Miniaturized, Microarray-Based Assays for Chemical Proteomic Studies of Protein Function

Blackburn, Jonathan M.; Shoko, Aubrey; Beeton-Kempen, Natasha

doi:10.1007/978-1-61779-349-3_10

Cited by 15 publications

(22 citation statements)

References 39 publications

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“…The full-length SARS-CoV-2 nucleocapsid (N) gene was synthesized (GeneArt, Regensburg, Germany) and cloned into a proprietary Escherichia coli/ Spodoptera frugiperda transfer vector, pPRO8, such that the construct encoded the full-length N protein as an in-frame fusion to a C-terminal Biotin Carboxyl Carrier Protein (BCCP) and c-Myc tag. pPRO8 is a derivative of pTriEx1.1 (Sigma, St Louis, MO, USA) and encodes the E. coli BCCP domain (amino acids 74–156 of the E. coli accB gene) downstream of a viral polyhedrin promoter and cloning sites; flanking this polh -BCCP expression cassette are the baculoviral 603 gene and the 1629 genes to enable subsequent homologous recombination of the construct into a replication-deficient baculoviral genome [ 17 ].…”

Section: Methodsmentioning

confidence: 99%

“…Following co-transfection of S. frugiperda Sf9 cells with a relevant pPRO8-derived transfer vector plus a linearized, replication deficient bacmid vector ( Autographa californica baculovirus vector pBAC10:KO 1629 [ 17 ]), baculovirus was amplified and recombinant proteins were expressed in S. frugiperda superSf9–3 strain (Oxford Expression Technologies, Oxford, UK) using previously published protocols [ 17 ]. Clarified cell lysates were prepared in insect lysis buffer (25 mM Hepes, 50 mM KCL, 20% glycerol, 0.1% Triton × 100, 1 × Halt™ Protease Inhibitor Cocktail, EDTA-free (Thermo Scientific, Waltham, MA, USA), 0.25% sodium deoxycholate acid, 25 U/mL Pierce Universal nuclease (Thermo Scientific), pH 8).…”

Section: Methodsmentioning

confidence: 99%

“…The array is based on the use of the biotin carboxyl carrier protein (BCCP), which acts as a marker for the correct folding of proteins, since only correctly folded proteins will be biotinylated. Therefore, it is possible to control the immobilization of antigens onto a streptavidin coated surface in an oriented manner [ 17 ]. Different prototype array designs, using various engineered SARS-CoV-2 nucleocapsid protein structural motifs, were tested on a cross-sectional convalescent COVID-19 cohort and pre-pandemic controls to determine cross-reactivity.…”

Section: Introductionmentioning

confidence: 99%

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Age, Disease Severity and Ethnicity Influence Humoral Responses in a Multi-Ethnic COVID-19 Cohort

Smith

Abdesselem

Mullins

et al. 2021

Viruses

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The COVID-19 pandemic has affected all individuals across the globe in some way. Despite large numbers of reported seroprevalence studies, there remains a limited understanding of how the magnitude and epitope utilization of the humoral immune response to SARS-CoV-2 viral anti-gens varies within populations following natural infection. Here, we designed a quantitative, multi-epitope protein microarray comprising various nucleocapsid protein structural motifs, including two structural domains and three intrinsically disordered regions. Quantitative data from the microarray provided complete differentiation between cases and pre-pandemic controls (100% sensitivity and specificity) in a case-control cohort (n = 100). We then assessed the influence of disease severity, age, and ethnicity on the strength and breadth of the humoral response in a multi-ethnic cohort (n = 138). As expected, patients with severe disease showed significantly higher antibody titers and interestingly also had significantly broader epitope coverage. A significant increase in antibody titer and epitope coverage was observed with increasing age, in both mild and severe disease, which is promising for vaccine efficacy in older individuals. Additionally, we observed significant differences in the breadth and strength of the humoral immune response in relation to ethnicity, which may reflect differences in genetic and lifestyle factors. Furthermore, our data enabled localization of the immuno-dominant epitope to the C-terminal structural domain of the viral nucleocapsid protein in two independent cohorts. Overall, we have designed, validated, and tested an advanced serological assay that enables accurate quantitation of the humoral response post natural infection and that has revealed unexpected differences in the magnitude and epitope utilization within a population.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Age, Disease Severity and Ethnicity Influence Humoral Responses in a Multi-Ethnic COVID-19 Cohort

Smith

Abdesselem

Mullins

et al. 2021

Viruses

Self Cite

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“…Each CT100 array was printed on a streptavidin‐coated microarray slide using a QArray2 robotic arrayer (Genetix, Berkshire, UK)) equipped with 8 × 300 μm flat‐tipped solid pins. In‐house streptavidin‐coated array surfaces were prepared essentially according to a previously published method (see Supporting Information, Section S2 for details). Each array consisted of eight sub‐arrays, with each sub‐array printed by a different pin.…”

Section: Methodsmentioning

confidence: 99%

Development of a novel, quantitative protein microarray platform for the multiplexed serological analysis of autoantibodies to cancer-testis antigens

et al. 2014

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The cancer-testis antigens are a group of unrelated proteins aberrantly expressed in various cancers in adult somatic tissues. This aberrant expression can trigger spontaneous immune responses, a phenomenon exploited for the development of disease markers and therapeutic vaccines. However, expression levels often vary amongst patients presenting the same cancer type, and these antigens are therefore unlikely to be individually viable as diagnostic or prognostic markers. Nevertheless, patterns of antigen expression may provide correlates of specific cancer types and disease progression. Herein, we describe the development of a novel, readily customizable cancer-testis antigen microarray platform together with robust bioinformatics tools, with which to quantify anti-cancer testis antigen autoantibody profiles in patient sera. By exploiting the high affinity between autoantibodies and tumor antigens, we achieved linearity of response and an autoantibody quantitation limit in the pg/mL range-equating to a million-fold serum dilution. By using oriented attachment of folded, recombinant antigens and a polyethylene glycol microarray surface coating, we attained minimal non-specific antibody binding. Unlike other proteomics methods, which typically use lower affinity interactions between monoclonal antibodies and tumor antigens for detection, the high sensitivity and specificity realized using our autoantibody-based approach may facilitate the development of better cancer biomarkers, as well as potentially enabling pre-symptomatic diagnosis. We illustrated the usage of our platform by monitoring the response of a melanoma patient cohort to an experimental therapeutic NY-ESO-1-based cancer vaccine; inter alia, we found evidence of determinant spreading in individual patients, as well as differential CT antigen expression and epitope usage.

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“…1−4 These techniques have been widely applied by miniaturization of conventionally analytical devices. 5 Antibody-based assays, 6 for example ELISA (enzyme-linked immunosorbent assay), are especially useful for detection and quantification of antigens. 7 High sensitivity is important for fluorescence-based high throughput screening of antigens or antibodies on a biosensor.…”

Section: Introductionmentioning

confidence: 99%

Sandwich Antibody Arrays Using Recombinant Antibody-Binding Protein L

Seo

Poulter

2014

Langmuir

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Antibody arrays are a useful for detecting antigens and other antibodies. This technique typically requires a uniform and well-defined orientation of antibodies attached to a surface for optimal performance. A uniform orientation can be achieved by modification of antibodies to include a single site for attachment. Thus, uniformly oriented antibody arrays require a bioengineered modification for the antibodies directly immobilization on the solid surface. In this study, we describe a “sandwich-type” antibody array where unmodified antibodies are oriented through binding with regioselectively immobilized recombinant antibody-binding protein L. Recombinant proL-CVIA bearing C-terminal CVIA motif is post-translationally modified with an alkyne group by protein farnesyltransferase (PFTase) at the cysteine residue in the CVIA sequence to give proL-CVIApf, which is covalently attached to an azido-modified glass slide by a Huisgen [3 + 2] cycloaddition reaction. Slides bearing antibodies bound to slides coated with regioselectively immobilized proL-CVIApf gave stronger fluorescence outputs and those where the antibody-binding protein was immobilized in random orientations on an epoxy-modified slide. Properly selected capture and detection antibodies did not cross-react with immobilized proL-CVIApf in sandwich arrays, and the proL-CVIApf slides can be used for multiple cycles of detected over a period of several months.

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Miniaturized, Microarray-Based Assays for Chemical Proteomic Studies of Protein Function

Cited by 15 publications

References 39 publications

Age, Disease Severity and Ethnicity Influence Humoral Responses in a Multi-Ethnic COVID-19 Cohort

Age, Disease Severity and Ethnicity Influence Humoral Responses in a Multi-Ethnic COVID-19 Cohort

Development of a novel, quantitative protein microarray platform for the multiplexed serological analysis of autoantibodies to cancer-testis antigens

Sandwich Antibody Arrays Using Recombinant Antibody-Binding Protein L

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