Masters Theses

Date of Award

3-1988

Degree Type

Thesis

Degree Name

Master of Science

Major

Life Sciences

Major Professor

Robert N. Moore

Committee Members

David A. Bemis, Barry T. Rouse

Abstract

Colony stimulating factors (CSFs) are a functionally defined class of glycoprotein growth factors that stimulate the proliferation and differentiation of immature precursor cells to mature granulocytes and/or macrophages and have an additional role as central regulators of inflammatory and immune responses. Human granulocyte/macrophage colony-stimulating factor (GM-CSF) and human macrophage colony-stimulating factor (M-CSF) have recently been cloned and the nucleotide sequence determined. The predicted amino acid sequences were analyzed to detect potential antigenic determinants by contiguous hexameric hydrophilicity estimates. Two 18 amino acid peptides were produced, one representing the major predicted epitope of human GM-CSF and the other representing a potentially significant epitope of human MCSF. The peptides were studied alone, and in combination with native CSFs to determine if they had any biological activity. The peptides were also used as antigens in the production of rabbit polyclonal antibodies.

Human GM-CSF peptide had no biological activity, as determined by tritiated thymidine (3H-TdR) incorporation, alone or in combination with native CSFs. The rabbit anti-human GM-CSF peptide antibody was shown to: 1) react with GM-CSF peptide by enzyme linked immunoassay (ELISA), 2) detect human as well as murine GM-CSF by ELISA and, 3) possess limited neutralizing activity, as determined by 3 H-TdR incorporation and soft agar assay, for murine GM-CSF. The human GM-CSF peptide, therefore, appears to have potential for the development of a heterogeneous ELISA for GM-CSF.

Human M-CSF peptide alone, like human GM-CSF peptide had no effect on the rate of 3H-TdR incorporation by murine marrow cells. However, in combination with murine L cell M-CSF and recombinant human M-CSF the M-CSF peptide caused an increase in the rate of DNA synthesis. Additional experiments indicated the increase in the rate of DNA synthesis was due to the presence of human M-CSF peptide. M-CSF peptide in combination with recombinant human G-CSF or murine GM-CSF did not result in an increase in 3H-TdR incorporation. The anti-human M-CSF peptide antibody did detect human M-CSF peptide by ELISA. However, the anti-human M-CSF did not detect, by ELISA, recombinant human M-CSF or L cell M-CSF. The M-CSF peptide appears to have potential for biological studies, but not for development of a heterogeneous ELISA for M-CSF.

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