The production and characterization of anti-bovine CD14 monoclonal antibodies

Eun J. Sohn; Max J. Paape; Robert R. Petersa; Raymond H. Fetterer; Neil C. Talbot; Douglas D. Bannerman

doi:doi:10.1051/vetres:2004035

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Volume 35 / No 5 (September-October 2004)

Vet. Res., 35 5 (2004) 597-608

Abstract

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Issue		Vet. Res. Volume 35, Number 5, September-October 2004


Page(s)		597 - 608
DOI		https://doi.org/10.1051/vetres:2004035
How to cite this article		Vet. Res. (2004) 597-608

Vet. Res. 35 (2004) 597-608
DOI: 10.1051/vetres:2004035

The production and characterization of anti-bovine CD14 monoclonal antibodies

Eun J. Sohn^a, Max J. Paape^b, Robert R. Petersa^a, Raymond H. Fetterer^c, Neil C. Talbot^d and Douglas D. Bannerman^b

^a Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA
^b Bovine Functional Genomics Laboratory, USDA-ARS, Beltsville, MD 20705, USA
^c Animal Parasitic Diseases Laboratory, USDA-ARS, Beltsville, MD 20705, USA
^d Biotechnology and Germplasm Laboratory, USDA-ARS, Beltsville, MD 20705, USA

(Received 12 January 2004; accepted 13 April 2004)

Abstract - To characterize further the chemical and biological properties of bovine soluble (bos) CD14, a panel of ten murine monoclonal antibodies (mAb) reactive with recombinant (r) bosCD14 were produced. A sandwich ELISA, using murine mAb and rabbit polyclonal antibodies reactive with rbosCD14 was developed. All the mAb were reactive by ELISA with baculovirus-derived rbosCD14 and they recognized rbosCD14 (40 kDa) by western blot analysis. The mAb also identified by western blot sCD14 (53 and 58 kDa) in milk and blood and sCD14 (47 kDa) in a lysate of macrophages obtained from involuted bovine mammary gland secretions. Analysis by ELISA of whey samples after intramammary injection of lipopolysaccharide (LPS) (10 µg) revealed increased sCD14 levels between 8 to 48 h after injection. Flow cytometric analysis showed that the mAb bound to macrophages isolated from involuted mammary gland secretions and mouse macrophages but not to swine or horse monocytes. Addition of anti-rbosCD14 mAb to monocytes stimulated with LPS reduced in vitro production of TNF- $\alpha$ . The anti-rbosCD14 antibodies generated in this study will be useful in studying CD14, an accessory molecule that contributes to host innate recognition of bacterial cell wall components in mammary secretions produced during mastitis.

Key words: CD14 / monoclonal antibodies / ELISA / LPS / mastitis

Corresponding author: Max J. Paape mpaape@anri.barc.usda.gov

© INRA, EDP Sciences 2004