Enzyme-linked immunosorbent assay and flow cytometric methods to screen hybridoma culture supernatants for antibodies to bovine neutrophil surface antigens, and monoclonal antibody production and characterization

Shashikumar K. Salgar From the Department of Animal Science, University of Maryland, College Park, MD 20742 (Salgar); Milk Secretion and Mastitis laboratory, USDA-ARS, Beltsville, MD 20705 (Paape); and Department of Animal Science, North Carolina State University, Raleigh, NC 27695 (Alston-Mills).

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Max J. Paape From the Department of Animal Science, University of Maryland, College Park, MD 20742 (Salgar); Milk Secretion and Mastitis laboratory, USDA-ARS, Beltsville, MD 20705 (Paape); and Department of Animal Science, North Carolina State University, Raleigh, NC 27695 (Alston-Mills).

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Brenda Alston-Mills From the Department of Animal Science, University of Maryland, College Park, MD 20742 (Salgar); Milk Secretion and Mastitis laboratory, USDA-ARS, Beltsville, MD 20705 (Paape); and Department of Animal Science, North Carolina State University, Raleigh, NC 27695 (Alston-Mills).

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Summary

Enzyme-linked immunosorbent assay and flow cytometric methods to screen hybridoma culture supernatants for antibodies to bovine neutrophils (surrace antigen-specific) were optimized. Sensitivity of the 2 methods was compared. A panel of 14 murine monoclonal antibodies (mab) to surface antigens of bovine polymorphonuclear neutrophilic leukocytes (neutrophils) was produced by hybridoma technology, and their isotypes were determined by wholecell elisa. Monoclonal antibody reactivity with neutrophils, eosinophils, and lymphocytes isolated on phosphate-buffered saline solution and on Ficoll-sodium diatrizoate were compared. Biochemical characterization of antigens recognized by mab was performed by immunoblot analysis. Neutrophil plasma membranes were isolated on sucrose gradients (20, 32, and 50%) and purified for polypeptide characterization. Neutrophil surface proteins were characterized by external labeling with 125I.

The flow cytometric method was proven to be more sensitive and rapid than elisa to screen hybridoma supernatants. This method allowed light-scatter gating of live neutrophil populations for analysis, which eliminated nonspecific binding of antibodies to contaminating cells and dead neutrophils. The optimal conditions for flow cytometric analyses were 5 × 105 neutrophils and 1 µg of fluorescein-labeled F(ab′)2/assay as the second antibody. The optimal conditions for hybridoma screening by elisa were neutrophil concentration of 2.5 × 105/well, using a 96-well polystyrene microtitration plate as solid support, and 2,2′-azino-di[3-ethyl-benzthiazoline sulfonate (6)] with H2O2 as the chromogenic substrate. Tissue culture plates as solid support and 3,3′, 5,5′- tetramethyl benzidine, with H2O2 as the chromogenic substrate, were equally as sensitive.

Panel mab reacted differently with neutrophils, eosinophils, and lymphocytes. Isolation of these cells from blood on Ficoll-sodium diatrizoate generally did not alter mab reactivity.

Coomassie blue-stained gels of neutrophil plasma membrane proteins contained about 25 polypeptide bands, 13 of which were major bands. Autoradiography revealed about 11 surface proteins, 5 of which were heavily labeled with Monoclonal antibody S7G8 identified a 65-kd protein and mab S8G10 identified 65- and 70-kd proteins. On the basis of molecular weight, mab S7G8 and S8G10 are comparable to human CD15, CDl6, and CD64 molecules. The mab generated in this study are potential candidates to discern bovine neutrophil function and heterogeneity.

Summary

Enzyme-linked immunosorbent assay and flow cytometric methods to screen hybridoma culture supernatants for antibodies to bovine neutrophils (surrace antigen-specific) were optimized. Sensitivity of the 2 methods was compared. A panel of 14 murine monoclonal antibodies (mab) to surface antigens of bovine polymorphonuclear neutrophilic leukocytes (neutrophils) was produced by hybridoma technology, and their isotypes were determined by wholecell elisa. Monoclonal antibody reactivity with neutrophils, eosinophils, and lymphocytes isolated on phosphate-buffered saline solution and on Ficoll-sodium diatrizoate were compared. Biochemical characterization of antigens recognized by mab was performed by immunoblot analysis. Neutrophil plasma membranes were isolated on sucrose gradients (20, 32, and 50%) and purified for polypeptide characterization. Neutrophil surface proteins were characterized by external labeling with 125I.

The flow cytometric method was proven to be more sensitive and rapid than elisa to screen hybridoma supernatants. This method allowed light-scatter gating of live neutrophil populations for analysis, which eliminated nonspecific binding of antibodies to contaminating cells and dead neutrophils. The optimal conditions for flow cytometric analyses were 5 × 105 neutrophils and 1 µg of fluorescein-labeled F(ab′)2/assay as the second antibody. The optimal conditions for hybridoma screening by elisa were neutrophil concentration of 2.5 × 105/well, using a 96-well polystyrene microtitration plate as solid support, and 2,2′-azino-di[3-ethyl-benzthiazoline sulfonate (6)] with H2O2 as the chromogenic substrate. Tissue culture plates as solid support and 3,3′, 5,5′- tetramethyl benzidine, with H2O2 as the chromogenic substrate, were equally as sensitive.

Panel mab reacted differently with neutrophils, eosinophils, and lymphocytes. Isolation of these cells from blood on Ficoll-sodium diatrizoate generally did not alter mab reactivity.

Coomassie blue-stained gels of neutrophil plasma membrane proteins contained about 25 polypeptide bands, 13 of which were major bands. Autoradiography revealed about 11 surface proteins, 5 of which were heavily labeled with Monoclonal antibody S7G8 identified a 65-kd protein and mab S8G10 identified 65- and 70-kd proteins. On the basis of molecular weight, mab S7G8 and S8G10 are comparable to human CD15, CDl6, and CD64 molecules. The mab generated in this study are potential candidates to discern bovine neutrophil function and heterogeneity.

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