title: Production and characterization of monoclonal antibodies (mAbs) against human serum albumin (HSA) for the development of an immunoaffinity system with oriented anti-HSA mAbs as immobilized ligand.
abstract: Proteins present in human serum are of immense importance in the field of biomarker discovery. But, the presence of high-abundant proteins like albumin makes the analysis more challenging because of masking effect on low-abundant proteins. Therefore, removal of albumin using highly specific monoclonal antibodies (mAbs) can potentiate the discovery of low-abundant proteins. In the present study, mAbs against human serum albumin (HSA) were developed and integrated in to an immunoaffinity based system for specific removal of albumin from the serum. Hybridomas were obtained by fusion of Sp2/0 mouse myeloma cells with spleen cells from the mouse immunized with HSA. Five clones (AHSA1-5) producing mAbs specific to HSA were established and characterized by enzyme linked immunosorbent assay (ELISA) and immunoblotting for specificity, sensitivity and affinity in terms of antigen binding. The mAbs were able to bind to both native albumin as well as its glycated isoform. Reactivity of mAbs with different mammalian sera was tested. The affinity constant of the mAbs ranged from 10(8) to 10(9)M(-1). An approach based on oriented immobilization was followed to immobilize purified anti-HSA mAbs on hydrazine activated agarose gel and the dynamic binding capacity of the column was determined.
authors.affiliation: Centre for Bioseparation Technology, VIT University, Vellore-632014, Tamil Nadu, India.
authors.forename: Poonam
authors.initials: P
authors.lastname: Rajak
authors.forename: M A
authors.initials: MA
authors.lastname: Vijayalakshmi
authors.forename: N S
authors.initials: NS
authors.lastname: Jayaprakash
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D012709:Serum Albumin / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D008024:Ligands; D012709:Serum Albumin