title: Fractionation of the human plasma proteome for monoclonal antibody proteomics-based biomarker discovery 2: antigen identification by dot-blot array screening.
abstract: Immunization with complex mixtures, like the human plasma resulted in the generation of cloned mAb libraries (PlasmaScan™ and QuantiPlasma™ libraries, with >1000 individual mAbs) reacting with a nonredundant set of antigenic epitopes. mAb proteomics refers to quasi-hypothesis-free profiling of plasma samples with nascent or cloned mAb libraries for the discovery of disease-specific biomarkers. Once mAbs with biomarker potential have been identified, the next task is the determination of cognate antigens recognized by the respective mAbs. To determine the cognate protein antigen corresponding to each individual mAbs in the cloned mAb libraries, we have separated human plasma by consecutive steps of desalting and various chromatography procedures. The process resulted in 783 fractions, which we termed "Analyte Library" (AL). The AL represents the human plasma proteome in relatively low-protein complexity fractions. Here, to determine the utility of the AL, we selected ten plasma proteins and checked for their presence in the fractions. Among the ten cases, the distribution of four selected plasma proteins matched expectations, as these proteins were present only in a few fractions corresponding to their physical, chemical, and biochemical properties. However, in six cases, we observed "smear" -like distribution or complete absence of the proteins, suggesting that protein-protein interactions or protein variants may alter the observed plasma distribution profiles. Nevertheless, we conclude that the AL is an efficient, high throughput tool to complement the mAb biomarker discovery process with cognate protein antigen identification for each mAbs.
authors.affiliation: Horváth Laboratory of Bioseparation Sciences, Research Centre for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
authors.forename: András
authors.initials: A
authors.lastname: Kovács
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Patai
authors.forename: András
authors.initials: A
authors.lastname: Guttman
authors.forename: János
authors.initials: J
authors.lastname: Kádas
authors.forename: László
authors.initials: L
authors.lastname: Takács
authors.forename: István
authors.initials: I
authors.lastname: Kurucz
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001798:Blood Proteins / Q000302:isolation & purification*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D040081:Protein Array Analysis / Q000379:methods; D020543:Proteome / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D020543:Proteome
keywords: Analyte Library; Biomarkers; Dot blot; Mass spectrometry; mAb proteomics