Immunofluorescence microscope assay of neutrophils and somatic cells in bovine milk

Abstract: Polyclonal antibody specific for bovine neutrophils was prepared. Serum titer was observed at 75th and 105th day of injection period. The first titer was 1250-fold serum dilution and the second – 31,250-fold serum dilution. Antibody was purified with affinity chromatography. Antibody reactivity to neutrophil fragments, Ficoll-separated bovine blood cells – granulocytes (neutrophils, basophils and eosinophils) and mononuclear cells, and bovine milk somatic cells (SC) were studied. The antibody has detection limit 5μg/mL neutrophil fragments and reacts only with neutrophils. The antibody was conjugated to fluorescein isothiocyanate (FITC). The specificity of the fluorescent conjugate was analyzed with Ficoll-separated blood cells and milk SC by fluorescence microscopic analysis. The microscope images prove the conjugate reactivity with blood and milk neutrophils. It was found that when SC count (SCC) was 100,000 cells/mL neutrophils were 30%, but when SCC exceeded 800,000 cells/mL neutrophils were 88%. The proposed antibody–FITC method was compared with commercial Giemsa method.

Keywords: Bovine milk; somatic cell count; neutrophil; FITC; conjugate; ELISA;