Design of recombinant antibody microarrays for complex proteome analysis: Choice of sample labeling‐tag and solid support

Abstract

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.

Identification of Serum Biomarker Signatures Associated with Pancreatic Cancer

“…3,11,13,14). Motivated by a recent study, where we indicated that affinity proteomics (15,16) could be used to pinpoint candidate PDAC serum biomarker signatures (17), the current study was undertaken to further analyze the serum proteome of pancreatic diseases. In the present study, we have for the first time prevalidated a serum biomarker signature for PDAC diagnosis, discriminating it from inflammatory states of the pancreas.…”

Molecular serum portraits in patients with primary breast cancer predict the development of distant metastases

“…We hypothesized that decoding patterns of immunoregulatory serum proteins could reveal important information about the risk of recurrence. Consequently, using minute amounts of nonfractionated serum (14) and a recombinant antibody microarray technology capable of analyzing large numbers of low-and high-abundance protein analytes, we screened samples from breast cancer patients collected over a 3-y period. The samples were collected from breast cancer patients before resection of the primary tumor and then postoperatively every 6-12 mo, resulting in three to five samples per patient.…”

Generation of miniaturized planar ecombinant antibody arrays using a microcantilever-based printer

“…The results showed that the CV values varied in a needle-, antibody-, and/or analyte-dependent manner, and were in the range of 1-36% (mean CV 17%) (serum diluted 450 times) and 15-50% (mean CV 31%) (serum diluted 1125 times). It should be noted that the preferred serum dilution is 450 times [41,44], for which acceptable mean CV value was observed, especially considering that this is the 1st generation of array platform produced with the Bioplume TM microdispenser. (A) the 10 scFv antibodies were printed at (i) 0.5 mg ml −1 (560 attomole) (6 antibodies) or at the maximum production concentration at hand, 0.38 mg ml −1 and 0.14 mg ml −1 (160 attomole), and/or (ii) titrated.…”

“…The specificity of these antibodies has previously been validated by using pure analytes; mixtures of pure analytes; well-characterized, standardized crude serum; and orthogonal methods, such as mass spectrometry (serum/tissue extract pull-down assays), ELISA, MesoScaleDiscovery (MSD) assay, immunohistochemistry, and/or cytometric bead assay; as well as by spiking and blocking experiments in crude sample formats (e.g. serum) [9,[40][41][42][43][44][45][46][47].…”