View Featured Offers >>
88005
SARS-CoV-2 Spike Protein Multi-Domain (S1-NTD, RBD, S1, S2) Serological IgG ELISA Kit
ELISA Kits
ELISA Kit
  1. Products
  2. ELISA Kits
  3. SARS-CoV-2 Spike Protein Multi-Domain (S1-NTD, RBD, S1, S2) Serological IgG ELISA Kit

SARS-CoV-2 Spike Protein Multi-Domain (S1-NTD, RBD, S1, S2) Serological IgG ELISA Kit #88005

Citations (0)
Patient Testing: Patient samples were tested using the SARS-CoV-2 Spike Protein Multi-Domain (S1-NTD, RBD, S1, S2) Serological IgG ELISA Kit #88005. Serum or plasma was obtained from donors who had been diagnosed with SARS-CoV-2 (diagnosed positive n=59*) or from presumed uninfected donors collected prior to the SARS-CoV-2 outbreak (presumed negative n=86). Samples were heat-inactivated (56°C for 30 min) and diluted 1:400 prior to running the assay, as described in the protocol. Samples were considered positive, negative, or inconclusive based on the criteria described in the “Data Analysis” section of the protocol for the kit. Positive percent agreement (PPA) and negative percent agreement (NPA) were calculated from these data.

*Positive reference samples were from patient donors with a positive SARS-CoV-2 diagnosis. However, a positive SARS-CoV-2 diagnosis will not always correlate with a positive response aganst each spike domain protein in this IgG Serological ELISA as differences in disease severity, timing of sample collection relative to disease onset, and patient profiles may affect presence and abundance of antibodies against each spike domain in the reference sample.

Note: We are continuing to test more samples as available. For the most up-to-date set of data, always refer to the product page for #88005 on the website.

Intra-Assay Precision: Three different serum samples were each tested in 16 replicates using a single assay kit of SARS-CoV-2 Spike Protein Multi-Domain (S1-NTD, RBD, S1, S2) Serological IgG ELISA Kit #88005. Intra-assay CV (%) was calculated for each sample, and each replicate was correctly identified as being positive or negative when compared to the Negative Control using the cutoff criteria described in the attached protocol.
Inter-Assay Precision: Six different assay kits from one lot of material were tested using 3 different serum samples, Positive Control, and Negative Control run in duplicate wells. Inter-assay CV (%) was calculated for each sample, and each assay kit correctly identified the samples as being positive or negative when compared to the Negative Control using the cutoff criteria described in the attached protocol.

*The included Positive Control in the kit is intended to give a positive signal for the RBD and S1 coated microwell strips, and is NOT a Positive Control for the S1-NTD or S2 microwells.

Plate map for SARS-CoV-2 Spike Protein Multi-Domain (S1-NTD, RBD, S1, S2) Serological IgG ELISA Kit #88005. Schematic representation of a 96-well plate depicting the color-code of the reagents used to detect binding of human IgG to four domains of SARS-CoV-2 spike protein (S-protein): the S1-NTD (blue, columns 1-3), RBD (orange, columns 4-6), S1 (gray, columns 7-9), and S2 (black, columns 10-12).
Spike Protein S1-NTD Serological IgG ELISA Patient Testing Results: Patient samples were tested using the SARS-CoV-2 Spike Protein Multi-Domain (S1-NTD, RBD, S1, S2) Serological IgG ELISA Kit #88005. Serum/plasma samples were heat-inactivated (56°C for 30 min) and diluted 1:400 prior to running the assay, as described in the protocol. Background subtracted absorbance values at 450 nm are plotted for each serum/plasma sample, with values corresponding to samples obtained from donors who had been diagnosed with SARS-CoV-2 (diagnosed positive n=59*) on the right, and values corresponding to samples from presumed uninfected donors collected prior to the SARS-CoV-2 outbreak (presumed negative n=86) on the left. Also shown is the cutoff line above which samples are considered positive (blue line), and the negative cutoff line (red line), below which samples are considered negative. Cutoff values were calculated as described in the “Data Analysis” section of the protocol for the kit.

*Positive reference samples were from patient donors with a positive SARS-CoV-2 diagnosis. However, a positive SARS-CoV-2 diagnosis will not always correlate with a positive response against each spike domain protein in this IgG Serological ELISA as differences in disease severity, timing of sample collection relative to disease onset, and patient profiles may affect presence and abundance of antibodies against each spike domain in the reference sample.

Spike Protein RBD Serological IgG ELISA Patient Testing Results: Patient samples were tested using the SARS-CoV-2 Spike Protein Multi-Domain (S1-NTD, RBD, S1, S2) Serological IgG ELISA Kit #88005. Serum/plasma samples were heat-inactivated (56°C for 30 min) and diluted 1:400 prior to running the assay, as described in the protocol. Background subtracted absorbance values at 450 nm are plotted for each serum/plasma sample, with values corresponding to samples obtained from donors who had been diagnosed with SARS-CoV-2 (diagnosed positive n=59*) on the right, and values corresponding to samples from presumed uninfected donors collected prior to the SARS-CoV-2 outbreak (presumed negative n=86) on the left. Also shown is the cutoff line above which samples are considered positive (blue line), and the negative cutoff line (red line), below which samples are considered negative. Cutoff values were calculated as described in the “Data Analysis” section of the protocol for the kit.

*Positive reference samples were from patient donors with a positive SARS-CoV-2 diagnosis. However, a positive SARS-CoV-2 diagnosis will not always correlate with a positive response against each spike domain protein in this IgG Serological ELISA as differences in disease severity, timing of sample collection relative to disease onset, and patient profiles may affect presence and abundance of antibodies against each spike domain in the reference sample.

Spike Protein S1 Domain Serological IgG ELISA Patient Testing Results: Patient samples were tested using the SARS-CoV-2 Spike Protein Multi-Domain (S1-NTD, RBD, S1, S2) Serological IgG ELISA Kit #88005. Serum/plasma samples were heat-inactivated (56°C for 30 min) and diluted 1:400 prior to running the assay, as described in the protocol. Background subtracted absorbance values at 450 nm are plotted for each serum/plasma sample, with values corresponding to samples obtained from donors who had been diagnosed with SARS-CoV-2 (diagnosed positive n=59*) on the right, and values corresponding to samples from presumed uninfected donors collected prior to the SARS-CoV-2 outbreak (presumed negative n=86) on the left. Also shown is the cutoff line above which samples are considered positive (blue line), and the negative cutoff line (red line), below which samples are considered negative. Cutoff values were calculated as described in the “Data Analysis” section of the protocol for the kit.

*Positive reference samples were from patient donors with a positive SARS-CoV-2 diagnosis. However, a positive SARS-CoV-2 diagnosis will not always correlate with a positive response against each spike domain protein in this IgG Serological ELISA as differences in disease severity, timing of sample collection relative to disease onset, and patient profiles may affect presence and abundance of antibodies against each spike domain in the reference sample.

Spike Protein S2 Domain Serological IgG ELISA Patient Testing Results: Patient samples were tested using the SARS-CoV-2 Spike Protein Multi-Domain (S1-NTD, RBD, S1, S2) Serological IgG ELISA Kit #88005. Serum/plasma samples were heat-inactivated (56°C for 30 min) and diluted 1:400 prior to running the assay, as described in the protocol. Background subtracted absorbance values at 450 nm are plotted for each serum/plasma sample, with values corresponding to samples obtained from donors who had been diagnosed with SARS-CoV-2 (diagnosed positive n=59*) on the right, and values corresponding to samples from presumed uninfected donors collected prior to the SARS-CoV-2 outbreak (presumed negative n=86) on the left. Also shown is the cutoff line above which samples are considered positive (blue line), and the negative cutoff line (red line), below which samples are considered negative. Cutoff values were calculated as described in the “Data Analysis” section of the protocol for the kit.

*Positive reference samples were from patient donors with a positive SARS-CoV-2 diagnosis. However, a positive SARS-CoV-2 diagnosis will not always correlate with a positive response against each spike domain protein in this IgG Serological ELISA as differences in disease severity, timing of sample collection relative to disease onset, and patient profiles may affect presence and abundance of antibodies against each spike domain in the reference sample.

Heatmap depicting SARS-CoV-2 Spike Protein Multi-Domain (S1-NTD, RBD, S1, S2) Serological IgG ELISA Kit #88005 patient testing results. Shown are the results for a subset of the patient serum/plasma samples that were tested for the presence of IgG antibodies against S1-NTD, RBD, S1, and/or S2 using kit #88005. Absorbance values (OD450 Value) corresponding to wells containing one of the four SARS-CoV-2 spike protein domains were used to generate the heatmap. Serum/plasma samples from a subset of donors who had been diagnosed with SARS-CoV-2 (diagnosed positive*) and from presumed uninfected donors collected prior to the SARS-CoV-2 outbreak (presumed negative) were heat-inactivated (56°C for 30 min) and diluted 1:400 prior to running the assay, as described in the protocol.

NOTE: Absorbance 450 nm values should not be directly compared across different spike domain proteins, as they are not absolute values.

*Positive reference samples were from patient donors with a positive SARS-CoV-2 diagnosis. However, a positive SARS-CoV-2 diagnosis will not always correlate with a positive response against each spike domain protein in this IgG Serological ELISA as differences in disease severity, timing of sample collection relative to disease onset, and patient profiles may affect presence and abundance of antibodies against each spike domain in the reference sample.

Image Gallery
Learn more about how we get our images
To Purchase # 88005
Cat. # Size Qty. Price
88005C		 1 Kit  (96 assays)

Custom Formulation

Important Ordering Details

Custom Ordering Details:

When ordering five or more kits, please contact us for processing time and pricing.

Looking for this ELISA kit in a 384-well format? Inquire for availability, processing time, and pricing.

Supporting Data

REACTIVITY H

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • GP-Guinea Pig
  • Rab-Rabbit
  • All-All Species Expected
Product Includes Volume Solution Color
Spike Multi-Domain Protein Coated Microwells 96 tests
Anti-Human IgG, HRP-linked Antibody (ELISA Formulated) 1 ea Red (Lyophilized)
Sample Diluent A 71637 25 ml
HRP Diluent 11 ml Red
ELISA Wash Buffer (20X) 9801 25 ml
TMB Substrate 7004 11 ml
STOP Solution 7002 11 ml
Sealing Tape 2 ea
ELISA Kit #88005 Positive Control 1 ea
ELISA Kit #88005 Negative Control 1 ea

Product Description

The SARS-CoV-2 Spike Protein Multi-Domain (S1-NTD, RBD, S1, S2) Serological IgG ELISA Kit is a solid-phase ELISA that detects binding of human IgG to four domains of SARS-CoV-2 spike protein (S-protein): the S1-NTD, RBD, S1, and S2 domains, individually. The four spike protein domains have each been individually coated onto microwells (see plate map for location/color-coding), such that twenty-four tests are provided for each spike protein domain (96 tests total). After incubation with sample, the human IgG specific for each spike protein domain is captured on the plate. The wells are then washed to remove unbound material. Anti-Human IgG, HRP-linked antibody is then used to recognize the bound IgG. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of IgG specific for each spike protein domain.

*Antibodies in this kit are custom formulations specific to kit.

Protocol

PRINT

View >Collapse >

SARS-CoV-2 Spike Protein Multi-Domain (S1-NTD, RBD, S1, S2) Serological IgG Sampler ELISA Protocol

This ELISA Kit Is Intended For Research Use Only. Not For Use in Diagnostic or Clinical Procedures.

A. Solutions and Reagents

NOTE: Prepare solutions with deionized/purified water or equivalent. Prepare only as much reagent as needed on the day of the experiment.

  1. Spike Multi-Domain Protein Coated Microwells: Bring all to room temperature before opening bag/use. Unused microwell strips should be returned to the original re-sealable bag containing the desiccant pack and stored at 4°C. Microwell strips are color-coded for each spike protein domain (see plate map below).

    Plate Map

  2. 1X ELISA Wash Buffer: Prepare by diluting ELISA Wash Buffer (20X) (included in each kit) to 1X with deionized water.
  3. Sample Diluent A: Diluent provided for dilution of samples and for reconstitution of Positive and Negative Controls included in kit.
  4. HRP Diluent: Red colored diluent for reconstitution and dilution of the Anti-Human IgG, HRP-linked Antibody (11 mL provided).
  5. Anti-Human IgG, HRP-linked Antibody (ELISA Formulated): Supplied lyophilized as a red colored cake or powder. Add 1.0 mL of HRP Diluent (red solution) to yield a concentrated stock solution. Incubate at room temperature for 5 min with occasional gentle mixing to fully reconstitute. To make the final working solution, add the full 1.0 mL volume of reconstituted HRP-linked Antibody to 10.0 mL of HRP Diluent in a clean tube and gently mix. For best results, use this working solution immediately. Unused working solution may be stored for up to 4 weeks at 4°C, although there may be some loss of signal compared to freshly made solution.

  6. Positive Control: Reconstitute the vial of lyophilized Positive Control with 1.0 mL Sample Diluent A. Mix thoroughly and gently, hold at room temperature for 1 minute and then follow the steps outlined below in the “Test Procedure” section. Positive Controls are recommended to be used immediately after reconstituting, however remaining material may be stored at -80°C (there may be some loss of the Positive Control signal if freeze/thawed). Positive Controls are supplied as a control reagent, not as an absolute quantitation measure.

    7. IMPORTANT: This control is intended to give a positive signal for the RBD and S1 coated microwell strips, and is NOT a positive control for the S1-NTD or S2 microwells.

  7. Negative Control: Reconstitute the vial of lyophilized Negative Control with 1.0 mL Sample Diluent A. Mix thoroughly and gently, hold at room temperature for 1 minute and then follow the steps outlined below in the “Test Procedure” section. Negative Controls are recommended to be used immediately after reconstituting, however remaining material may be stored at -80°C (there may be some loss of the Negative Control signal if freeze/thawed).
  8. TMB Substrate (#7004): Bring to room temperature before use.
  9. STOP Solution (#7002): Bring to room temperature before use.

B. Test Procedure

NOTE: Equilibrate all materials and prepared reagents to room temperature prior to running the assay.

  1. Prepare all reagents as indicated above (Section A).

  2. Human-sourced samples should be handled in accordance with accepted safety practices. Samples should be diluted at least 1:400 with Sample Diluent A and can be further serially diluted if relative quantification is needed by the user. Positive and Negative Controls do NOT need to be diluted after reconstitution. Refer to the datasheet which shows typical results observed for the Positive Control, Negative Control, serum from uninfected individuals, and serum from SARS-CoV-2 patients. When using the cutoff criteria described below to determine if a sample is positive for anti-CoV-2 Spike Protein antibodies, samples diluted 1:400 must be compared to the undiluted Negative Control.

    3. NOTE: Sample storage/handling, including heat-inactivation of samples, can potentially affect observed signals. Therefore, it is strongly recommended that in addition to the Positive and Negative Controls included with the kit, the user includes their own negative and positive patient samples as controls when running the assay in order to establish an appropriate cutoff value.

  3. Add 100 µL of each diluted sample, Positive Control, Negative Control, and blank (Sample Diluent A only) to the appropriate wells. Seal the plate with the supplied sealing tape and incubate for 1 hour at 37°C.
  4. Gently remove the tape and wash wells:
    1. Discard plate contents into a receptacle.
    2. Wash 4 times with 1X ELISA Wash Buffer, 200 µL each time for every well. After each wash, aspirate or decant from wells. Invert the plate and blot it against clean paper towels to remove the residual solution in each well, but do not allow wells to completely dry at any time.
    3. Clean the underside of all wells with a lint-free tissue.
  5. Add 100 µL of reconstituted Anti-Human IgG, HRP-linked Antibody (ELISA Formulated). Seal with tape and incubate the plate for 30 min at 37°C.
  6. Repeat wash procedure (Section B, Step 4).
  7. Add 100 µL of TMB Substrate to each well. Seal with tape and incubate the plate in the dark for 10 min at 37°C.

  8. Add 100 µL of STOP Solution to each well. Shake gently for a few seconds.

    9. NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.

  9. Read results:
    1. Visual Determination: Read within 30 min after adding STOP Solution.
    2. Spectrophotometric Determination: Wipe underside of wells with a lint-free tissue. Read absorbance at 450 nm within 30 min after adding STOP Solution.
  10. Data Analysis:
    1. Subtract “blank” well (Sample Diluent A only) absorbance 450 nm values from sample, Positive, and Negative Control values.
    2. Positive Control Values should be > 1.0 for S1 and RBD microwell strips.
    3. Samples (1:400 dilution) are considered positive, negative, or inconclusive by using the following multipliers relative to the blank subtracted Negative Control absorbance 450 nm values.

      Positive Criteria Negative Criteria Inconclusive Criteria
      S1-NTD > 1.7 x Negative Control < 1.35 x Negative Control > 1.35 x Negative Control and < 1.7 x Negative Control
      RBD > 3.4 x Negative Control < 2.7 x Negative Control > 2.7 x Negative Control and < 3.4 x Negative Control
      S1 > 4 x Negative Control < 3 x Negative Control > 3 x Negative Control and < 4 x Negative Control
      S2 > 4.1 x Negative Control < 3.1 x Negative Control > 3.1 x Negative Control and < 4.1 x Negative Control

    4. Limitations: Experimental cutoffs were determined by assaying a set of SARS-CoV-2 positive samples (from donors with positive SARS-CoV-2 diagnosis) and uninfected donor serum collected prior to the SARS-CoV-2 pandemic.

      NOTE: Positive reference samples were from patient donors with a positive SARS-CoV-2 diagnosis. However, a positive SARS-CoV-2 diagnosis will not always correlate with a positive response against each spike domain protein in this IgG Serological ELISA as differences in disease severity, timing of sample collection relative to disease onset, and patient profiles may affect presence and abundance of antibodies against each spike domain in the reference sample.

      Researchers can establish or modify this cutoff using additional samples. Positive or negative results from this assay should not be the sole basis for determining the infection status of a sample. A negative result can occur in SARS-CoV-2 patient samples due to:

      • improper sample handling/storage
      • timing of sample collection post-infection
      • patients having impaired immune function

      NOTE: Absorbance 450 nm values should not be directly compared across different spike domain proteins, as they are not absolute values.

posted September 2020

Protocol Id: 2306

Specificity / Sensitivity

The SARS-CoV-2 Spike Protein Multi-Domain (S1-NTD, RBD, S1, S2) Serological IgG ELISA Kit detects endogenous levels of human IgG directed to the S1-NTD (16-316), RBD (318-541), S1 (16-681), and S2 (686-1208) domains of SARS-CoV-2 spike protein (S-protein).

Background

The cause of the COVID-19 pandemic is a novel and highly pathogenic coronavirus, termed SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2). SARS-CoV-2 is a member of the Coronaviridae family of viruses (1). The genome of SARS-CoV-2 is similar to other coronaviruses, and is comprised of four key structural proteins: S, the spike protein, E, the envelope protein, M, the membrane protein, and N, the nucleocapsid protein (2). Coronavirus spike proteins are class I fusion proteins and harbor an ectodomain, a transmembrane domain, and an intracellular tail (3,4). The highly glycosylated ectodomain projects from the viral envelope surface and facilitates attachment and fusion with the host cell plasma membrane. The ectodomain can be further subdivided into host receptor-binding domain (RBD) (S1) and membrane-fusion (S2) subunits, which are produced upon proteolysis by host proteases at S1/S2 and S2’ sites. S1 and S2 subunits remain associated after cleavage and assemble into crown-like homotrimers (2,4). In humans, both SARS-CoV and SARS-CoV-2 spike proteins utilize the angiotensin-converting enzyme 2 (ACE2) protein as a receptor for cellular entry (5-7). Spike protein subunits represent a key antigenic feature of coronavirus virions, and therefore represent an important target of vaccines, novel therapeutic antibodies, and small-molecule inhibitors (8,9).

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not for Use in Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit our Trademark Information page.