Design of recombinant antibody microarrays for serum protein profiling: targeting of complement proteins.
Antibody-based microarrays is a novel technology with great promise for high-throughput proteomics. The process of designing high-performing arrays has, however, turned out to be challenging. Here, we have designed the next generation of a human recombinant scFv antibody microarray platform for protein expression profiling of nonfractionated biotinylated human plasma and serum proteomes. The setup, based on black polymer Maxisorb slides interfaced with a fluorescent-based read-out system, was found to provide specific, sensitive (subpicomolar (pM) range) and reproducible means for protein profiling. Further, a chip-to-chip normalization protocol critical for comparing data generated on different chips was devised. Finally, the microarray data were found to correlate well with clinical laboratory data obtained using conventional methods, as demonstrated for a set of medium abundant (micromolar (microM) to nanomolar (nM) range) protein analytes in serum and plasma samples derived from healthy and complement-deficient individuals.
Ingvarsson
Johan
J
Department of Immunotechnology, BMC D13, Lund University, SE-221 84 Lund, Sweden.
Larsson
Anette
A
Sjöholm
Anders G
AG
Truedsson
Lennart
L
Jansson
Bo
B
Borrebaeck
Carl A K
CA
Wingren
Christer
C
D019921:Biotinylation; D001798:Blood Proteins / Q000737:chemistry; D003165:Complement System Proteins / Q000737:chemistry; D006801:Humans; D008956:Models, Chemical; D040081:Protein Array Analysis / Q000379:methods*; D021381:Protein Transport; D011506:Proteins / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001798:Blood Proteins; D011506:Proteins; D020543:Proteome; D011994:Recombinant Proteins; D003165:Complement System Proteins