Highly Multiplexed Antibody Suspension Bead Arrays for Plasma Protein Profiling

Drobin, Kimi; Nilsson, Peter; Schwenk, Jochen M.

doi:10.1007/978-1-4614-7209-4_8

Cited by 60 publications

(69 citation statements)

References 9 publications

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“…The bead arrays were created as previously described 42, 43 by diluting 1.6 μg of each antibody into 100 μL of antibody dilution buffer. All antibodies were immobilized onto color-coded magnetic beads (MagPlex-C, Luminex Corp.) with each bead identity corresponding to a unique antibody.…”

Section: Methodsmentioning

confidence: 99%

Discovery of circulating proteins associated to knee radiographic osteoarthritis

Lourido

Ayoglu

Fernández‐Tajes

et al. 2017

Sci Rep

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Currently there are no sufficiently sensitive biomarkers able to reflect changes in joint remodelling during osteoarthritis (OA). In this work, we took an affinity proteomic approach to profile serum samples for proteins that could serve as indicators for the diagnosis of radiographic knee OA. Antibody suspension bead arrays were applied to analyze serum samples from patients with OA (n = 273), control subjects (n = 76) and patients with rheumatoid arthritis (RA, n = 244). For verification, a focused bead array was built and applied to an independent set of serum samples from patients with OA (n = 188), control individuals (n = 83) and RA (n = 168) patients. A linear regression analysis adjusting for sex, age and body mass index (BMI) revealed that three proteins were significantly elevated (P < 0.05) in serum from OA patients compared to controls: C3, ITIH1 and S100A6. A panel consisting of these three proteins had an area under the curve of 0.82 for the classification of OA and control samples. Moreover, C3 and ITIH1 levels were also found to be significantly elevated (P < 0.05) in OA patients compared to RA patients. Upon validation in additional study sets, the alterations of these three candidate serum biomarker proteins could support the diagnosis of radiographic knee OA.

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Section: Methodsmentioning

confidence: 99%

Discovery of circulating proteins associated to knee radiographic osteoarthritis

Lourido

Ayoglu

Fernández‐Tajes

et al. 2017

Sci Rep

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“…Bead arrays were created as previously described . Antibodies were diluted to 17.5 µg mL –1 in 100 µL 0.1 m 2[N‐Morpholino]ethanesulfonic acid (MES)‐buffer (M2933, Sigma–Aldrich), pH 4.5, using a pipetting robot (TECAN EVO150), and then coupled to carboxylated color‐coded magnetic beads (MagPlex‐C, Luminex Corp).…”

Section: Methodsmentioning

confidence: 99%

Systematic Development of Sandwich Immunoassays for the Plasma Secretome

et al. 2019

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The plasma proteome offers a clinically useful window into human health. Recent advances from highly multiplexed assays now call for appropriate pipelines to validate individual candidates. Here, a workflow is developed to build dual binder sandwich immunoassays (SIA) and for proteins predicted to be secreted into plasma. Utilizing suspension bead arrays, ≈1800 unique antibody pairs are first screened against 209 proteins with recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies, dilution‐dependent curves in plasma and concentration‐dependent curves of full‐length proteins for 102 (49%) of the targets are obtained. For 22 protein assays, the longitudinal, interindividual, and technical performance is determined in a set of plasma samples collected from 18 healthy subjects every third month over 1 year. Finally, 14 of these assays are compared with with SIAs composed of other binders, proximity extension assays, and affinity‐free targeted mass spectrometry. The workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, and the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.

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“…Multiplexed antibody suspension bead arrays also contained one bead population processed in protein‐free buffer. For sample analysis, plasma was labeled at a 1:10 dilution in PBS with activated biotin (Pierce), and processed in accordance to previous protocols 13, 14. Liquids were transferred between plates using a liquid handler (CyBi‐SELMA, CyBio), beads were washed using a plate washer (EL406, Biotek), and assay plates (Greiner) incubated at 23°C for 16 h on a plate rotator (Grant).…”

Section: Methodsmentioning

confidence: 99%

Affinity proteomics discovers decreased levels of AMFR in plasma from Osteoporosis patients

Qundos

Drobin

Mattsson

et al. 2015

Proteomics Clinical Apps

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PurposeAffinity proteomic approaches by antibody bead arrays enable multiplexed analysis of proteins in body fluids. In the presented study, we investigated blood plasma within osteoporosis to discovery differential protein profiles and to propose novel biomarkers candidates for subsequent studies.Experimental designStarting with 4608 antibodies and plasma samples from 22 women for an untargeted screening, a set of 72 proteins were suggested for further analysis. Complementing these with targets from literature and other studies, a targeted bead array of 180 antibodies was built to profile for 92 proteins in plasma samples of 180 women from two independent population‐based studies.ResultsDifferential profiles between osteoporosis patients and matched controls were discovered for 12 proteins in at least one of the two study sets. Among these targets, the levels of autocrine motility factor receptor (AMFR) were concordantly lower in plasma of female osteoporosis patients. Subsequently, verification of anti‐AMFR antibody selectivity was conducted using high‐density peptide and protein arrays, and Western blotting.Conclusions and clinical relevanceFurther validation in additional study sets will be needed to determine the clinical value of the observed decrease in AMFR plasma levels in osteoporosis patients, but AMFR may aid our understanding of disease mechanisms and could support existing tools for diagnosis and monitoring of patient mobility within osteoporosis.

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Highly Multiplexed Antibody Suspension Bead Arrays for Plasma Protein Profiling

Cited by 60 publications

References 9 publications

Discovery of circulating proteins associated to knee radiographic osteoarthritis

Discovery of circulating proteins associated to knee radiographic osteoarthritis

Systematic Development of Sandwich Immunoassays for the Plasma Secretome

Affinity proteomics discovers decreased levels of AMFR in plasma from Osteoporosis patients

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