Design of recombinant antibody microarrays for complex proteome analysis: choice of sample labeling-tag and solid support.
Antibody-based microarray is a novel technology with great potential within high-throughput proteomics. The process of designing high-performing antibody (protein) microarrays has, however, turned out to be a challenging process. In this study, we have developed further our human recombinant single-chain variable-fragment (scFv) antibody microarray methodology by addressing two crucial technological issues, choice of sample labeling-tag and solid support. We examined the performance of a range of dyes in a one- or two-color approach on a selection of solid supports providing different surface and coupling chemistries, and surface structures. The set-ups were evaluated in terms of sensitivity, specificity, and selectivity. The results showed that a one-color approach, based on NHS-biotin (or ULS-biotin) labeling, on black polymer Maxisorb slides (or Nexterion slide H) was the superior approach for targeting low-abundant (pg/mL) analytes in nonfractionated, complex proteomes, such as human serum or crude cell supernatants. Notably, microarrays displaying adequate spot morphologies, high S/Ns, minimized nonspecific binding, and most importantly a high selectivity, specificity, and sensitivity (>or=fM range) were obtained. Taken together, we have designed the first generation of a high-performing recombinant scFv antibody microarray technology platform on black polymer Maxisorb slides for sensitive profiling of low-abundant analytes in nonfractionated biotinylated complex proteomes.
Wingren
Christer
C
Department of Immunotechnology, BMC D13, Lund University, Lund, Sweden. christer.wingren@immun.ith.se
Ingvarsson
Johan
J
Dexlin
Linda
L
Szul
Dominika
D
Borrebaeck
Carl A K
CA
D019921:Biotinylation; D002232:Carbocyanines / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D020543:Proteome / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D012680:Sensitivity and Specificity