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Will a sandwich assay work with a GST 26 antibody/protein?

Will a sandwich assay work with a GST 26 antibody/protein?


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I have a GST 26 kda antibody, and want to use it in a sandwich lateral flow assay. Are there enough epitope regions for both antibodies to bind to the protein?


This is completely dependent on the type of antibody you are using and the epitope specificity of that antibody (or antibodies). Because you haven't given any more detail, it's very hard to give you a definitive answer.

GST is known to dimerise, so even a monoclonal antibody might work in your sandwich assay, as long as the (identical) epitopes are far enough apart in the dimeric structure to allow two antibodies to bind.

If you're dealing with a polyclonal antibody mixture, then your chances are better. But even still, you won't know until you try it.


A Simple Test Tube-Based ELISA Experiment for the High-School Classroom *

Immunology is gaining prominence both in the media as well as on the Advanced Placement (AP) exam in Biology. One of the challenges of teaching modern biological topics such as immunology and biochemistry in the high-school setting is the increased reliance on expensive technology in the research world. To begin to bridge this widening gap, we devised an experiment using a novel macroscale enzyme-linked immunosorbent assay that is suitable for AP-level high-school biology classrooms as well as entry-level collegiate laboratories. This novel method does not require a plate reader for data analysis, but instead relies on more common and inexpensive equipment such as a clinical test tube centrifuge and a simple test tube spectrophotometer. The experimental plan focuses on students measuring antibody concentrations in “unknown” samples and includes the collection and analysis of a standard curve using reagents prepared by the instructor. Students will be introduced to enzyme action, quantitative laboratory technique, antibodies, and the immune system, with the overall goal being to explore and highlight the inherent connections within the fields of biochemistry and immunology.

Enzyme-linked immunosorbent assays (ELISA) are biochemical techniques designed to quantitatively detect small amounts of many molecules including proteins, hormones, and antibodies originally developed by Perlmann and coworker in the early 1970s [1]. Although there are some variations in how these are conducted (e.g., direct ELISA vs. sandwich ELISA), ELISA remains a fundamental technique that is extremely common within both clinical [2𠄵] and investigative laboratories [6, 7]. In the clinic, and even at home, ELISA is the basis for a host of molecular diagnostics. At home, these include pregnancy test strips that detect human chorionic gonadotropin and fertility tests strips that detect luteinizing hormone in urine. Within the clinic, ELISA is instrumental in the detection of a host of disease biomarkers. There are hundreds of examples, including prostate-specific antigen (PSA) found in patients with prostate cancer [2, 3], anti-nuclear antibody (ANA) in Lupus patients [4], and glycoprotein 120 (GP120) on the surface of the human immunodeficiency virus in AIDS patients [5]. As such, the ELISA method is omnipresent in the lives of nearly everyone and therefore serves as an outstanding platform for a laboratory exercise at the high-school level.

In a direct ELISA, the target antigen is typically immobilized to a 96-well microtiter plate via a chemical reaction that results in the covalent attachment of the antigen, most commonly a protein, through free amino groups. Detection is then performed in three simple steps, which include probing the coated well with a primary antibody specific for the target antigen, probing the bound antibody using a secondary antibody specific for the constant region of the primary antibody, and then detection using a number of options. The secondary antibody is typically conjugated to either horseradish peroxidase or alkaline phosphatase. These enzymes are then readily detectable using a colorimetric substrate reaction, thus amplifying the signal and providing the technique with its name: enzyme-linked. The color change is then analyzed in a plate reader equipped to detect changes in absorbance as a measure of either target or primary antibody concentration.

ELISA is performed in a plate to facilitate the analysis of large numbers of samples and primary antibody dilutions. Known standards, typically in triplicate, are needed each time the assay is performed to generate a linear absorbance versus antigen or antibody concentration standard curve which is then used to calculate the concentrations of the unknown samples based on their absorbance values. Completion of an ELISA is therefore dependent on a rudimentary understanding of protein–protein binding and specificity, enzyme action, basic chemistry, and the function of antibodies.

One variation in ELISA can be a constant target antigen coated onto the plate, but with unknown antibodies from patient samples. The ANA test serves as an appropriate example in that the purpose is to detect specific antibody in the patient’s blood stream rather than the target of those antibodies [8]. As such, the plate-immobilized antigen remains constant, and the unknown factor becomes the antibody concentration. This arrangement is the basis for the present laboratory exercise.

The most common limitation for high-school classrooms is equipment to carry out modern experimental methods. In the case of ELISA, a plate reader is required, which costs thousands of dollars and is well outside the reach of most high-school budgets. This has lead to useful but less satisfying web-based simulations to illustrate how ELISA is done. In this manuscript, we describe a novel macroscale ELISA that relies solely on a clinical-style test tube centrifuge and a basic single-chamber spectrophotometer capable of reading absorbance at 450 nm. Class II major histocompatibility complex (MHCII) is immobilized to resin particles as a substitute for the typical microtiter ELISA plate, which enables all subsequent incubations and data collection to take place in a glass test tube. Standard and “unknown” dilutions of an anti-MHCII monoclonal antibody (mAb) serves as the experimental sample, and detection was accomplished via standard horseradish peroxidase (HRP)-conjugated secondary antibody. We report results from a high-school Advanced Placement (AP) Biology classroom, which show the effectiveness and forgiveness of the protocol in the hands of inexperienced students. This laboratory exercise successfully integrates fundamental laboratory skills such as centrifugation, micropipette technique, and spectrophotometry with the introduction of key concepts in critical AP Biology curriculum areas such as biochemistry of molecular interactions, antibodies, and enzyme action in the context of the immune response.


Clinical laboratory testing routinely provides actionable results, which help direct patient care in the inpatient and outpatient settings. Since December 2019, a novel coronavirus (SARS-CoV-2) has been causing disease (COVID-19 [coronavirus disease 2019]) in patients, beginning in China and now extending worldwide. In this context of a novel viral pandemic, clinical laboratories have developed multiple novel assays for SARS-CoV-2 diagnosis and for managing patients afflicted with this illness. These include molecular and serologic-based tests, some with point-of-care testing capabilities. Herein, we present an overview of the types of testing available for managing patients with COVID-19, as well as for screening of potential plasma donors who have recovered from COVID-19.

Beginning in December 2019, an outbreak of “pneumonia of unknown cause” was detected in Wuhan City, Hubei Province, China. Ultimately, the 2019 novel coronavirus, or SARS-CoV-2, was identified as the causative agent and subsequently isolated and sequenced. 1 Since that time, SARS-CoV-2 has spread worldwide, causing a severe illness known as COVID-19 (coronavirus disease 2019), which led the World Health Organization (WHO) to declare it a pandemic on March 11, 2020. 2 Since the beginning of the outbreak, clinical laboratories have been developing various assays to aid in detecting SARS-CoV-2 and clinically managing patients with COVID-19.

The 3 categories of tests used to detect current or past viral infection are molecular, serologic, and antigen-detection assays ( Table 1). In this context, a molecular assay is used to determine whether a patient is actively infected with the pathogen of interest. Reverse transcription polymerase chain reaction (RT-PCR) is a common laboratory technique used to detect respiratory viral pathogens, such as influenza and respiratory syncytial virus (RSV). 3 Currently, this is the main type of test being utilized to determine whether patients are infected with SARS-CoV-2.

Clinical Laboratory Testing Types

Test Type . Patient Specimen Type . Detection . Clinical Utility . Necessary Reagents . Development Time .
Nucleic acid amplification (ie, RT-PCR, isothermal amplification) NP swab a RNA Active infection Oligo primers Fastest: oligonucleotide production and molecular assay development (takes days to weeks)
Serology Serum IgM and/or IgG, or total antibodies Past exposure immune status Recombinant/purified protein Intermediate: production of viral protein (recombinant/purified) and assay development/optimization (takes 2 to several weeks)
Protein detection NP swab and/or other clinical fluids a Viral antigen Active infection Antibody to viral protein(s) Slowest: requires antibody production, assay development, and optimization (takes several weeks to months)
Test Type . Patient Specimen Type . Detection . Clinical Utility . Necessary Reagents . Development Time .
Nucleic acid amplification (ie, RT-PCR, isothermal amplification) NP swab a RNA Active infection Oligo primers Fastest: oligonucleotide production and molecular assay development (takes days to weeks)
Serology Serum IgM and/or IgG, or total antibodies Past exposure immune status Recombinant/purified protein Intermediate: production of viral protein (recombinant/purified) and assay development/optimization (takes 2 to several weeks)
Protein detection NP swab and/or other clinical fluids a Viral antigen Active infection Antibody to viral protein(s) Slowest: requires antibody production, assay development, and optimization (takes several weeks to months)

Abbreviations: RT-PCR, reverse transcription polymerase chain reaction NP, nasopharyngeal Ig, immunoglobulin.

a NP swabs are difficult to access using this method.

Clinical Laboratory Testing Types

Test Type . Patient Specimen Type . Detection . Clinical Utility . Necessary Reagents . Development Time .
Nucleic acid amplification (ie, RT-PCR, isothermal amplification) NP swab a RNA Active infection Oligo primers Fastest: oligonucleotide production and molecular assay development (takes days to weeks)
Serology Serum IgM and/or IgG, or total antibodies Past exposure immune status Recombinant/purified protein Intermediate: production of viral protein (recombinant/purified) and assay development/optimization (takes 2 to several weeks)
Protein detection NP swab and/or other clinical fluids a Viral antigen Active infection Antibody to viral protein(s) Slowest: requires antibody production, assay development, and optimization (takes several weeks to months)
Test Type . Patient Specimen Type . Detection . Clinical Utility . Necessary Reagents . Development Time .
Nucleic acid amplification (ie, RT-PCR, isothermal amplification) NP swab a RNA Active infection Oligo primers Fastest: oligonucleotide production and molecular assay development (takes days to weeks)
Serology Serum IgM and/or IgG, or total antibodies Past exposure immune status Recombinant/purified protein Intermediate: production of viral protein (recombinant/purified) and assay development/optimization (takes 2 to several weeks)
Protein detection NP swab and/or other clinical fluids a Viral antigen Active infection Antibody to viral protein(s) Slowest: requires antibody production, assay development, and optimization (takes several weeks to months)

Abbreviations: RT-PCR, reverse transcription polymerase chain reaction NP, nasopharyngeal Ig, immunoglobulin.

a NP swabs are difficult to access using this method.

RT-PCR is a sensitive technique for RNA detection, whereby RNA is reverse transcribed into complementary DNA (cDNA) and cDNA targets specific for the pathogen of interest are amplified. If SARS-CoV-2 RNA is present in a patient specimen, typically collected as a nasopharyngeal (NP) or anterior nasal swab, 4 it will be detected by this assay. Depending on the platform, these assays can be completed in less than 1 hour to several hours, once the specimen arrives in the laboratory and is loaded onto the platform.

The caveats to interpreting results from this assay type are that doing so does not inform us whether a patient previously had the infection rather, this type of assay only detects patients actively shedding virus (current infection or carriage state) or those who have residual viral RNA present. Therefore, these assays are most useful in acute settings to detect patients with COVID-19, where the results can inform appropriate isolation protocols and ensure that appropriate personal protective equipment (PPE) protocols are utilized when treating these patients. As of the date of publication of this article, of the 102 commercial laboratories and/or test kit manufacturers approved for emergency use by the United States Food and Drug Administration (FDA) for SARS-CoV-2 testing, 81 of them were molecular assays ( Table 2). We note that many of these commercial assays require a laboratory to have vendor-specific instrumentation and equipment to utilize these test kits. The FDA also has authorized 37 molecular-based laboratory developed tests (LDTs) that can be used in the single laboratory that developed the test.

Current FDA Emergency Use Authorized SARS-CoV-2 Assays, as of May 26, 2020

Molecular . . .
Manufacturer . Test Name . Assay Type .
1drop Inc. 1copy COVID-19 qPCR Multi Kit RT-PCR
Abbott Diagnostics Scarborough, Inc. ID NOW COVID-19 Isothermal nucleic acid amplification
Abbott Molecular Abbott RealTime SARS-CoV-2 assay RT-PCR
Abbott Molecular Inc. Alinity m SARS-CoV-2 assay RT-PCR
Altona Diagnostics GmbH RealStar SARS-CoV02 RT-PCR Kits U.S. RT-PCR
Applied BioCode, Inc. BioCode SARS-CoV-2 Assay RT-PCR
Applied DNA Sciences, Inc. Linea COVID-19 Assay Kit RT-PCR
Assurance Scientific Laboratories Assurance SARS-CoV-2 Panel RT-PCR
Atila BioSystems, Inc. iAMP COVID-19 Detection Kit Isothermal amplification test
Avellino Lab USA, Inc. AvellinoCoV2 test RT-PCR
Becton, Dickinson & Company BD SARS-CoV-2Reagents for BD MAX System RT-PCR
Becton, Dickinson & Company (BD) BioGX SARS-CoV-2 Reagents for BD MAX System RT-PCR
BGI Genomics Co. Ltd Real-Time Fluorescent RT-PCR Kit for Detecting SARS-2019-nCoV RT-PCR
BioCore Co., Ltd. BioCore 2019-nCoV Real Time PCR Kit RT-PCR
Bio-Rad Laboratories, Inc Bio-Rad SARS-CoV-2 ddPCR Test RT-PCR
BioFire Defense, LLC BioFire COVID-19 Test RT-PCR
BioFire Defense, LLC BioFire Respiratory Panel 2.1 (RP2.1) *panel includes 20 other viral or bacterial pathogens RT-PCR
BioMérieux SA SARS-COV-2 R-GENE RT-PCR
Centers for Disease Control and Prevention’s (CDC) CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel (CDC) RT-PCR
Cepheid Xpert Xpress SARS-CoV-2 test RT-PCR
ChromaCode Inc. HDPCR SARS-CoV-2 Assay RT-PCR
Co-Diagnostics, Inc. Logix Smart Coronavirus Disease 2019 (COVID-19) Kit RT-PCR
Cue Health Inc. Cue COVID-19 Test Isothermal nucleic acid amplification
dba SpectronRx Hymon SARS-CoV-2 Test Kit RT-PCR
DiaCarta, Inc QuantiVirus SARS-CoV-2 Test kit RT-PCR
DiaSorin Molecular LLC Simplexa COVID-19 Direct assay RT-PCR
Everlywell, Inc. Everlywell COVID-19 Test Home Collection Kit RT-PCR
Euroimmun US Inc. EURORealTime SARS-CoV-2 RT-PCR
Fast Track Diagnostics Luxembourg S.á.r.l. FTD SARS-CoV-2 RT-PCR
Fosun Pharma USA Inc. Fosun COVID-19 RT-PCR Detection Kit RT-PCR
Fulgent Therapeutics, LLC Fulgent COVID-19 by RT-PCR Test RT-PCR
GeneMatrix, Inc. NeoPlex COVID-19 Detection Kit RT-PCR
Genetron Health (Beijing) Co., Ltd. Genetron SARS-CoV-2 RNA Test RT-PCR
GenMark Diagnostics, Inc. ePlex SARS-CoV-2 Test PCR, electrochemical detection (voltammetry)
GenoSensor, LLC GS™ COVID-19 RT-PCR KIT RT-PCR
Gnomegen LLC Gnomegen COVID-19 RT-Digital PCR Detection Kit RT-PCR
Gnomegen LLC Gnomegen COVID-19-RT-qPCR Detection Kit RT-PCR
Gravity Diagnostics, LLC Gravity Diagnostics COVID-19 Assay RT-PCR
Hologic, Inc. Aptima SARS-CoV-2 assay Target capture, transcription mediated amplification and dual kinetic assay
Hologic, Inc. Panther Fusion SARS-CoV-2 RT-PCR
Illumina, Inc. Illumina COVIDSeq Test Next-Generation Sequencing (NGS)
InBios International, Inc Smart Detect SARS-CoV-2 rRT-PCR Kit RT-PCR
Ipsum Diagnostics, LLC COV-19 IDx assay RT-PCR
Kaiser Permanente Mid-Atlantic States KPMAS COVID-19 Test RT-PCR
KorvaLabs Inc. Curative-Korva SARS-Cov-2 Assay RT-PCR
LabGenomics Co., Ltd. LabGun COVID-19 RT-PCR Kit RT-PCR
Laboratory Corporation of America (LabCorp) COVID-19 RT-PCR Test RT-PCR
Luminex Corporation ARIES SARS-CoV-2 Assay RT-PCR
Luminex Molecular Diagnostics, Inc. NxTAG CoV Extended Panel Assay RT-PCR
Maccura Biotechnology (USA) LLC SARS-CoV-2 Fluorescent PCR Kit RT-PCR
Mesa Biotech Inc. Accula SARS-Cov-2 Test PCR and lateral flow assay
NeuMoDx Molecular, Inc. NeuMoDx SARS-CoV-2 Assay RT-PCR
OPTI Medical Systems, Inc. OPTI SARS-CoV-2 RT PCR Test RT-PCR
OSANG Healthcare GeneFinder COVID-19 Plus RealAmp Kit RT-PCR
P23 Labs, LLC. P23 Labs TaqPath SARS-CoV-2 Assay RT-PCR
PerkinElmer, Inc. PerkinElmer New Coronavirus Nucleic Acid Detection Kit RT-PCR
Phosphorus Diagnostics LLC Phosphorus COVID-19 RT-qPCR Test RT-PCR
Primerdesign Ltd. Primerdesign Ltd COVID-19 genesig Real-Time PCR assay RT-PCR
PrivaPath Diagnostics, Inc. LetsGetChecked Coronavirus (COVID-19) Test RT-PCR
QIAGEN GmbH QIAstat-Dx Respiratory SARS-CoV-2 Panel *panel includes 22 other viral or bacterial pathogens RT-PCR
Quest Diagnostics Infectious Disease, Inc. Quest SARS-CoV-2 rRT-PCR RT-PCR
Quidel Corporation Lyra SARS-CoV-2 Assay RT-PCR
Quidel Corporation Lyra Direct SARS-CoV-2 Assay RT-PCR
Rheonix, Inc. Rheonix COVID-19 MDx Assay RT-PCR
Roche Molecular Systems, Inc. (RMS) cobas SARS-CoV-2 RT-PCR
RTA Laboratories Biological Products Pharmaceutical and Machinery Industry Diagnovital SARS-CoV-2 Real-Time PCR Kit RT-PCR
Rutgers Clinical Genomics Laboratory at RUCDR Infinite Biologics - Rutgers University Rutgers Clinical Genomics Laboratory TaqPath SARS-CoV-2-Assay RT-PCR
Sansure BioTech Inc. Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing) RT-PCR
ScienCell Research Laboratories ScienCell SARS-CoV-2 Coronavirus Real-time RT-PCR (RT-qPCR) Detection Kit RT-PCR
SD Biosensor, Inc. STANDARD M nCoV Real-Time Detection Kit RT-PCR
Seasun Biomaterials U-TOP COVID-19 Detection Kit RT-PCR
Seasun Biomaterials, Inc. AQ-TOP COVID-19 Rapid Detection Kit RT-LAMP
Seegene, Inc. Allplex 2019-nCoV Assay RT-PCR
Sherlock BioSciences, Inc. Sherlock CRISPR SARS-CoV-2 Kit CRISPR
SolGent Co., Ltd. DiaPlexQ Novel Coronavirus (2019-nCoV) Detection Kit RT-PCR
TBG Biotechnology Corp. ExProbe SARS-CoV-2 Testing Kit RT-PCR
Thermo Fisher Scientific, Inc. TaqPath COVID-19 Combo Kit RT-PCR
Tide Laboratories, LLC DTPM COVID-19 RT-PCR Test RT-PCR
Trax Management Services Inc. PhoenixDx 2019-CoV RT-PCR
Wadsworth Center, New York State Department of Public Health’s (CDC) New York SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Panel RT-PCR
Zymo Research Corporation Quick SARS-CoV-2rRT-PCR Kit RT-PCR
Serology
ManufacturerTest NameAssay Type
Abbott Laboratories Inc. SARS-CoV-2 IgG assay Chemiluminescent microparticle immunoassay
Autobio Diagnostics Co. Ltd. Anti-SARS-CoV-2 Rapid Test IgM and IgG Lateral flow immunoassay
Bio-Rad Laboratories, Inc Platelia SARS-CoV-2 Total Ab assay Enzyme-Linked Immunosorbent Assays (ELISA)
Cellex Inc. qSARS-CoV-2 IgG/IgM Rapid Test Lateral flow immunoassay
Chembio Diagnostic System, Inc. DPP COVID-19 IgM/IgG System Immunochromatography
DiaSorin Inc. LIAISON SARS-CoV-2 S1/S2 IgG Chemiluminescent immunoassay
Emory Medical Laboratories SARS-CoV-2 RBD IgG test Enzyme-Linked Immunosorbent Assays (ELISA)
EUROIMMUN US Inc. Anti-SARS-CoV-2 ELISA (IgG) Enzyme-Linked Immunosorbent Assays (ELISA)
Hangzhou Biotest Biotech Co., Ltd. RightSign COVID-19 IgG/IgM Rapid Test Cassette Lateral flow chromatographic immunoassay
Healgen Scientific LLC COVID-19 IgG/IgM Rapid Test Cassette Lateral flow immunoassay
InBios International, Inc. SCoV-2 Detect IgG ELISA ELISA
Mount Sinai Laboratory COVID-19 ELISA IgG Antibody Test Enzyme-Linked Immunosorbent Assays (ELISA)
Ortho Clinical Diagnostics, Inc. VITROS Immunodiagnostic Products Anti-SARS-CoV-2 Total Reagent Pack Immunometric luminescence
Ortho-Clinical Diagnostics, Inc. VITROS Immunodiagnostic Products Anti-SARS-CoV-2 IgG Reagent Pack Immunometric luminescence
Roche Diagnostics Elecsys Anti-SARS-CoV-2 Electrochemiluminescence Immunoassay
Siemens Healthcare Diagnostics Inc. Dimension Vista SARS-CoV-2 Total antibody assay (COV2T) Chemiluminescent immunoassay
Dimension EXL SARS-CoV-2 Total antibody assay (CV2T) Chemiluminescent immunoassay
Atellica IM SARS-CoV-2 Total (COV2T) Chemiluminescent immunoassay
ADVIA Centaur SARS-CoV-2 Total (COV2T) Chemiluminescent immunoassay
Vibrant America Clinical Labs Vibrant COVID-19 Ab Assay Chemiluminescence immunoassay
Wadsworth Center, New York State Department of Health New York SARS-CoV Microsphere Immunoassay for Antibody Detection Microsphere Immunoassay
Antigen
ManufacturerTest NameAssay Type
Quidel Corporation Sofia 2 SARS Antigen FIA Lateral flow immunofluorescent sandwich assay
Molecular . . .
Manufacturer . Test Name . Assay Type .
1drop Inc. 1copy COVID-19 qPCR Multi Kit RT-PCR
Abbott Diagnostics Scarborough, Inc. ID NOW COVID-19 Isothermal nucleic acid amplification
Abbott Molecular Abbott RealTime SARS-CoV-2 assay RT-PCR
Abbott Molecular Inc. Alinity m SARS-CoV-2 assay RT-PCR
Altona Diagnostics GmbH RealStar SARS-CoV02 RT-PCR Kits U.S. RT-PCR
Applied BioCode, Inc. BioCode SARS-CoV-2 Assay RT-PCR
Applied DNA Sciences, Inc. Linea COVID-19 Assay Kit RT-PCR
Assurance Scientific Laboratories Assurance SARS-CoV-2 Panel RT-PCR
Atila BioSystems, Inc. iAMP COVID-19 Detection Kit Isothermal amplification test
Avellino Lab USA, Inc. AvellinoCoV2 test RT-PCR
Becton, Dickinson & Company BD SARS-CoV-2Reagents for BD MAX System RT-PCR
Becton, Dickinson & Company (BD) BioGX SARS-CoV-2 Reagents for BD MAX System RT-PCR
BGI Genomics Co. Ltd Real-Time Fluorescent RT-PCR Kit for Detecting SARS-2019-nCoV RT-PCR
BioCore Co., Ltd. BioCore 2019-nCoV Real Time PCR Kit RT-PCR
Bio-Rad Laboratories, Inc Bio-Rad SARS-CoV-2 ddPCR Test RT-PCR
BioFire Defense, LLC BioFire COVID-19 Test RT-PCR
BioFire Defense, LLC BioFire Respiratory Panel 2.1 (RP2.1) *panel includes 20 other viral or bacterial pathogens RT-PCR
BioMérieux SA SARS-COV-2 R-GENE RT-PCR
Centers for Disease Control and Prevention’s (CDC) CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel (CDC) RT-PCR
Cepheid Xpert Xpress SARS-CoV-2 test RT-PCR
ChromaCode Inc. HDPCR SARS-CoV-2 Assay RT-PCR
Co-Diagnostics, Inc. Logix Smart Coronavirus Disease 2019 (COVID-19) Kit RT-PCR
Cue Health Inc. Cue COVID-19 Test Isothermal nucleic acid amplification
dba SpectronRx Hymon SARS-CoV-2 Test Kit RT-PCR
DiaCarta, Inc QuantiVirus SARS-CoV-2 Test kit RT-PCR
DiaSorin Molecular LLC Simplexa COVID-19 Direct assay RT-PCR
Everlywell, Inc. Everlywell COVID-19 Test Home Collection Kit RT-PCR
Euroimmun US Inc. EURORealTime SARS-CoV-2 RT-PCR
Fast Track Diagnostics Luxembourg S.á.r.l. FTD SARS-CoV-2 RT-PCR
Fosun Pharma USA Inc. Fosun COVID-19 RT-PCR Detection Kit RT-PCR
Fulgent Therapeutics, LLC Fulgent COVID-19 by RT-PCR Test RT-PCR
GeneMatrix, Inc. NeoPlex COVID-19 Detection Kit RT-PCR
Genetron Health (Beijing) Co., Ltd. Genetron SARS-CoV-2 RNA Test RT-PCR
GenMark Diagnostics, Inc. ePlex SARS-CoV-2 Test PCR, electrochemical detection (voltammetry)
GenoSensor, LLC GS™ COVID-19 RT-PCR KIT RT-PCR
Gnomegen LLC Gnomegen COVID-19 RT-Digital PCR Detection Kit RT-PCR
Gnomegen LLC Gnomegen COVID-19-RT-qPCR Detection Kit RT-PCR
Gravity Diagnostics, LLC Gravity Diagnostics COVID-19 Assay RT-PCR
Hologic, Inc. Aptima SARS-CoV-2 assay Target capture, transcription mediated amplification and dual kinetic assay
Hologic, Inc. Panther Fusion SARS-CoV-2 RT-PCR
Illumina, Inc. Illumina COVIDSeq Test Next-Generation Sequencing (NGS)
InBios International, Inc Smart Detect SARS-CoV-2 rRT-PCR Kit RT-PCR
Ipsum Diagnostics, LLC COV-19 IDx assay RT-PCR
Kaiser Permanente Mid-Atlantic States KPMAS COVID-19 Test RT-PCR
KorvaLabs Inc. Curative-Korva SARS-Cov-2 Assay RT-PCR
LabGenomics Co., Ltd. LabGun COVID-19 RT-PCR Kit RT-PCR
Laboratory Corporation of America (LabCorp) COVID-19 RT-PCR Test RT-PCR
Luminex Corporation ARIES SARS-CoV-2 Assay RT-PCR
Luminex Molecular Diagnostics, Inc. NxTAG CoV Extended Panel Assay RT-PCR
Maccura Biotechnology (USA) LLC SARS-CoV-2 Fluorescent PCR Kit RT-PCR
Mesa Biotech Inc. Accula SARS-Cov-2 Test PCR and lateral flow assay
NeuMoDx Molecular, Inc. NeuMoDx SARS-CoV-2 Assay RT-PCR
OPTI Medical Systems, Inc. OPTI SARS-CoV-2 RT PCR Test RT-PCR
OSANG Healthcare GeneFinder COVID-19 Plus RealAmp Kit RT-PCR
P23 Labs, LLC. P23 Labs TaqPath SARS-CoV-2 Assay RT-PCR
PerkinElmer, Inc. PerkinElmer New Coronavirus Nucleic Acid Detection Kit RT-PCR
Phosphorus Diagnostics LLC Phosphorus COVID-19 RT-qPCR Test RT-PCR
Primerdesign Ltd. Primerdesign Ltd COVID-19 genesig Real-Time PCR assay RT-PCR
PrivaPath Diagnostics, Inc. LetsGetChecked Coronavirus (COVID-19) Test RT-PCR
QIAGEN GmbH QIAstat-Dx Respiratory SARS-CoV-2 Panel *panel includes 22 other viral or bacterial pathogens RT-PCR
Quest Diagnostics Infectious Disease, Inc. Quest SARS-CoV-2 rRT-PCR RT-PCR
Quidel Corporation Lyra SARS-CoV-2 Assay RT-PCR
Quidel Corporation Lyra Direct SARS-CoV-2 Assay RT-PCR
Rheonix, Inc. Rheonix COVID-19 MDx Assay RT-PCR
Roche Molecular Systems, Inc. (RMS) cobas SARS-CoV-2 RT-PCR
RTA Laboratories Biological Products Pharmaceutical and Machinery Industry Diagnovital SARS-CoV-2 Real-Time PCR Kit RT-PCR
Rutgers Clinical Genomics Laboratory at RUCDR Infinite Biologics - Rutgers University Rutgers Clinical Genomics Laboratory TaqPath SARS-CoV-2-Assay RT-PCR
Sansure BioTech Inc. Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing) RT-PCR
ScienCell Research Laboratories ScienCell SARS-CoV-2 Coronavirus Real-time RT-PCR (RT-qPCR) Detection Kit RT-PCR
SD Biosensor, Inc. STANDARD M nCoV Real-Time Detection Kit RT-PCR
Seasun Biomaterials U-TOP COVID-19 Detection Kit RT-PCR
Seasun Biomaterials, Inc. AQ-TOP COVID-19 Rapid Detection Kit RT-LAMP
Seegene, Inc. Allplex 2019-nCoV Assay RT-PCR
Sherlock BioSciences, Inc. Sherlock CRISPR SARS-CoV-2 Kit CRISPR
SolGent Co., Ltd. DiaPlexQ Novel Coronavirus (2019-nCoV) Detection Kit RT-PCR
TBG Biotechnology Corp. ExProbe SARS-CoV-2 Testing Kit RT-PCR
Thermo Fisher Scientific, Inc. TaqPath COVID-19 Combo Kit RT-PCR
Tide Laboratories, LLC DTPM COVID-19 RT-PCR Test RT-PCR
Trax Management Services Inc. PhoenixDx 2019-CoV RT-PCR
Wadsworth Center, New York State Department of Public Health’s (CDC) New York SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Panel RT-PCR
Zymo Research Corporation Quick SARS-CoV-2rRT-PCR Kit RT-PCR
Serology
ManufacturerTest NameAssay Type
Abbott Laboratories Inc. SARS-CoV-2 IgG assay Chemiluminescent microparticle immunoassay
Autobio Diagnostics Co. Ltd. Anti-SARS-CoV-2 Rapid Test IgM and IgG Lateral flow immunoassay
Bio-Rad Laboratories, Inc Platelia SARS-CoV-2 Total Ab assay Enzyme-Linked Immunosorbent Assays (ELISA)
Cellex Inc. qSARS-CoV-2 IgG/IgM Rapid Test Lateral flow immunoassay
Chembio Diagnostic System, Inc. DPP COVID-19 IgM/IgG System Immunochromatography
DiaSorin Inc. LIAISON SARS-CoV-2 S1/S2 IgG Chemiluminescent immunoassay
Emory Medical Laboratories SARS-CoV-2 RBD IgG test Enzyme-Linked Immunosorbent Assays (ELISA)
EUROIMMUN US Inc. Anti-SARS-CoV-2 ELISA (IgG) Enzyme-Linked Immunosorbent Assays (ELISA)
Hangzhou Biotest Biotech Co., Ltd. RightSign COVID-19 IgG/IgM Rapid Test Cassette Lateral flow chromatographic immunoassay
Healgen Scientific LLC COVID-19 IgG/IgM Rapid Test Cassette Lateral flow immunoassay
InBios International, Inc. SCoV-2 Detect IgG ELISA ELISA
Mount Sinai Laboratory COVID-19 ELISA IgG Antibody Test Enzyme-Linked Immunosorbent Assays (ELISA)
Ortho Clinical Diagnostics, Inc. VITROS Immunodiagnostic Products Anti-SARS-CoV-2 Total Reagent Pack Immunometric luminescence
Ortho-Clinical Diagnostics, Inc. VITROS Immunodiagnostic Products Anti-SARS-CoV-2 IgG Reagent Pack Immunometric luminescence
Roche Diagnostics Elecsys Anti-SARS-CoV-2 Electrochemiluminescence Immunoassay
Siemens Healthcare Diagnostics Inc. Dimension Vista SARS-CoV-2 Total antibody assay (COV2T) Chemiluminescent immunoassay
Dimension EXL SARS-CoV-2 Total antibody assay (CV2T) Chemiluminescent immunoassay
Atellica IM SARS-CoV-2 Total (COV2T) Chemiluminescent immunoassay
ADVIA Centaur SARS-CoV-2 Total (COV2T) Chemiluminescent immunoassay
Vibrant America Clinical Labs Vibrant COVID-19 Ab Assay Chemiluminescence immunoassay
Wadsworth Center, New York State Department of Health New York SARS-CoV Microsphere Immunoassay for Antibody Detection Microsphere Immunoassay
Antigen
ManufacturerTest NameAssay Type
Quidel Corporation Sofia 2 SARS Antigen FIA Lateral flow immunofluorescent sandwich assay

Abbreviations: FDA, United States Food and Drug Administration qPCR, quantitative polymerase chain reaction RT-PCR, reverse transcriptase polymerase chain reaction ddPCR, droplet digital polymerase chain reaction CDC, Centers for Disease Control and Prevention PCR, polymerase chain reaction Ig, immunoglobulin ELISA, enzyme-linked immunosorbent assay FIA, fluorescence immunoassay analyzer.

a As of May 26, 2020. For the most up-to-date list, please refer to: https://www.fda.gov/medical-devices/emergency-situations-medical-devices/emergency-use-authorizations. We also note that based on the FDA policy for Diagnostic Tests for Coronavirus Disease–2019 during the Public Health Emergency issued on March 16, 2020, commercial manufacturers can develop and distribute serology tests without an emergency-use authorization (EUA), as long as the test has been validated and the FDA is notified.

b Panel includes 20 other viral or bacterial pathogens.

Current FDA Emergency Use Authorized SARS-CoV-2 Assays, as of May 26, 2020

Molecular . . .
Manufacturer . Test Name . Assay Type .
1drop Inc. 1copy COVID-19 qPCR Multi Kit RT-PCR
Abbott Diagnostics Scarborough, Inc. ID NOW COVID-19 Isothermal nucleic acid amplification
Abbott Molecular Abbott RealTime SARS-CoV-2 assay RT-PCR
Abbott Molecular Inc. Alinity m SARS-CoV-2 assay RT-PCR
Altona Diagnostics GmbH RealStar SARS-CoV02 RT-PCR Kits U.S. RT-PCR
Applied BioCode, Inc. BioCode SARS-CoV-2 Assay RT-PCR
Applied DNA Sciences, Inc. Linea COVID-19 Assay Kit RT-PCR
Assurance Scientific Laboratories Assurance SARS-CoV-2 Panel RT-PCR
Atila BioSystems, Inc. iAMP COVID-19 Detection Kit Isothermal amplification test
Avellino Lab USA, Inc. AvellinoCoV2 test RT-PCR
Becton, Dickinson & Company BD SARS-CoV-2Reagents for BD MAX System RT-PCR
Becton, Dickinson & Company (BD) BioGX SARS-CoV-2 Reagents for BD MAX System RT-PCR
BGI Genomics Co. Ltd Real-Time Fluorescent RT-PCR Kit for Detecting SARS-2019-nCoV RT-PCR
BioCore Co., Ltd. BioCore 2019-nCoV Real Time PCR Kit RT-PCR
Bio-Rad Laboratories, Inc Bio-Rad SARS-CoV-2 ddPCR Test RT-PCR
BioFire Defense, LLC BioFire COVID-19 Test RT-PCR
BioFire Defense, LLC BioFire Respiratory Panel 2.1 (RP2.1) *panel includes 20 other viral or bacterial pathogens RT-PCR
BioMérieux SA SARS-COV-2 R-GENE RT-PCR
Centers for Disease Control and Prevention’s (CDC) CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel (CDC) RT-PCR
Cepheid Xpert Xpress SARS-CoV-2 test RT-PCR
ChromaCode Inc. HDPCR SARS-CoV-2 Assay RT-PCR
Co-Diagnostics, Inc. Logix Smart Coronavirus Disease 2019 (COVID-19) Kit RT-PCR
Cue Health Inc. Cue COVID-19 Test Isothermal nucleic acid amplification
dba SpectronRx Hymon SARS-CoV-2 Test Kit RT-PCR
DiaCarta, Inc QuantiVirus SARS-CoV-2 Test kit RT-PCR
DiaSorin Molecular LLC Simplexa COVID-19 Direct assay RT-PCR
Everlywell, Inc. Everlywell COVID-19 Test Home Collection Kit RT-PCR
Euroimmun US Inc. EURORealTime SARS-CoV-2 RT-PCR
Fast Track Diagnostics Luxembourg S.á.r.l. FTD SARS-CoV-2 RT-PCR
Fosun Pharma USA Inc. Fosun COVID-19 RT-PCR Detection Kit RT-PCR
Fulgent Therapeutics, LLC Fulgent COVID-19 by RT-PCR Test RT-PCR
GeneMatrix, Inc. NeoPlex COVID-19 Detection Kit RT-PCR
Genetron Health (Beijing) Co., Ltd. Genetron SARS-CoV-2 RNA Test RT-PCR
GenMark Diagnostics, Inc. ePlex SARS-CoV-2 Test PCR, electrochemical detection (voltammetry)
GenoSensor, LLC GS™ COVID-19 RT-PCR KIT RT-PCR
Gnomegen LLC Gnomegen COVID-19 RT-Digital PCR Detection Kit RT-PCR
Gnomegen LLC Gnomegen COVID-19-RT-qPCR Detection Kit RT-PCR
Gravity Diagnostics, LLC Gravity Diagnostics COVID-19 Assay RT-PCR
Hologic, Inc. Aptima SARS-CoV-2 assay Target capture, transcription mediated amplification and dual kinetic assay
Hologic, Inc. Panther Fusion SARS-CoV-2 RT-PCR
Illumina, Inc. Illumina COVIDSeq Test Next-Generation Sequencing (NGS)
InBios International, Inc Smart Detect SARS-CoV-2 rRT-PCR Kit RT-PCR
Ipsum Diagnostics, LLC COV-19 IDx assay RT-PCR
Kaiser Permanente Mid-Atlantic States KPMAS COVID-19 Test RT-PCR
KorvaLabs Inc. Curative-Korva SARS-Cov-2 Assay RT-PCR
LabGenomics Co., Ltd. LabGun COVID-19 RT-PCR Kit RT-PCR
Laboratory Corporation of America (LabCorp) COVID-19 RT-PCR Test RT-PCR
Luminex Corporation ARIES SARS-CoV-2 Assay RT-PCR
Luminex Molecular Diagnostics, Inc. NxTAG CoV Extended Panel Assay RT-PCR
Maccura Biotechnology (USA) LLC SARS-CoV-2 Fluorescent PCR Kit RT-PCR
Mesa Biotech Inc. Accula SARS-Cov-2 Test PCR and lateral flow assay
NeuMoDx Molecular, Inc. NeuMoDx SARS-CoV-2 Assay RT-PCR
OPTI Medical Systems, Inc. OPTI SARS-CoV-2 RT PCR Test RT-PCR
OSANG Healthcare GeneFinder COVID-19 Plus RealAmp Kit RT-PCR
P23 Labs, LLC. P23 Labs TaqPath SARS-CoV-2 Assay RT-PCR
PerkinElmer, Inc. PerkinElmer New Coronavirus Nucleic Acid Detection Kit RT-PCR
Phosphorus Diagnostics LLC Phosphorus COVID-19 RT-qPCR Test RT-PCR
Primerdesign Ltd. Primerdesign Ltd COVID-19 genesig Real-Time PCR assay RT-PCR
PrivaPath Diagnostics, Inc. LetsGetChecked Coronavirus (COVID-19) Test RT-PCR
QIAGEN GmbH QIAstat-Dx Respiratory SARS-CoV-2 Panel *panel includes 22 other viral or bacterial pathogens RT-PCR
Quest Diagnostics Infectious Disease, Inc. Quest SARS-CoV-2 rRT-PCR RT-PCR
Quidel Corporation Lyra SARS-CoV-2 Assay RT-PCR
Quidel Corporation Lyra Direct SARS-CoV-2 Assay RT-PCR
Rheonix, Inc. Rheonix COVID-19 MDx Assay RT-PCR
Roche Molecular Systems, Inc. (RMS) cobas SARS-CoV-2 RT-PCR
RTA Laboratories Biological Products Pharmaceutical and Machinery Industry Diagnovital SARS-CoV-2 Real-Time PCR Kit RT-PCR
Rutgers Clinical Genomics Laboratory at RUCDR Infinite Biologics - Rutgers University Rutgers Clinical Genomics Laboratory TaqPath SARS-CoV-2-Assay RT-PCR
Sansure BioTech Inc. Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing) RT-PCR
ScienCell Research Laboratories ScienCell SARS-CoV-2 Coronavirus Real-time RT-PCR (RT-qPCR) Detection Kit RT-PCR
SD Biosensor, Inc. STANDARD M nCoV Real-Time Detection Kit RT-PCR
Seasun Biomaterials U-TOP COVID-19 Detection Kit RT-PCR
Seasun Biomaterials, Inc. AQ-TOP COVID-19 Rapid Detection Kit RT-LAMP
Seegene, Inc. Allplex 2019-nCoV Assay RT-PCR
Sherlock BioSciences, Inc. Sherlock CRISPR SARS-CoV-2 Kit CRISPR
SolGent Co., Ltd. DiaPlexQ Novel Coronavirus (2019-nCoV) Detection Kit RT-PCR
TBG Biotechnology Corp. ExProbe SARS-CoV-2 Testing Kit RT-PCR
Thermo Fisher Scientific, Inc. TaqPath COVID-19 Combo Kit RT-PCR
Tide Laboratories, LLC DTPM COVID-19 RT-PCR Test RT-PCR
Trax Management Services Inc. PhoenixDx 2019-CoV RT-PCR
Wadsworth Center, New York State Department of Public Health’s (CDC) New York SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Panel RT-PCR
Zymo Research Corporation Quick SARS-CoV-2rRT-PCR Kit RT-PCR
Serology
ManufacturerTest NameAssay Type
Abbott Laboratories Inc. SARS-CoV-2 IgG assay Chemiluminescent microparticle immunoassay
Autobio Diagnostics Co. Ltd. Anti-SARS-CoV-2 Rapid Test IgM and IgG Lateral flow immunoassay
Bio-Rad Laboratories, Inc Platelia SARS-CoV-2 Total Ab assay Enzyme-Linked Immunosorbent Assays (ELISA)
Cellex Inc. qSARS-CoV-2 IgG/IgM Rapid Test Lateral flow immunoassay
Chembio Diagnostic System, Inc. DPP COVID-19 IgM/IgG System Immunochromatography
DiaSorin Inc. LIAISON SARS-CoV-2 S1/S2 IgG Chemiluminescent immunoassay
Emory Medical Laboratories SARS-CoV-2 RBD IgG test Enzyme-Linked Immunosorbent Assays (ELISA)
EUROIMMUN US Inc. Anti-SARS-CoV-2 ELISA (IgG) Enzyme-Linked Immunosorbent Assays (ELISA)
Hangzhou Biotest Biotech Co., Ltd. RightSign COVID-19 IgG/IgM Rapid Test Cassette Lateral flow chromatographic immunoassay
Healgen Scientific LLC COVID-19 IgG/IgM Rapid Test Cassette Lateral flow immunoassay
InBios International, Inc. SCoV-2 Detect IgG ELISA ELISA
Mount Sinai Laboratory COVID-19 ELISA IgG Antibody Test Enzyme-Linked Immunosorbent Assays (ELISA)
Ortho Clinical Diagnostics, Inc. VITROS Immunodiagnostic Products Anti-SARS-CoV-2 Total Reagent Pack Immunometric luminescence
Ortho-Clinical Diagnostics, Inc. VITROS Immunodiagnostic Products Anti-SARS-CoV-2 IgG Reagent Pack Immunometric luminescence
Roche Diagnostics Elecsys Anti-SARS-CoV-2 Electrochemiluminescence Immunoassay
Siemens Healthcare Diagnostics Inc. Dimension Vista SARS-CoV-2 Total antibody assay (COV2T) Chemiluminescent immunoassay
Dimension EXL SARS-CoV-2 Total antibody assay (CV2T) Chemiluminescent immunoassay
Atellica IM SARS-CoV-2 Total (COV2T) Chemiluminescent immunoassay
ADVIA Centaur SARS-CoV-2 Total (COV2T) Chemiluminescent immunoassay
Vibrant America Clinical Labs Vibrant COVID-19 Ab Assay Chemiluminescence immunoassay
Wadsworth Center, New York State Department of Health New York SARS-CoV Microsphere Immunoassay for Antibody Detection Microsphere Immunoassay
Antigen
ManufacturerTest NameAssay Type
Quidel Corporation Sofia 2 SARS Antigen FIA Lateral flow immunofluorescent sandwich assay
Molecular . . .
Manufacturer . Test Name . Assay Type .
1drop Inc. 1copy COVID-19 qPCR Multi Kit RT-PCR
Abbott Diagnostics Scarborough, Inc. ID NOW COVID-19 Isothermal nucleic acid amplification
Abbott Molecular Abbott RealTime SARS-CoV-2 assay RT-PCR
Abbott Molecular Inc. Alinity m SARS-CoV-2 assay RT-PCR
Altona Diagnostics GmbH RealStar SARS-CoV02 RT-PCR Kits U.S. RT-PCR
Applied BioCode, Inc. BioCode SARS-CoV-2 Assay RT-PCR
Applied DNA Sciences, Inc. Linea COVID-19 Assay Kit RT-PCR
Assurance Scientific Laboratories Assurance SARS-CoV-2 Panel RT-PCR
Atila BioSystems, Inc. iAMP COVID-19 Detection Kit Isothermal amplification test
Avellino Lab USA, Inc. AvellinoCoV2 test RT-PCR
Becton, Dickinson & Company BD SARS-CoV-2Reagents for BD MAX System RT-PCR
Becton, Dickinson & Company (BD) BioGX SARS-CoV-2 Reagents for BD MAX System RT-PCR
BGI Genomics Co. Ltd Real-Time Fluorescent RT-PCR Kit for Detecting SARS-2019-nCoV RT-PCR
BioCore Co., Ltd. BioCore 2019-nCoV Real Time PCR Kit RT-PCR
Bio-Rad Laboratories, Inc Bio-Rad SARS-CoV-2 ddPCR Test RT-PCR
BioFire Defense, LLC BioFire COVID-19 Test RT-PCR
BioFire Defense, LLC BioFire Respiratory Panel 2.1 (RP2.1) *panel includes 20 other viral or bacterial pathogens RT-PCR
BioMérieux SA SARS-COV-2 R-GENE RT-PCR
Centers for Disease Control and Prevention’s (CDC) CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel (CDC) RT-PCR
Cepheid Xpert Xpress SARS-CoV-2 test RT-PCR
ChromaCode Inc. HDPCR SARS-CoV-2 Assay RT-PCR
Co-Diagnostics, Inc. Logix Smart Coronavirus Disease 2019 (COVID-19) Kit RT-PCR
Cue Health Inc. Cue COVID-19 Test Isothermal nucleic acid amplification
dba SpectronRx Hymon SARS-CoV-2 Test Kit RT-PCR
DiaCarta, Inc QuantiVirus SARS-CoV-2 Test kit RT-PCR
DiaSorin Molecular LLC Simplexa COVID-19 Direct assay RT-PCR
Everlywell, Inc. Everlywell COVID-19 Test Home Collection Kit RT-PCR
Euroimmun US Inc. EURORealTime SARS-CoV-2 RT-PCR
Fast Track Diagnostics Luxembourg S.á.r.l. FTD SARS-CoV-2 RT-PCR
Fosun Pharma USA Inc. Fosun COVID-19 RT-PCR Detection Kit RT-PCR
Fulgent Therapeutics, LLC Fulgent COVID-19 by RT-PCR Test RT-PCR
GeneMatrix, Inc. NeoPlex COVID-19 Detection Kit RT-PCR
Genetron Health (Beijing) Co., Ltd. Genetron SARS-CoV-2 RNA Test RT-PCR
GenMark Diagnostics, Inc. ePlex SARS-CoV-2 Test PCR, electrochemical detection (voltammetry)
GenoSensor, LLC GS™ COVID-19 RT-PCR KIT RT-PCR
Gnomegen LLC Gnomegen COVID-19 RT-Digital PCR Detection Kit RT-PCR
Gnomegen LLC Gnomegen COVID-19-RT-qPCR Detection Kit RT-PCR
Gravity Diagnostics, LLC Gravity Diagnostics COVID-19 Assay RT-PCR
Hologic, Inc. Aptima SARS-CoV-2 assay Target capture, transcription mediated amplification and dual kinetic assay
Hologic, Inc. Panther Fusion SARS-CoV-2 RT-PCR
Illumina, Inc. Illumina COVIDSeq Test Next-Generation Sequencing (NGS)
InBios International, Inc Smart Detect SARS-CoV-2 rRT-PCR Kit RT-PCR
Ipsum Diagnostics, LLC COV-19 IDx assay RT-PCR
Kaiser Permanente Mid-Atlantic States KPMAS COVID-19 Test RT-PCR
KorvaLabs Inc. Curative-Korva SARS-Cov-2 Assay RT-PCR
LabGenomics Co., Ltd. LabGun COVID-19 RT-PCR Kit RT-PCR
Laboratory Corporation of America (LabCorp) COVID-19 RT-PCR Test RT-PCR
Luminex Corporation ARIES SARS-CoV-2 Assay RT-PCR
Luminex Molecular Diagnostics, Inc. NxTAG CoV Extended Panel Assay RT-PCR
Maccura Biotechnology (USA) LLC SARS-CoV-2 Fluorescent PCR Kit RT-PCR
Mesa Biotech Inc. Accula SARS-Cov-2 Test PCR and lateral flow assay
NeuMoDx Molecular, Inc. NeuMoDx SARS-CoV-2 Assay RT-PCR
OPTI Medical Systems, Inc. OPTI SARS-CoV-2 RT PCR Test RT-PCR
OSANG Healthcare GeneFinder COVID-19 Plus RealAmp Kit RT-PCR
P23 Labs, LLC. P23 Labs TaqPath SARS-CoV-2 Assay RT-PCR
PerkinElmer, Inc. PerkinElmer New Coronavirus Nucleic Acid Detection Kit RT-PCR
Phosphorus Diagnostics LLC Phosphorus COVID-19 RT-qPCR Test RT-PCR
Primerdesign Ltd. Primerdesign Ltd COVID-19 genesig Real-Time PCR assay RT-PCR
PrivaPath Diagnostics, Inc. LetsGetChecked Coronavirus (COVID-19) Test RT-PCR
QIAGEN GmbH QIAstat-Dx Respiratory SARS-CoV-2 Panel *panel includes 22 other viral or bacterial pathogens RT-PCR
Quest Diagnostics Infectious Disease, Inc. Quest SARS-CoV-2 rRT-PCR RT-PCR
Quidel Corporation Lyra SARS-CoV-2 Assay RT-PCR
Quidel Corporation Lyra Direct SARS-CoV-2 Assay RT-PCR
Rheonix, Inc. Rheonix COVID-19 MDx Assay RT-PCR
Roche Molecular Systems, Inc. (RMS) cobas SARS-CoV-2 RT-PCR
RTA Laboratories Biological Products Pharmaceutical and Machinery Industry Diagnovital SARS-CoV-2 Real-Time PCR Kit RT-PCR
Rutgers Clinical Genomics Laboratory at RUCDR Infinite Biologics - Rutgers University Rutgers Clinical Genomics Laboratory TaqPath SARS-CoV-2-Assay RT-PCR
Sansure BioTech Inc. Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing) RT-PCR
ScienCell Research Laboratories ScienCell SARS-CoV-2 Coronavirus Real-time RT-PCR (RT-qPCR) Detection Kit RT-PCR
SD Biosensor, Inc. STANDARD M nCoV Real-Time Detection Kit RT-PCR
Seasun Biomaterials U-TOP COVID-19 Detection Kit RT-PCR
Seasun Biomaterials, Inc. AQ-TOP COVID-19 Rapid Detection Kit RT-LAMP
Seegene, Inc. Allplex 2019-nCoV Assay RT-PCR
Sherlock BioSciences, Inc. Sherlock CRISPR SARS-CoV-2 Kit CRISPR
SolGent Co., Ltd. DiaPlexQ Novel Coronavirus (2019-nCoV) Detection Kit RT-PCR
TBG Biotechnology Corp. ExProbe SARS-CoV-2 Testing Kit RT-PCR
Thermo Fisher Scientific, Inc. TaqPath COVID-19 Combo Kit RT-PCR
Tide Laboratories, LLC DTPM COVID-19 RT-PCR Test RT-PCR
Trax Management Services Inc. PhoenixDx 2019-CoV RT-PCR
Wadsworth Center, New York State Department of Public Health’s (CDC) New York SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Panel RT-PCR
Zymo Research Corporation Quick SARS-CoV-2rRT-PCR Kit RT-PCR
Serology
ManufacturerTest NameAssay Type
Abbott Laboratories Inc. SARS-CoV-2 IgG assay Chemiluminescent microparticle immunoassay
Autobio Diagnostics Co. Ltd. Anti-SARS-CoV-2 Rapid Test IgM and IgG Lateral flow immunoassay
Bio-Rad Laboratories, Inc Platelia SARS-CoV-2 Total Ab assay Enzyme-Linked Immunosorbent Assays (ELISA)
Cellex Inc. qSARS-CoV-2 IgG/IgM Rapid Test Lateral flow immunoassay
Chembio Diagnostic System, Inc. DPP COVID-19 IgM/IgG System Immunochromatography
DiaSorin Inc. LIAISON SARS-CoV-2 S1/S2 IgG Chemiluminescent immunoassay
Emory Medical Laboratories SARS-CoV-2 RBD IgG test Enzyme-Linked Immunosorbent Assays (ELISA)
EUROIMMUN US Inc. Anti-SARS-CoV-2 ELISA (IgG) Enzyme-Linked Immunosorbent Assays (ELISA)
Hangzhou Biotest Biotech Co., Ltd. RightSign COVID-19 IgG/IgM Rapid Test Cassette Lateral flow chromatographic immunoassay
Healgen Scientific LLC COVID-19 IgG/IgM Rapid Test Cassette Lateral flow immunoassay
InBios International, Inc. SCoV-2 Detect IgG ELISA ELISA
Mount Sinai Laboratory COVID-19 ELISA IgG Antibody Test Enzyme-Linked Immunosorbent Assays (ELISA)
Ortho Clinical Diagnostics, Inc. VITROS Immunodiagnostic Products Anti-SARS-CoV-2 Total Reagent Pack Immunometric luminescence
Ortho-Clinical Diagnostics, Inc. VITROS Immunodiagnostic Products Anti-SARS-CoV-2 IgG Reagent Pack Immunometric luminescence
Roche Diagnostics Elecsys Anti-SARS-CoV-2 Electrochemiluminescence Immunoassay
Siemens Healthcare Diagnostics Inc. Dimension Vista SARS-CoV-2 Total antibody assay (COV2T) Chemiluminescent immunoassay
Dimension EXL SARS-CoV-2 Total antibody assay (CV2T) Chemiluminescent immunoassay
Atellica IM SARS-CoV-2 Total (COV2T) Chemiluminescent immunoassay
ADVIA Centaur SARS-CoV-2 Total (COV2T) Chemiluminescent immunoassay
Vibrant America Clinical Labs Vibrant COVID-19 Ab Assay Chemiluminescence immunoassay
Wadsworth Center, New York State Department of Health New York SARS-CoV Microsphere Immunoassay for Antibody Detection Microsphere Immunoassay
Antigen
ManufacturerTest NameAssay Type
Quidel Corporation Sofia 2 SARS Antigen FIA Lateral flow immunofluorescent sandwich assay

Abbreviations: FDA, United States Food and Drug Administration qPCR, quantitative polymerase chain reaction RT-PCR, reverse transcriptase polymerase chain reaction ddPCR, droplet digital polymerase chain reaction CDC, Centers for Disease Control and Prevention PCR, polymerase chain reaction Ig, immunoglobulin ELISA, enzyme-linked immunosorbent assay FIA, fluorescence immunoassay analyzer.

a As of May 26, 2020. For the most up-to-date list, please refer to: https://www.fda.gov/medical-devices/emergency-situations-medical-devices/emergency-use-authorizations. We also note that based on the FDA policy for Diagnostic Tests for Coronavirus Disease–2019 during the Public Health Emergency issued on March 16, 2020, commercial manufacturers can develop and distribute serology tests without an emergency-use authorization (EUA), as long as the test has been validated and the FDA is notified.

b Panel includes 20 other viral or bacterial pathogens.

Given that true NP specimens are often difficult to obtain, the FDA has stated that oropharyngeal (OP), nasal midturbinate, and anterior nares swabs are acceptable when using an NP swab is not possible. 4 Also, the FDA recently granted an Emergency-Use Authorization (EUA) to Rutgers Clinical Genomics Laboratory-Rutgers University for an RT-PCR LDT for qualitative detection of SARS-CoV-2 in saliva specimens as well as OP, NP, anterior nasal, and midturbinate nasal swabs. Saliva testing presents potential benefits of eliminating the need for swabs and decreasing the risk posed to the health-care workers collecting these specimens. However, this specimen type might require additional dilution or pretreatment due to its viscosity. Also, viral RNA might be more difficult to detect in this specimen type, although the results of a previous study 5 found that saliva and NP specimens were comparable for detection of respiratory viruses by RT-PCR.

The possibility of false negative results with molecular assays should also be considered. One report 6 documents a patient with multiple RT-PCR NP/OP specimens that tested negative this patient ultimately had SARS-CoV-2 detected in a bronchoalveolar lavage (BAL) fluid specimen. For lower respiratory tract specimens, currently, the FDA recommends testing BAL fluid only under certain clinical circumstances such as invasive mechanical ventilation. Sputum should be tested from patients who develop a productive cough, although the FDA does not recommend induction of sputum for SARS-CoV-2 testing. 4

The other main type of assay is serological. These assays determine the exposure history and/or immune status of a patient. They detect the presence of antibodies against SARS-CoV-2 antigens in serum, plasma, or whole blood specimens. After initial viral infection, there is a delay before the production of antibodies by the immune system ( Figure 1). During this time, known as the window period, a patient who is infected with SARS-CoV-2 but has no detectable antibodies, would have negative test results on a serologic assay. Typically, when the immune system mounts a response against a virus, short-lived immunoglobulin (Ig)M antibodies are initially produced, followed by a more durable IgG antibody response. However, there are limited data thus far in the literature regarding the longevity of anti–SARS-CoV-2 antibodies.

SARS-CoV-2 estimated seroconversion rates.

SARS-CoV-2 estimated seroconversion rates.

The graph in Figure 1 demonstrates estimated viral RNA, IgM, and IgG detection levels for SARS-CoV-2 based on the limited published literature to date. The estimated median seroconversion time is 7 to 12 days most patients with COVID-19 have detectable antibodies approximately 15 days after the onset of symptoms. Due to the subjectivity in determining symptom onset, these dates can be highly variable. Also, viral RNA has been shown to peak in the first week of illness and then gradually decline.

Serological tests can be unique to one class of immunoglobulins can detect IgM and IgG antibodies simultaneously or can be total antibody tests, which detect IgA antibodies as well. Depending on the exact protocol and platform, such assays can typically be completed in 1 to 2 hours once a specimen arrives in the laboratory and is loaded onto the relevant platform. We note, however, that at the time of publication of this article, there are a few commercial assays available on large, automated analyzers, from diagnostic manufacturers including Roche Diagnostics, Abbott Diagnostics, and Ortho Clinical Diagnostics (with offerings more recently available from Beckman Coulter, Inc and Siemens AG). However, to our knowledge, there are no objective, peer-reviewed data on their performance characteristics.

Many available commercial serological assays use a lateral flow assay format for many of these assays, there are unsubstantiated, or even false, claims about test performance. 7 The estimated median seroconversion time is 7 to 12 days, with virtually all patients with COVID-19 having detectable antibodies approximately 15 days after onset of symptoms. 8–10 However, the results of a recent acute antibody response study 11 demonstrated simultaneous or sequential IgM and IgG seroconversion, with a slight decrease in IgM antibody titers 3 weeks after symptom onset. Therefore, these assays will be most helpful in determining the exposure status of an individual and in assessing the immune response of that person to SARS-CoV-2.

Because SARS-CoV-2 is a new virus, it is not clear whether an immune response confers immunity and how long the immune response will last—that is, whether it is durable and sustained (years) or if it is short-lived (1–2 months). These assays can be particularly helpful for individuals who may have had symptoms consistent with COVID-19 but who were never tested with an RT-PCR test (due to the severe limitations in testing capabilities in many areas) and now have recovered from their illness. Currently, the Centers for Disease Control and Prevention (CDC) does not recommend using serology testing to determine eligibility to return to the in-person workforce. Individuals who were symptomatic can stop self-isolation as long as their symptoms have improved with 3 days of no fever and at least 10 days have elapsed since the onset of their symptoms. 12

Finally, these tests can also be used in serosurveillance, in which data are obtained to calculate the prevalence of anti–SARS-CoV-2 antibodies in the community. Such information can help epidemiologists better understand the true burden of disease and to model continued viral-transmission dynamics based on the percentage of the population that is immune vs susceptible. Given that approximately 80% of COVID-19 cases are mild to moderate in severity 10, 13 and that molecular testing has been restricted to the most severely ill patients, the true number of cases has likely not been revealed by molecular-based assays. Thus, serological testing can provide a more accurate enumeration of the number of past infections. As with all laboratory testing, however, the results of these assays will only be accurate and maximally useful based on their performance characteristics, including sensitivity and specificity.

COVID-19 testing is also important for identifying potential patients who have recovered from COVID-19 and have detectable antibodies against SARS-CoV-2 (convalescent donors) for clinical trials. Currently, studies are underway in which convalescent donors can donate plasma, which can then be transfused into critically ill patients with COVID-19. A review of the published literature to date 14 indicates that treatment with plasma from convalescent donors demonstrates beneficial effects, although further evaluation with clinical trials remains imperative. Potential donors require a serologic assay to detect the presence of anti–SARS-CoV-2 antibodies in their plasma. For this type of plasma to be beneficial, the antibodies present should have neutralizing activity (ie, the antibodies bind to and neutralize infection by active SARS-CoV-2 virus). Such testing is not currently performed in clinical laboratories but rather in research laboratories. Ideally, convalescent plasma donors would be noninfectious (symptom-free for >14 days) and have high titers of virus-neutralizing antibodies (as determined by serologic testing).

Point-of-care (POC) testing is beginning to be available for SARS-CoV-2. POC testing refers to a broad category of diagnostic tests that can be performed where patient care occurs. Functionally, these tests have a rapid turnaround time (TAT) and can potentially be performed by various nonlaboratory clinical personnel. These assays can be molecular or serologic. One molecular POC test by Abbott Diagnostics uses isothermal nucleic acid amplification (a technique similar to polymerase chain reaction [PCR]) to detect SARS-CoV-2 in approximately 15 minutes. The results of recent studies 15–17 have demonstrated low sensitivity for the Abbott ID Now assay with specimens collected in transport media. Thus, the EUA for this test was modified for testing only from direct/dry swabs.

The Cellex serologic POC qSARS-CoV-2 IgG/IgM Rapid Test (Cellex, Inc.) utilizes a lateral flow immunoassay, which qualitatively detects IgM and/or IgG antibodies from whole-blood specimens. A blood specimen flows by capillary action along the cassette and, if anti–SARS-CoV-2 IgM or IgG antibodies are present, they will bind to recombinant SARS-CoV-2 antigens present on the test strip. The presence of these antibody-antigen complexes are then detected by a colorimetric change, which is revealed when the complexes are captured by anti–human IgG or anti–human IgM antibodies. The results are available in approximately 15 to 20 minutes. Of note, currently POC serology tests must be performed in conjunction with a CLIA (Clinical Laboratory Improvement Amendments)–licensed laboratory and cannot be performed in locations with only a CLIA waiver, such as a physician office.

Finally, there is now an antigen detection assay available from Quidel Corporation (Sofia 2 SARS Antigen FIA) that uses a lateral flow immunofluorescent sandwich assay technique for detection of the nucleocapsid protein antigen of SARS-CoV and SARS-CoV-2. In theory, viral proteins can be detected using one of a number of antigen capture methods (eg, antibodies, aptamers). Such tests are used routinely for other viral assays (eg, human immunodeficiency virus [HIV] p24 antigen as part of 4 th - and 5 th -generation HIV tests), and also for hepatitis B surface antigen. 18 Like molecular assays, antigen detection tests can be used to detect active SARS-CoV-2 infection.

In summary, molecular and serological tests provide meaningful data for treating patients with COVID-19. However, each category has different clinical utility, different characteristics, and different limitations. Clinical laboratories continue to develop new assays and implement increased testing capabilities to meet the high demands for patient testing during this pandemic.


Assay development, high throughput and high content screening in pre-clinical drug discovery

Assays that are screened against libraries of compounds to identify chemical starting points in the early stages of drug discovery can be classified as being biochemical or cell-based in nature. The exact assay that is utilized in a screen is decided upon a case-by-case basis after taking into account a number of factors such as provision of reagents, throughput, cost, and many others that have been discussed extensively in the literature [17]. The biochemical target-based (reductionist) approach was largely adopted in the post-Human Genome Project era where specific genes were identified and cloned and the corresponding proteins expressed in sufficient quantity with acceptable activity for screening [18]. This was a marked shift from earlier cell-based assays where modulation of specific targets did not occur, but instead relevant cellular phenotypic responses were measured [19, 20]. Significant effort has been expended to mimic these physiologically relevant cell-based systems with a significantly higher throughput [21] and advances have been made using a variety of these and subsequently deployed in cancer drug discovery in particular [22�] as well as being expanded to areas such as predictive toxicology [25].

For any given protein target class, a variety of fully validated screening compatible assay kits are commercially available. These offer the potential to reduce cycle times significantly for hit identification and beyond. Alternatively, it may be possible to exploit specific commercial reagents to build de novo assays and this isrelevant when investigating newly identified proteins and their substrates. Where appropriate, schematic representations of assays are provided (Figs.  1 , ​ ,2, 2 , ​ ,3, 3 , and ​ and4). 4 ). The ultimate decision as to which assay to use in a screening campaign is usually considered on a case-by-case basis when initiating a drug discovery project since all assays have specific advantages and disadvantages. For example, in the case of the protein kinases, biochemical assays are often utilized and more than 20 of such assays are commercially available [26, 27] whereas in the case of G-protein-coupled receptors, cell-based assays are more commonly employed [28�]. It is prudent to develop a panel of assays with different readout modes, as these are suitable for the hit validation stage thus allowing confirmation as to whether the activities of compounds translate to more than one assay format thereby adding confidence that they are not assay artefacts [33�]. This is important as it is now known that assays that make use of specific tagged proteins in the AlphaScreen™ assay format often yield specific interfering compounds as false positive hits [37, 38]. Assay formats that have enhanced the capabilities relative to phenotypic assays include label-free impedance-based [39, 40] dynamic mass redistribution [41, 42] and multiplex assays [43, 44], and these have been successfully applied in screening against small-molecule libraries. More recent state-of-the-art screening compatible assays use three-dimensional spheroids that offer the potential to represent the microenvironment of cells in the body [45].

AlphaLISA® histone deacetylate assay that detects Histone H3-K9(Ac) or Histone H3-K27(Ac). The acetylated histones are detected using a biotinylated anti-H3 antibody and AlphaLISA®-acceptor beads conjugated specific to the acetylated lysine. Streptavidin-donor beads then capture the biotinylated antibody, bringing the acceptor and donor beads into proximity. Upon laser irradiation of the donor beads at 680 nm, short-lived singlet oxygen molecules produced by the donor beads can reach the acceptor beads in proximity to generate an amplified chemiluminescent signal at 615 nm

a Colorimetric coupled histone deacetylate assay that makes use of a chromogenic peptide substrate (proprietary Color de Lys® Substrate) containing a ε-acetylated lysine residue. When an HDAC enzyme acts upon the substrate and the sidechain of a ε-acetylated lysine residue is deacetylated, it becomes susceptible to further degradation by an enzyme in the developer reagent (proprietary Color de Lys® Developer). The action of the enzyme within the developer reagent results in the release of a chromophore detected by measuring the absorbance of the reaction at 405 nm. b Fluorometric coupled histone deacetylate assay that makes use of a fluorogenic peptide substrate (proprietary Fluor de Lys® Substrate) containing a ε-acetylated lysine residue. When an HDAC enzyme acts upon the substrate and the sidechain of a ε-acetylated lysine residue is deacetylated, it becomes susceptible to further degradation by an enzyme in the developer reagent (proprietary Fluor de Lys® Developer) resulting in the release of 7-amino-4-methylcoumarin fluorophore which undergoes excitation at 360 nm and emits at 460 nm

Luminescence coupled histone deacetylate assay that makes use of specific amino-luciferin labelled ε-acetylated lysine peptide substrates for HDAC Class I/II enzymes. When the substrate undergoes deacetylation by the HDAC enzyme, the product becomes susceptible to the Developer reagent and results in the release of amino-luciferin. This amino-luciferin is the substrate for a luciferase enzyme (also in the Developer reagent) and yields a glow-type luminescence

Time-resolved fluorescence resonance energy transfer histone deacetylase assay. A signal is generated when the deacetylated peptides are captured by the Europium-labelled antibody donor and streptavidin-ULight™-acceptor thus bringing the Europium-donor and ULight™-acceptor molecules into close proximity. Upon irradiation at 340 nm, the energy from the Europium-donor is transferred to the ULight™-acceptor, which, in turn, generates a signal at 665 nm

The pre-requisites for high throughput screening (HTS) are access to a suitable assay as briefly described above and a suitable compound library. Compound libraries are usually stored in pure DMSO at concentrations between 1 mM and 10 mM as this will allow for a range of final assay concentrations of compound whilst retaining ρ% DMSO (v/v) in the final assay. The extent of automation when embarking upon an HTS campaign will depend upon the numbers of compounds screened and it would be reasonable to screen a compound library composed of a few thousand compounds manually in miniaturized formats (e.g. 384- or 1536-well microtiter plates). However, where � compounds are screened (in 384-well microtiter plates), it would be prudent to use some degree of automation such as stand-alone reagent dispensers or a robotic screening system [46�]. One way to minimize the consumption of reagents when screening very large numbers of compounds is to miniaturize and parallelize an assay into 1536-well microtiter plates [50]. However, such miniaturization requires the addition of very small volumes of compound stock solutions and technologies such as the contactless acoustic dispenser from Labcyte Inc. makes this possible [51].

High content screening (HCS) is now an established technique that is routinely utilized in chemical biology and drug discovery and has made a significant impact upon understanding the output of phenotypic screening. This is a cell-based approach that can offer a multi-parameter readout detecting simultaneously a multitude of cellular changes that are subsequently attributed to specific targets [52�]. This approach is particularly relevant in epigenetics as the discovery of Romidepsin and Vorinostat as anti-cancer drugs originates from phenotypic assays [57].


Abstract

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.


Will a sandwich assay work with a GST 26 antibody/protein? - Biology

Enzyme linked immunosorbent assay, or ELISA, is a powerful qualitative and quantitative tool for measuring proteins in biologic samples. Multiple variants of ELISA immunoassays exist, and are chosen based on the samples being tested.

What is ELISA?

Since its development during the 1960s and 1970s 1 , ELISA has become established as a method that uses a specific antibody to detect the presence of a protein using chromogenic, fluorescent, or luminescent readout 2 . Because ELISA kits and ELISA sets come with standards &ndash solutions of the target protein at known concentrations &ndash ELISAs can also be used to calculate the absolute concentration of a protein in test samples.

How Does it Work?

Direct ELISA is used to detect antigen that is directly bound to a plate &ndash for example, on whole cells or viruses &ndash while indirect ELISA, which usually takes the format of a sandwich ELISA, uses a multi-step protocol to capture the target protein using a second antibody to detect. This method is used for crude liquid samples including serum and urine.

In a sandwich ELISA, a 96-well plate is coated with an antibody specific to the protein of interest. The plate is then incubated with a blocking buffer containing irrelevant proteins, to block non-specific binding of proteins from the test sample to the plate. Samples, plus appropriate controls and standards, are then added to the plate, where the target protein is captured by the antibody coating the plate. Next, the detection process begins. A primary &ldquodetection antibody&rdquo &ndash also specific to the protein of interest &ndash is added. This antibody is either directly conjugated to, or then bound by a secondary antibody conjugated to, an enzyme &ndash such as HRP for a chromogenic reaction &ndash followed by the addition of its corresponding substrate.

The combination of enzyme with substrate initiates the reaction whose product is detected by a plate reader to give the readout used to calculate the absolute protein concentration. For chromogenic reactions, the readout is in OD, or optical density &ndash a measure of the absorbance of a specific wavelength of light by the solution in each well. To convert the OD values into protein concentrations, the OD values for the standards are plotted against their known concentrations, giving a standard curve unique to the plate from which it was read. Next, the OD values for the samples are plotted along the linear portion of that standard curve, and the protein concentrations extrapolated from the corresponding point along the second axis.

Why ELISA?

Because of its qualitative and quantitative applications, ELISA is a robust technique that allows scientists to both detect the presence of a specific protein in samples and determine the amount of protein present. Experimentally, this means that ELISA can be used to compare differences in protein concentration between control and experimental groups in an in vivo or in vitro experiment, or between healthy donor and patient samples in clinical studies.

Bethyl Laboratories sells high quality ELISA kits. These products have recently been used to study:


22 comments on &ldquoCrappy Antibodies: Available Now, and for the Foreseeable Future&rdquo

Most competent immunologists don’t depend on one antibody. Rather, they do sandwich assays or immunoprecipitation followed by Westerns that rely on recognition of two different epitopes. Even monoclonals have cross-reactivity issues hence the need for looking at two separate epitopes. As with any assay, you need to validate before you can depend on the results.

@1 Steve: true but, how many times does that complicate the issue for each time it reduces it?

a scientist who doesn’t believe in validating the quality of their reagents is not a scientist.

It goes beyond what is described, however. At least according to folks I’ve talked to since it’s not my field.
Not only is there variability in reactivity and selectivity between vendors for nominally similar mABs, but batch to batch variability from the same vendor and intra-batch variability depending on storage length and conditions.
The only real recourse seemingly is to test your antibodies prior to every experiment. Short of that you are asking for real trouble in terms of any reproducibility. Of course, just because it’s reproducible doesn’t mean it’s “correct” either. It could be reproducibly unselective.

Minimally people should be using universal IDs (like Research Resource ID, RRID) so that the exact vendor, part, and lot can be traced back for reproducing work- e.g.:
https://scicrunch.org/resources/Antibodies/search
There’s a link for each reagent registered that autogenerates the text you need to insert into a publication protocol for referencing the RRID. Some publishers have started requiring the use of them. (Incidentally that site supports Derek’s estimate, they have 2.4 million registered.)

From my experience, biomedical research has largely adopted a kit-based, ready-to-use mentality. Although one should expect high quality when spending $400+ on 100 micrograms on a primary antibody, research customers should still verify product performance before committing to expensive experiments. What disappoints me the most is that various open-access, crowd-sourced evaluation tools exist, yet hardly any biomedical researcher uses them.
While many can regurgitate acronyms in a signal transduction pathway, very few understand the underlying chemical mechanisms. The pursuit of Big Data should not excuse inattention to detail or neglecting best practices.

I don’t remember whether it was in 11th grade chem or 12th grade organic chem lab where the teacher substituted solvents on us – replacing methanol with water, maybe – and the grade for that day was based on how long it took you to figure out that something wasn’t right. I always figured it was one of the most valuable lessons I learned.
But then we walked five miles to school, barefoot in the snow, uphill both ways, so that sort of thing probably doesn’t apply today.

The lack of antibody validation is a problem of huge proportions. Just look at this paper:
http://www.ncbi.nlm.nih.gov/pubmed/25613900
(Proteomics. Tissue-based map of the human proteome.)
The antibody validation is risible at best (spotted array), and their own westerns invalidate quite a few of their antibodies that I have looked at. Yet this has been no barrier to publish a paper in Science.
@SP Having resource ID will help only if companies maintain consistent quality. My experience with SCBT and Abcam shows that there is big batch to bath variability.

GPCR ab are particularly crappy & widely abused. I prefer Westerns as the first test of their potential utility. GPCR are glycoproteins & therefore appear as wide, fuzzy bands on Western blots. If you don’t see this in a recombinant (+ the signal disappears in the nontransfected parental), the ab is junk. If a figure in a paper, shows a GPCR as sharp band on a Western, the ab data is beyond suspect.
See Fig 5 in Gu & Schonbrunn, 1997 (no firewall Mol Endocrinol. 1997 May11(5):527-37)- that is what a GPCR looks like in a Western.

There’s a reason why a certain vendor is affectionately referred to as “SantaCrap”, right!

@10: it used to be that Santa Cruz was the one company you knew to stay away from. Now the problem is that there are 10+ companies as crappy as them (who are probably just reselling the SC stuff).
My rule of thumb is to stay away from the companies that offer antibodies to essentially every protein known to man. The best companies (eg Cell Signaling) have much smaller catalogues, and for a reason.

Yeah, well it’s a lot like generic drugs, that are “identical”……but they are not. And now even pharmacies can force the use of a generic even when the patient asks for a brand name and is willing to pay more for the original.

@10 Virgil: That would be the speed-of-delivery issue… if your business model depends on being first-to-market for every new protein, and you get there by selling the first antisera/hybridoma that shows any detectable binding in any measure (western, flow, IHC), then your catalog will be an enriched collection of questionable products.

I think it goes without saying that you have to validate your reagents for your specific assay. With monoclonals you have to understand the science behind them as well. What was used as antigen to elicit them? For proteins, do they recognize conformational vs. linear epitopes? I suspect that has a lot to do with the “less than 50%” result. There are many perfectly good monoclonals that don’t work on fixed sections.

This is why we run controls. Lots of them.

@14 RickW: 100% this.
I have seen a reasonably nice cars worth wasted on a monoclonal that reliably recognized a denatured protein. Native form? Not a twitch. Turns out the vendor had used the same batch of antigen for both immunisation and reference material. This contained some of the denatured stuff… Also: that fancy new sepsis marker? Actually was hemoglobin all along.
tldr
Purity and Identity are as important in biology as in chemistry. Sometimes more difficult.

@2- Sorry, I have no idea what you’re talking about.

Yes!! SantaCrap, I completely forgot that is how we referred to those guys.

A very pertinent article- thanks Derek.
People tend to miss out on a very important sector when discussing antibodies- the technology transfer offices (TTOs) who bring the antibodies out from labs to the commercial suppliers.
Picking up from this article- so even if you buy half a dozen antibodies against the same protein from different outfits, you may have only bought two. Or one. Who knows?
We are trying to tackle exactly this- if we could link inventors, publications and institutes to each antibody- at least some of these issues can be resolved.
If this is of interest to you please visit us on http://www.ximb.io

Apart from the quality issue, this points to a different issue- namely that basic scientists have a very hazy idea of what validation of an assay entails. Look at the amount of data which is required to validate a standard small molecule LCMS assay ( where the samples are cleaner and the method of detection is straightforward and predictable) and try to ask for something similar from a bench pharmacologist. You will be met with a blank stare.

@20, Emjeff
I’m a bench biochemist, but I’ve also done some forensic toxicology, and I know what you mean about the differences in validating assays in the two fields. And I agree that biologists should generally be more careful in that regard.
However, an important difference in the two situations is that the result of a validated small molecule LCMS assay should be dispositive on its own. The result of, say, a Western or immunocytochemical experiment *should* be just one of a number of lines of evidence.


DEVELOPING AFFINITY TAG TECHNOLOGIES

Some newer affinity tags have yet to be rigorously evaluated and consequently cannot be recommended above the tried-and-true affinity tags described above (see Commonly Used Affinity Tags). Still, many of these novel affinity tags contain unique properties that may make them quite useful in cases of troublesome purifications.

Small Ubiquitin-Like Modifier

Small ubiquitin-like modifier (SUMO) family proteins are

100 amino acids in length and are highly conserved in eukaryotes. They are essential for normal cell functions such as nuclear transport, signal transduction, and protein stabilization. SUMO proteins function by forming a covalent bond with a target protein, typically at a lysine residue, which is later cleaved by SUMO protease. SUMO protease recognizes the sequence x-Gly-Gly-x, and therefore doesn’t cleave the target protein. This aspect of SUMO proteases make SUMO an attractive affinity tag because no recombinant linker region needs to be constructed and the native N-terminus of the target protein is maintained. Other advantages of SUMO are that it increases protein expression and solubility. Several commercial kits are available that utilize both yeast and human SUMO proteins and proteases (Life Technologies and LifeSensors). Since SUMO is not expressed in prokaryotes, most kits use E. coli to express the protein and typically include a polyhistidine tag. After SUMO is cleaved, the protein can be isolated using polyhistidine antibodies, Ni-NTA or NI-IMAC or chromatography. One kit, SUMOstar, allows for the expression and purification of SUMO-tagged proteins in insect and mammalian cells.

HaloTag®

The HaloTag® (Promega) is a 33-kDa protein based on a modified haloalkane dehalogenase found in bacteria. In bacteria, native dehalogenase acts through a nucleophilic displacement reaction to remove halides from aliphatic hydrocarbons (Los et al., 2008). Asp106 is responsible for forming the enzyme-substrate complex while His272 is responsible for hydrolysis of the intermediate. A point mutation, His272Phe, impairs hydrolysis leading to a covalent bond between the enzyme and substrate. The commercially available HaloTag® has the His272Phe point mutation along with other amino acid substitutions that lead to increased ligand binding.

Although other hydrolytic enzymes could be modified, haloalkane dehalogenase has several advantages: it is a small protein that does not demand the use of cofactors or post-translational modifications for its enzymatic reaction. Also, ligands of haloalkane dehalogenase may be designed to diffuse through cell membranes, be nontoxic, and avoid cross-reactivity with other cellular proteins. (Los et al. 2008). For protein purification, an expression vector is transfected into a cell line in order to create a fusion protein. Synthetic HaloTag® ligand is then added to the cells to label the protein. The synthetic ligand can have one of many functional groups attached, including various epitope tags (e.g., GST). This versatility allows HaloTag® to be used not only for in vitro protein labeling and purification but also in vivo labeling. Cells are then lysed and the fusion protein captured on a purification matrix. The mutated hydryolase HaloTag® creates a covalent attachment to the HaloLink® resin via an immobilized chloroalkane. This covalent bond allows for harsher wash conditions on the matrix. After washing, the protein of interest is cleaved from HaloTag with the use of TEV protease. HisLink® resin is utilized to remove TEV Protease from the protein, and the result is a highly pure, tag-free protein.

Profinity eXact

The Profinity eXact™ system (Bio-Rad) is based on the serine protease subtilisin from Bacillus amyloliquefaciens. Subtilisin, a member of the subtilase protein family, is somewhat promiscuous in its substrate choices. The action of subtilisin is to bind to residues P1-P4 of its substrate, resulting in the interpolation of the substrate’s backbone between β-strands 100� and 125� of subtilisin (Biao et al. 2004). Engineering of subtilisin has resulted in a mutant subtilisin protein with reconstructed P1 and P4 binding pockets in order to increase sequence selectivity. Biosynthesis of subtilisin is dependent on a 75-amino acid N-terminal prodomain (Ruan et al., 2004). It is this sequence that has been modified to act as a tag. Using an expression vector, recombinant protein is created that contains the subtilisin prodomain tag on the N-terminus of the protein of interest. The recombinant protein is then applied to a column with immobilized subtilisin protease. Cleavage at the C-terminus of the tag by mature subtilisin protease is initiated by fluoride buffers and allows for the release of the purified target protein. The result is a protein with its native N-terminal that is tag-free.

Major advantages of the Profinity eXact™ system include that it does not require the use of any proteases. It allows for a quick 30 minute on-column purification and cleavage that can be performed in one step, enabling the purified proteins to maintain their native structure. Additionally, the column can be regenerated by removing the bound prodomain at pH = 2.1 (Biao et al., 2004). The subtilisin prodomain tag is known to work well with soluble heterotrimeric G-protein α-subunits, which are often problematic when attempting to purify or over-express them. A hydrophobic portion of the tag itself increases the solubility of the fusion G protein, and it has been shown that no structure or activation dynamics are altered during the purification process using the Profinity eXact™ system (Abdulaev et al., 2005).

PDZ domain-based tags

Post-synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 protein (zo-1) were the first three proteins discovered to share the PDZ domain, a small (

10 kDa) structural domain common across species. PDZ domains bind to the C-termini of their protein targets, and are often found in signal transduction pathways where they scaffold different signaling components together. The N-terminal PDZ domain (PDZ1) of the Drosophila protein InaD has been suggested as a potential protein tagging system, as it binds the C-terminus of its target, NorpA, via an intermolecular disulfide bond. Proteins with a NorpA tag could be eluted from a PDZ-coupled resin (or vice versa) via reduction of the disulfide bond (Kimple et al., 2001 Kimple and Sondek 2002).

The PDZ/C-terminal peptide interaction has recently been harnessed for other applications. A human papilloma virus (HPV) E6 protein dipstick test is being developed that utilizes the binding affinity between E6 PDZ and its C-terminal targets (Arbor Vita). Like the InaD/NorpA interaction, technique could also potentially be utilized to tag and detect heterologous proteins.


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