SARS-CoV-2 HuProt™: The Novel Coronavirus Microarray

There have been other protein microarrays, but none were made from a protein library as comprehensive or thoroughly validated as HuProt. HuProt arrays allow for pan-isotype autoantibody biomarker profiling versus properly folded, three-dimensional human proteins. Now with SARS-CoV-2 proteins.

SARS-CoV-2 HuProt™: The Novel Coronavirus Microarray

There have been other protein microarrays, but none were made from a protein library as comprehensive or thoroughly validated as HuProt. HuProt arrays allow for pan-isotype autoantibody biomarker profiling versus properly folded, three-dimensional human proteins. Now with SARS-CoV-2 proteins.

The world’s largest human protein library. Now with SARS-CoV-2.

All of our futures now depend on the human body’s ability to make antibodies to SARS-CoV-2, the virus that causes pandemic COVID-19 disease. And while it is now clear that recovered patients make protective antibodies against the disease – surprisingly it is the sickest patients making the most anti-SARS-CoV-2 IgG antibodies.

The question is…why?

To help researchers answer this urgent question CDI Labs has created SARS-CoV-2 HuProt™: The Human Proteome Microarray with SARS-CoV-2 proteins.  SARS-CoV-2 HuProt™ contains >21,000 full-length human proteins and is the most powerful tool ever created for mapping antigen-specific autoimmunity.  SARS-CoV-2 HuProt™ microarrays are available for direct purchase or an all-inclusive service. HuProt™ enables multi-isotype serum profiling of antibodies against three-dimensional antigens with a quantitative readout. It’s also useful for testing monoclonal antibodies, proteins, DNA, RNA, and even some small molecules.  With SARS-CoV-2 HuProt™, researchers can finally discover new antigen-specific immune responses that accompany different features of SARS-CoV-2 disease progression, clinical trial treatment response, and vaccine development.

Most fluids contain antibodies and we can analyze them (i.e. peripheral serum/plasma, cerebrospinal fluid, tissue lysates, stool, saliva), or in a synthesized sample, for any isotype (i.e. IgM, IgG, IgA, IgD, IgE). Secondary antibody binding is then quantified using fluorescence intensity measurements of features on the array. We’ll provide you with those RAW readouts – and optionally – normalized readouts and group-comparison statistics.

The two SARS-CoV-2 proteins on these arrays are:

SARS-CoV-2 S1 Receptor-Binding Domain: This S1 ACE2 receptor binding spike is essential for entry of the novel coronavirus into human cells. Our S1 matches the sequence published here, is expressed in human cells & has confirmed binding to ACE2.

SARS-CoV-2 Nucleocapsid: The nucleocapsid is the most abundant protein of a coronavirus. This version is purified from E.coli.

Download the protocols followed by this SARS-CoV-2 service.

CDI Antygen™ HuProt™ arrays can be purchased directly or provided as a complete analysis service.

HuProt™ v4.0 Protein Microarray Technical Manual

HuProt™ technical details.

CDI Labs’ HuProt™ Human Proteome Microarray is the most comprehensive human full-length protein array ever created (Jeong et al, 2012). Our latest version, HuProt™v4.0, contains over 21,000 human proteins and protein isoforms. This covers over 81% of human proteins in each major functional category of the proteome, as defined by the Human Protein Atlas (Venkataraman et al, 2018), and allows hundreds of interactions to be profiled at once in a wide range of applications, including mapping antigen-specific immunity (multi-isotype serum profiling), determining monoclonal antibody specificity, and to study protein-protein interaction, substrate identification, protein-DNA binding, protein-RNA binding, and binding of some small molecules.

The HuProt™ expression library was created by inserting full-length human open reading frames (ORFs) inserted into a yeast high-copy expression vector that produces GST-His6 fusion proteins when induced in yeast (Hu S et al 2009; Jeong J et al 2012).  Each human ORF expression vector was verified for integrity by Sanger sequencing (Venkataraman et al, 2018).  These sequence-verified expression vectors encoding human ORFs were transformed into yeast to create our master library; this eukaryotic expression system ensures HuProt™ proteins maintain both function and proper conformation (Hu S, poster).

As required about once per year this yeast library is used to synthesize human proteins in high-throughput. Full-length synthesized proteins are purified via their GST-His6 fusion tags as previously described (Hu S et al, 2009).  These proteins are then frozen in aliquots in a 384-well format for later use in printing on HuProt™ microarrays.

To print individual batches of HuProt™ arrays, the purified human proteins are then printed as duplicate spot pairs using an Arrayjet UltraMarathon printer (Arrayjet, UK) on PATH nitrocellulose slides (GraceBio, USA) .  Controls on HuProt™ include titrated GST protein, titrated human IgG, other human antibody isotypes, histones, mouse and rabbit anti-biotin, mouse IgM, and biotin-tagged control for streptavidin detection.  HuProt™ also contains landmark control spots, including Alex Fluor 555/647 (Jeong et al, 2012). Success of each batch of HuProt™ microarrays is validated by anti-GST staining. This confirms full-length GST-His6 fusion proteins were successfully expressed, synthesized, purified, and printed across our clone collection.

HuProt™ service details & data deliverables.

A typical HuProt™ service involves case and control serum samples diluted 1:1000 and reacted with individual microarrays with BSA blocking. After incubation, samples are stained for one or two desired fluorescent secondary detection antibodies (ie IgG / IgA / IgM). Arrays are scanned and raw TIF images are created using Molecular Devices Genepix microarray scanners. Spots are then aligned to CDI library (.gal) files, and spot readings are output as raw Genepix results (.gpr) files. These raw results are then delivered both as .gpr files and combined data tables. If the samples are blinded to CDI, this is the final output. If sample blinding is not a requirement, CDI can additionally prepare basic quantile normalized case / control group statistics with ranked p-values from Student’s t-tests, Wilcoxon signed-rank tests, and other statistical outputs.

HuProt™ service sample requirements.

Human serum or plasma: Send two 10μL aliquots per sample.

Antibody isolates: At least 3μg per sample (conc. >0.1mg / mL).

Human (CSF) cerebrospinal fluid: At least 1.5mL per sample.

Protein / peptide / small molecule: At least 3μg per sample.

DNA / RNA: At least 300μL per sample (conc. >2μM).

Other fluids: Please contact us.

Cohort balancing (studies >48 samples):  Individual samples are run on individual HuProt slides; these are produced in batches of 600 and our lab team typically processes x48 samples per day.  While data from multiple days and print batches is easily pooled by our informatics pipeline, customers running larger studies may wish to consider balancing case/control and replicate samples within and across both processing days and print batches.

Additional information.

To add custom controls to HuProt™: We can print up to 10 custom controls per order for $500. Please send at least 10 μL of each antigen (conc. >0.2 mg /mL) in a buffer WITHOUT detergent.

Sample shipping & sample return.

We will include shipping details in your quote – you must cover the cost of shipping samples to CDI. Typically, after you receive your report, CDI keeps the remaining samples for two months and then disposes of them. When shipping to us, please let us know if you want the remaining samples returned after the study is complete. We will charge for return shipping.

Contact us to learn more.

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