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Fractionation of the human plasma proteome for monoclonal antibody proteomics‐based biomarker discovery 2: Antigen identification by dot‐blot array screening
Author(s) -
Kovács András,
Patai Zoltán,
Guttman András,
Kádas János,
Takács László,
Kurucz István
Publication year - 2013
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201200677
Subject(s) - proteome , monoclonal antibody , antigen , epitope , biomarker discovery , proteomics , blood proteins , western blot , human proteome project , antibody , microbiology and biotechnology , chemistry , epitope mapping , computational biology , biology , biochemistry , immunology , gene
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.