isolates of Pseudomonas aeruginosa, and to compare this technique with the traditional gel method for DNA separation. METHODS One hundred clinical strains of P. aeruginosa obtained from a hospital in northwestern Ohio were tested for reactivity to 3 serotype-specific monoclonal antibodies by enzyme-linked immunosorbent assay. Twelve strains (4 from each serogroup) were selected for DNA analysis by polymerase chain reaction (PCR)-based, single primer DNA fingerprinting methods with 3 different primers: 1 enterobacterial repetitive intergenic consensus PCR and 2 arbitrarily primed PCRs. The PCR products were analyzed by agarose slab gel and microchip gel electrophoresis. RESULTS Of the 100 clinical isolates tested, 39% (4%, 14%, and 21%) were found to be serotypes 0:3, 0:6, and 0:11, respectively. Twelve strains were chosen for DNA analysis by PCR. The PCR products were analyzed by agarose slab gel electrophoresis and on microchips to determine interspecies diversity. Both methods demonstrated that different serotypes exhibited different electrophoretic patterns. Two strains (clinical strains 6 and 7, serotype 0:6) showed identical patterns, indicating a high degree of relatedness. CONCLUSION In all cases, there was concordance between the electrophoretic patterns detected by the two methods. The capability of conducting both PCR and microchip gel electrophoresis offers an opportunity for an automated and rapid method for genetic analysis and differentiation among strains of P. aeruginosa and other microorganisms.

Monoclonal antibodies to CD44 epitopes on mouse endothelium.

CD44 is a widely expressed, plasma membrane protein. Many molecular forms of CD44 are possible as it is encoded by a gene with multiple exons that can be alternatively spliced and its deduced protein sequence contains numerous glycosylation sites. Through its role as an adhesion molecule, CD44 is involved in many and diverse biological processes, including angiogenesis, lymphogenesis, wound healing, inflammation, and cancer metastasis. We have developed a new panel of rat monoclonal antibodies (MAbs) to murine CD44 by immunization with mouse lung endothelial cells (LEII cells). The antibodies were characterized using immunoprecipitation, mass spectrometry, competition binding, and cross Western blot experiments with MAb 133-13A, which recognizes CD44 expressed on tumor cells. The new MAbs recognize three distinct epitope groups. MAbs 531-2A and 531-32A compete for binding with the MAb 133-13A that was described previously. MAb 531-30A identifies a CD44 epitope found on low molecular weight forms expressed in vivo, while MAb 531-22A appears to recognize an epitope specific for endothelial cells. This novel panel of anti-CD44 antibodies has potential for investigating the role of CD44 in disease pathogenesis models in the mouse. They may be particularly useful for examining the role of endothelial cells in these models.

A monoclonal antibody to a carbohydrate epitope expressed on glycolipid and on α3β1 integrin on human esophageal carcinoma

A mouse monoclonal antibody (MAb-9) produced by immunization with a human esophageal carcinoma cell line, TE-2 (derived from undifferentiated squamous cell carcinoma) reacted specifically with about 30% of esophageal carcinoma cell lines and tissue sections from clinical samples. MAb-9 showed minimal reactivity with normal esophageal tissue. 125I, fluorescent or gold particle labeled MAb-9 bound to TE-2 cell surfaces. 125I-radiolabeled MAb-9 was used to detect reactive material from cell extracts in Western blot. Treatment of TE-2 membrane proteins with neuraminidase, N-glycanase or O-glycanase reduced antigen detection. Treatment of cells with periodic acid destroyed antibody binding in ELISA. Lipid extracts from cell membranes, containing glycolipids, also reacted with MAb-9. MAb-9 was used to purify target antigen from detergent solubilized membrane proteins and the prominent bands from subsequent gel electrophoresis were trypsin digested and analyzed by mass spectrometry. Peptides from α3 and β1 integ...

Monoclonal Antibodies in Cancer Detection and Therapy

The advent of monoclonal antibodies has revolutionized thinking about cancer therapy management. In this article, the authors review some of the applications of monoclonal antibodies in cancer research and identify requirements for monoclonal antibody to be useful in cancer detection or therapy. Empirical tests of antibody circulation in vivo are necessary to establish specificity. 68 references, 3 figures.

Endogenous type C viral gene expression in cultures of fetal diploid baboon cells treated with 5′-bromodeoxyuridine1,2

Abstract Cultures of fetal diploid baboon fibroblasts treated with 5-bromodeoxyuridine synthesized protein antigenically related to baboon endogenous type C viral gag gene product, p28. Cellular immunofluorescence studies revealed induction of viral p28 in greater than 10% of treated cells in two baboon cell lines. Radioimmunoassays detected p28 antigenic specificities indistinguishable from those of purified virus [M7 strain of baboon endogenous virus (BEV)]. However, viral RNA-dependent DNA polymerase was not detected in culture fluids, and infectious virus was rarely recovered by cocultivation with susceptible heterologous cells. Extracellular particles containing p28 were not readily detected, further indicating that viral gag gene-coded proteins were synthesized independently of whole virus. Normal cultures of the same baboon cells exhibited endogenous expression of a glycoprotein antigenically related to BEV gp70, suggesting differential regulation of the endogenous gag and env gene-coded products. Baboon cell cultures exogenously infected with BEV produced extracellular particles having viral p28 and gp70 as measured by radioimmunoassays of culture fluids. Virus-infected cultures expressed about 0.01% of total lysate protein as p28, whereas bromodeoxyuridine-treated cultures expressed about 0.001% of their protein as p28. Since induced cultures have about 10% positive cells versus close to 100% for infected cultures, the amount of p28 per producing cell was about the same in both cell populations.