title: Characterization of monoclonal antibodies against human lactoferrin.
abstract: The iron-binding glycoprotein human lactoferrin (hLF) is involved in the host defense against infection and is a modulator of inflammatory reactions. We generated monoclonal antibodies (mAbs) to hLF as tools to assist both structure-function studies and the development of recombinant human lactoferrin for applications in human health care. Binding experiments with ten distinct anti-hLF mAbs to tryptic and recombinant hLF fragments in ELISA and/or on immunoblots revealed that five mAbs bound to conformational epitopes residing in the N-lobe (residues 1 to 334), whereas the other five bound to C-lobe conformational epitopes (residues 335 to 692). None of the mAbs bound to hLF denatured upon reduction. Monoclonal antibody E11 appeared to bind to the arginine-rich N-terminus of hLF, which is the binding site for heparin, bacterial lipopolysaccharide, human lysozyme, DNA and receptors. The dissociation constant of the distinct mAbs for hLF ranged from 0.5 to 18 nM, without differences in affinity for unsaturated or iron-saturated hLF, indicating that the conformational changes subject to incorporation of iron do not seem to affect the exposure and/or conformation of the antibody epitopes. The mAbs did not bind to human transferrin, a protein closely related to hLF in size, primary amino acid sequence and structure. Two C-lobe specific mAbs, E2 and E8, cross-reacted with bovine and/or porcine lactoferrin, indicating that human, bovine and porcine lactoferrin share antigenic determinants. This panel of mAbs will be used to develop quantitative and qualitative immunoassays for hLF and to delineate which regions of hLF are relevant to its anti-infective and anti-inflammatory properties.
authors.affiliation: Pharming, Archimedesweg 4, 2333 CN, PO Box 451, 2300 AL Leiden, The Netherlands.
authors.forename: Patrick H C
authors.initials: PH
authors.lastname: van Berkel
authors.forename: Harrie A
authors.initials: HA
authors.lastname: van Veen
authors.forename: Marlieke E J
authors.initials: ME
authors.lastname: Geerts
authors.forename: Jan H
authors.initials: JH
authors.lastname: Nuijens
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000918:Antibody Specificity; D001665:Binding Sites; D002417:Cattle; D003429:Cross Reactions; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D007781:Lactoferrin / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D013045:Species Specificity; D013552:Swine; D014357:Trypsin
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007781:Lactoferrin; D014357:Trypsin