Development of cell-specific rat monoclonal antibodies and their use as probes to study mechanisms of toxic lung injury in the Bald/c mouse

Author

Jon Allison Hotchkiss

Date of Award

8-1986

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Stephen J. Kennel

Committee Members

Kai-Lin Lee, Peter Witschi, John Cook, Peter Lalley

Abstract

Rat monoclonal antibodies (MoAb) were developed that bind to specific subpopulations of cells in lungs of adult BALB/c mice. MoAb are synthesized by clonal hybridoma cell lines produced through fusion of mouse myeloma line SP2/0 and splenic lymphocytes from Fisher 344 rats which have been immunized with normal mouse lung homogenate. Antibody-producing hybrids were selected for specific binding to enzyme-dissociated lung cells (DLC) using a solid phase radioimmunoassay. Immunohistochemistry (IHC), laser flow cytometry and SDS-PAGE analysis of radiolabeled proteins immunoprecipitated by specific MoAb were used to characterize the binding specificity of six MoAb. MoAb 411-155B and MoAb 411-E3 both precipitate proteins of approximately 140 K dalton M_r. and bind to cells of similar size; however, MoAb 411-155B binds to a larger fraction of normal DLC (24.1 ± 0.5%; mean ± S.D.) than does MoAb 41I-E3 (7.9 ± 0.7%). MoAb 411-41E binds to 33.3 ± 2.4% of DLC while MoAb 411-3E binds to 21.6 ± 2.1% of DLC.

IHC analysis of formalin fixed, deparaffinized sections of adult BALB/c lung reveals that MoAb 411-201B binds to capillary endothelial cells and yields a linear staining pattern throughout all alveolar septae identical to the pattern produced by Raghu et al. (1985) using anti-human type IV collagen antisera. The cell associated binding of MoAb 411-201B is rapidly lost upon treatment with trypsin and collagenase. This antibody precipitates a radiolabeled surface protein of 112K dalton M_r. Taken together, the data suggest that the epitope recognized by MoAb 411-201B is associated with an extracellular matrix component produced by capillary endothelial cells.

MoAb 411-52 binds to an epitope present on 4.6 ± 0.5% of normal adult BALB/c DLC. IHC analysis indicates that 1) MoAb 411-52 binds exclusively to squamous epithelial cells lining alveoli (type I cells); 2) the epitope recognized by MoAb 411-52 is first detected approximately 21 days after birth; and 3) MoAb 411-52 binding is abolished following administration of the type I necrotizing agent butylated hydroxytoluene (BHT). Electronraicrographs of DLC isolated by means of fluorescence-activated cell sorting on the basis of MoAb 411-52 binding confirmed that MoAb 411-52 binds to murine type I pneumocytes. This MoAb was used to monitor the destruction and regeneration of type I cells following BHT- and bleomycin-induced lung damage.

These monoclonal antibodies will be useful as cell specific probes to detect primary lung damage and may aid in the study of mechanisms of toxic lung injury.

Recommended Citation

Hotchkiss, Jon Allison, "Development of cell-specific rat monoclonal antibodies and their use as probes to study mechanisms of toxic lung injury in the Bald/c mouse. " PhD diss., University of Tennessee, 1986.
https://trace.tennessee.edu/utk_graddiss/12267