COVID-19 IgG Antibodies: Precise Measurement with the Kantaro COVID-SeroKlir Kit

The Kantaro COVID-SeroKlir kit from EKF Diagnostics is a direct enzyme-linked immunosorbent assay (ELISA) kit that enables quantitative identification of human antibodies to the COVID-19 virus in plasma (Li-Heparin/K2-EDTA) and serum specimens.

After receiving a CE mark in October 2020, the COVID-SeroKlir had also received the Emergency Use Authorization (EUA) from the FDA in November 2020. This kit has shown a sensitivity of 97.8% and a specificity of 99.6% for identifying COVID-19-specific IgG antibodies against a couple of virus antigens—the full-length spike protein and its receptor-binding domain.

The COVID-SeroKlir is a two-step ELISA that can be employed by all CLIA-certified laboratories without the necessity for proprietary devices.

The SeroKlir ELISA kit uses regular ELISA procedures and equipment and includes components to test as many as 630 samples from patients. The test involves a two-phase ELISA—the preliminary plate screens for RBD negative or positive specimen and the second plate uses full-length Spike protein to offer a quantitative result of the antibody titer or concentration.

Clinicians from the Icahn School of Medicine at Mount Sinai Health System, New York1 have developed the SeroKlir ELISA kit. The test has been trialed on a group of over 70,000 patients, including more than 30,000 who were diagnosed with the COVID-19 infection.2

Key features

  • Reduces false negatives and false positives
  • A sensitivity of 97.8% and a specificity of 99.6%
  • Includes 30,000 COVID-19 patient test data set
  • Has received FDA EUA and CE mark
  • Peer-reviewed in the Science and Nature journals

Kit specifications

Table 1. Source: EKF Diagnostics

Kit component Specification
7 x RBD plates 96 well polystyrene microplate coated with recombinant SARS-CoV-2 Spike protein RBD antigen sufficient for 630 screening tests
3 x spike plates 96 well polystyrene microplate coated with full length recombinant SARS-CoV-2 Spike protein sufficient for 228 quantitative tests
Controls RBD Positive Control
RBD Negative Control
Spike Low Control
Spike Mid Control
Spike High Control
Calibrators 8 calibrators (range 0-200 AU/mL)
Consumables RBD conjugate concentrate - IgG ELISA
Spike conjugate concentrate - IgG ELISA
Conjugate buffer - IgG ELISA
Sample buffer - IgG ELISA
TMB substrate - IgG ELISA
Stop solution - IgG ELISA
Wash buffer - IgG ELISA

 

Product Code: CVD2-2019

Performance data

Table 2. Source: EKF Diagnostics

  Performance specifications
Assay type Solid phase sandwich ELISA
Format 96 well one-piece plate
Assay length 3.5 hours (RBD ELISA)
3.5 hours (spike ELISA)
Sample types Serum (20 uL)
EDTA plasma (20 uL)
Heparin plasma (20 uL)
Assay range 3.2 – 160 AU/mL
Specificity 99.6%
Sensitivity 97.8%

 

Method

The two phase ELISA interrogates the full length Spike protein as well as its Receptor Binding Domain (RBD):

  • RBD is utilized in the first phase to detect antibody-negative samples
  • Full-length Spike protein is employed in the second phase to validate positive specimens and yield a quantitative antibody titer
COVID-19 IgG Antibodies: Precise Measurement with the Kantaro COVID-SeroKlir Kit

Image Credit: EKF Diagnostics

Analytical sensitivity

Analytical sensitivity—Limit of Quantitation (LoQ), Limit of Detection (LoD) and Limit of Blank (LoB) were defined in accordance with the recommendations in CLSI guideline EP17-A2. A summary data on Spike ELISA and RBD ELISA is given below.

Positive Percent Agreement—In the case of positive specimens validated with a familiar EUA-authorized molecular test, PPA was found to be 97.8%. Moreover, a couple of specimens that tested negative with the COVID-SeroKlir Kantaro Quantitative SARS-CoV-2 IgG Antibody Kit also tested negative on a prevalent EUA-approved serology test, indicating that these were real negative specimens.

Negative Percent Agreement—In the case of negative specimens, the NPA was noted to be 99.6%. A total of 14 specimens had tested positive on the RBD ELISA. Among these, 13 specimens have later tested negative on the Spike ELISA. Hence, the number of negative specimens was 281 out of 282.

Table 3. Source: EKF Diagnostics

Sensitivity RBD ELISA
(CI)
Spike ELISA
(AU/mL)
LoB LoD LoQ
0.70 0.82 ---
1.98 2.61 3.20

 

Video Credit: EKF Diagnostics

Clinical utility

A preliminary qualitative (screening) ELISA is conducted against the recombinant Receptor Binding Domain (RBD) of COVID-19. From this screen, positive specimens are studied with a quantitative ELISA against the full-length SARS-CoV-2 Spike protein.

The assay helps determine the quantitative amounts of neutralizing antibodies, suggesting an adaptive immune reaction to SARS-CoV-2 in patients assumed to have prior SARS-CoV-2 infection, or for identifying IgG seroconversion in patients after the known recent COVID-19 infection.

Finding out the number of people who are shown to have developed the SARS-CoV-2-specific antibodies helps determine seroprevalence in any group of exposed individuals or geographic area and may indicate the probable risk of reinfection. The outcomes of the assay correspond with the neutralization of the SARS-CoV-2 virus in vitro.

But the results obtained from the COVID-SeroKlir Kantaro Quantitative SARS-CoV-2 IgG Antibody Kit must not be used as the only means for diagnosis and must not be used for diagnosing people who have severe COVID-19 infection.

Results are only meant for identifying SARS-CoV-2 IgG antibodies. SARS-CoV-2-specific IgG antibodies are usually detected at 10 to 14 days subsequent to infection but are likely to occur later. The existence of IgG antibodies, after the earlier negative testing, defines IgG antibody seroconversion post the SARS-CoV-2 infection.

Furthermore, negative results do not preclude severe SARS-CoV-2 infection and must not be taken as the only basis to reach decisions on patient management.

IgG antibodies are not likely to be present for over two weeks after infection and patients may continue to remain infectious at the time of severe acute infection even in the presence of the IgG antibody. Outcomes should be integrated with patient history, clinical observations and epidemiological data. The sensitivity of the COVID-SeroKlir Kantaro Quantitative SARS-CoV-2 IgG Antibody Kit early after the infection is not known.

References

  1. Amanat et al. Nature Medicine 26 2033-1036 (2020).
  2. Wajnberg et al. Science 10.1126/science.abd7728 (2020).