COVID-SeroIndex, Kantaro SARS-COV-2 IgG Antibody RUO Kit

RUO Quantitative ELISA. Patent Pending

COVID-SeroIndex

COVID-SeroIndex is a quantitative ELISA kit that enables an objective measurement of SARS-CoV-2 IgG antibodies which are indicative of a prior COVID-19 infection. This kit is configured and optimized to support research associated with the development of vaccines for COVID-19, serving as a tool to provide performance data during research and development phases. Validation studies have demonstrated a specificity of 99.8% and a sensitivity of 97.8%.
SARS-CoV-2 IgG serological ELISA Kit
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COVID-SeroIndex, Kantaro SARS-COV-2 IgG Antibody RUO Kit Summary

Sample Type & Volume Required Per Well Serum (10 uL), EDTA Plasma (10 uL), Heparin Plasma (10 uL)
Sensitivity 3.2 AU/mL
Assay Range 3.2 - 160 AU/mL (Serum, EDTA Plasma, Heparin Plasma)
Specificity Natural and recombinant human anti-SARS-CoV-2 IgG
Cross-reactivity < 0.5% cross-reactivity observed with available related molecules.< 50% cross-species reactivity observed with species tested.
Interference No significant interference observed with available related molecules.

Product Summary

COVID-SeroIndex is a 2-phase ELISA optimized for accurate detection of SARS-CoV-2 IgG antibodies, present in human plasma or serum samples. Validation studies have demonstrated a specificity of 99.8% and a sensitivity of 97.8%. Sufficient reagents are provided to test 360 samples per kit.Phase 1 is a qualitative ELISA in which negative samples are eliminated from further analysis. Phase 2 is a quantitative ELISA confirming positive samples and providing an antibody titer for SARS-CoV-2 IgG antibodies present in the sample.

Precision

Intra-Assay Precision (Precision within an assay)
Three samples of known concentration were tested on one plate to assess intra-assay precision.

Inter-Assay Precision (Precision between assays)
Three samples of known concentration were tested in separate assays to assess inter-assay precision.

RBD ELISA

  Intra-Assay Precision Inter-Assay Precision
Sample 1 2 3 1 2 3
n 3 3 3 18 18 18
Mean (CI) 0.565 0.914 1.58 0.616 0.862 1.53
Standard Deviation 0.029 0.011 0.020 0.047 0.039 0.123
CV (%) 5.1 1.2 1.3 7.6 4.5 8.0

Spike ELISA

  Intra-Assay Precision Inter-Assay Precision
Sample 1 2 3 1 2 3
n 3 3 3 18 18 18
Mean (AU/mL)* 3.44 57.2 109 3.47 58.5 109
Standard Deviation 0.080 3.03 7.49 0.093 5.09 12.7
CV (%) 2.3 5.3 6.9 2.7 8.7 11.7
*Spike values were not multiplied by the dilution factor.

Linearity

Linearity was demonstrated according to recommendations in CLSI guideline EP06-A. Three individual samples were proportionally diluted with blank serum samples. The blank serum samples used to make the dilutions were preCOVID-19 samples collected prior to September 2019.

The linear range is 3.1–160 AU/mL and the Analytical Measuring Range (AMR) is 3.2-161 AU/mL.

Sample # Dilution Levels
in the Linear Range
Linear Range (AU/mL) Regression Equation Correlation
Coefficient (R2)

1

10 4.16-161 AU/mL = 0.00819 + 226.9 x (Dilution Factor) 0.98
2 10 8.19-145 AU/mL = 4.228 + 245.6 x (Dilution Factor) 0.99
3 9 3.08-75.1 AU/mL = 0.215 + 173.7 x (Dilution Factor) 0.98

Background: SARS-CoV-2

SARS-CoV-2, which causes the global pandemic coronavirus disease 2019 (Covid-19), belongs to a family of viruses known as coronaviruses that are commonly comprised of four structural proteins: Spike protein(S), Envelope protein (E), Membrane protein (M), and Nucleocapsid protein (N) (1). SARS-CoV-2 Spike Protein (S Protein) is a glycoprotein that mediates membrane fusion and viral entry. The S protein is homotrimeric, with each ~180-kDa monomer consisting of two subunits, S1 and S2 (2). In SARS-CoV-2, as with most coronaviruses, proteolytic cleavage of the S protein into two distinct peptides, S1 and S2 subunits, is required for activation. The S1 subunit is focused on attachment of the protein to the host receptor while the S2 subunit is involved with cell fusion (3-5). Based on structural biology studies, the receptor binding domain (RBD), located in the C-terminal region of S1, can be oriented either in the up/standing or down/lying state (6). The standing state is associated with higher pathogenicity and both SARS-CoV-1 and MERS can access this state due to the flexibility in their respective RBDs. A similar two-state structure and flexibility is found in the SARS-CoV-2 RBD (7). Based on amino acid (aa) sequence homology, the SARS-CoV-2 S1 subunit RBD has 73% identity with the RBD of the SARS-CoV-1 S1 RBD, but only 22% homology with the MERS S1 RBD. The low aa sequence homology is consistent with the finding that SARS and MERS bind different cellular receptors (8). The S Protein of the SARS-CoV-2 virus, like the SARS-CoV-1 counterpart, binds Angiotensin-Converting Enzyme 2 (ACE2), but with much higher affinity and faster binding kinetics (9). Before binding to the ACE2 receptor, structural analysis of the S1 trimer shows that only one of the three RBD domains in the trimeric structure is in the "up" conformation. This is an unstable and transient state that passes between trimeric subunits but is nevertheless an exposed state to be targeted for neutralizing antibody therapy (10). Polyclonal antibodies to the RBD of the SARS-CoV-2 protein have been shown to inhibit interaction with the ACE2 receptor, confirming RBD as an attractive target for vaccinations or antiviral therapy (11). There is also promising work showing that the RBD may be used to detect presence of neutralizing antibodies present in a patient's bloodstream, consistent with developed immunity after exposure to the SARS-CoV-2 virus (12). Lastly, it has been demonstrated the S Protein can invade host cells through the CD147/EMMPRIN receptor and mediate membrane fusion (13, 14).

COVID-SeroIndex is a 2-phase ELISA optimized for accurate detection of SARS-CoV-2 IgG antibodies, present in human plasma or serum samples. Validation studies have demonstrated a specificity of 99.8% and a sensitivity of 97.8%. Sufficient reagents are provided to test 360 samples per kit.Phase 1 is a qualitative ELISA in which negative samples are eliminated from further analysis. Phase 2 is a quantitative ELISA confirming positive samples and providing an antibody titer for SARS-CoV-2 IgG antibodies present in the sample.

Specifications

Source
N/A
Shipping Conditions
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Applications
ELISA
Species
SARS-CoV-2

Product Datasheets

Data Example

COVID-SeroIndex, Kantaro Quantitative SARS-CoV-2 IgG Antibody RUO Kit Standard Curve

COVID-SeroIndex correlation of quantitative Spike IgG antibodies to viral neutralization. A study was conducted to correlate the quantitative levels of anti-Spike protein IgG antibodiesto viral neutralization in a microneutralization (MN) assay. 120 patient samples with levels ofantibodies across the AMR of the assay were evaluated in a MN assay. V Information on the format and interpretation of the MNassay can be found in the following reference: Amanat, F., et. al., “A Serological Assay to DetectSARS-CoV-2 Seroconversion in Humans”; Nature Medicine. 2020 May 12. PMID: 32398876.

COVID-SeroIndex Detection Antibody Class Specificity Class specificity of the monoclonal detection antibody was evaluated in an antigen-downELISA study. Ten antigens, including seven different human IgG samples, were diluted to25 ng/mL or 100 ng/mL (not shown) and coated on a plate. A dilution series of the monoclonaldetection antibody was incubated on the plate prior to detection. Summary data indicates thatthe monoclonal detection antibody detects human IgG isotypes and has minimal detection ofhuman IgA or IgM that approaches level of the blank with titration.

Background: Spike RBD

References
1.   Wu, F. et al. (2020) Nature 579:265.
2.   Tortorici, M.A. and D. Veesler (2019). Adv. Virus Res. 105:93.
3.   Bosch, B.J. et al. (2003). J. Virol. 77:8801.
4.   Belouzard, S. et al. (2009) Proc. Natl. Acad. Sci. 106:5871.
5.   Millet, J.K. and G. R. Whittaker (2015) Virus Res. 202:120.
6.   Yuan, Y. et al. (2017) Nat. Commun. 8:15092.
7.   Walls, A.C. et al. (2010) Cell 180:281.
8.   Jiang, S. et al. (2020) Trends. Immunol. 
      https://doi.org/10.1016/j.it.2020.03.007.
9.   Ortega, J.T. et al. (2020) EXCLI J. 19:410.
10. Wrapp, D. et al. (2020) Science 367:1260.
11. Tai, W. et al. (2020) Cell. Mol. Immunol. 
      https://doi.org/10.1016/j.it.2020.03.007.
12. Okba, N. M. A. et al. (2020). Emerg. Infect. Dis. 
      https://doi.org/10.3201/eid2607.200841.
13. Wang, X. et al. (2020) https://doi.org/10.1038/s41423-020-0424-9.
14. Wang, K. et al. (2020) bioRxiv 
      https://www.biorxiv.org/content/10.1101/2020.03.14.988345v1.
Long Name
Spike Receptor Binding Domain
Entrez Gene IDs
3200426 (HCoV-HKU1); 1489668 (SARS-CoV); 43740568 (SARS-CoV-2)
Alternate Names
2019-nCoV Peplomer protein; 2019-nCoV S Protein; 2019-nCoV Spike; COVID-19 Spike; E2; Human coronavirus spike glycoprotein; Peplomer protein; S glycoprotein; S protein; SARS-COV-2 E2; SARS-COV-2 Peplomer protein; SARS-COV-2 S protein; SARS-COV-2 Spike glycoprotein; SARSCOV2 Spike protein; SARS-CoV-2; Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein; Spike glycoprotein; Spike RBD; Spike S1 RBD; Spike S1; Spike; surface glycoprotein

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