Quantitative antibody titers without multiple dilutions
Fully validated for both single well and duplicate well samples
- SARS-CoV-2 spike protein Receptor Binding Domain (RBD)
- Target antigen selected to correlate with neutralizing antibody
- Measurement against pre-coated standards
- 96-well plate with 76 patient samples (singlet); 38 patient samples (duplicate)
- Quantitative antibody titers with positive-negative determination, no multiple dilutions
- For use with human serum, citrate plasma or EDTA plasma
- Manual or automated plate processing
The test is listed on the FDA website and may be viewed there by scrolling to “What Laboratories and Manufacturers are Offering Tests for COVID-19“, then opening “Q: What commercial manufacturers are distributing serology test kits under the policy outlined in Section IV.D of the Policy for Coronavirus Disease-2019 Tests?“, then scrolling down the list to Akston Biosciences. The AntiCoV-ID™ IgG ELISA is registered with the FDA under product code QKO, with the Device Listing number D409900 under Akston Biosciences’ Establishment Registration Number 3010002636.
Akston has submitted a request for emergency use authorization (“EUA”) for the Anti-CoV-ID™ IgG ELISA Kit, but FDA has not completed review of the test. Pending this review, it is distributed as an in vitro diagnostic device (“IVD”) in the United States under Section IV.D of the Policy for Diagnostic Tests for Coronavirus Disease-2019 during the Public Health Emergency.
AntiCoV-ID™ IgG ELISA is an indirect, enzyme linked immunosorbent assay (ELISA) designed to measure anti-spike protein RBD antibodies against the SARS-CoV-2 virus (COVID-19 virus, 2019 Novel Coronavirus) in human patient serum and plasma samples, including heat-inactivated serum or heat-inactivated plasma. The indirect immunoassay uses a recombinant SARS-CoV-2 spike protein RBD immobilized on ELISA plates as the capture antigen to bind the SARS-CoV-2 specific anti-spike RBD antibodies in the serum samples when incubated in the microplate wells. A simple wash step removes all unbound proteins, leaving the anti-SARS-CoV-2 spike protein RBD antibodies bound to the plate.
A second incubation is performed where the anti-spike protein RBD antibodies are detected by an anti-human IgG antibody (not cross-reactive to IgM) that is conjugated to horseradish peroxidase (HRP). After a second simple wash step to remove the unbound enzyme-conjugate, the assay plate wells are incubated with 3,3’,5,5’-trimethylbenzidine which causes a colorimetric change that is proportional to the amount of bound enzyme conjugate in each well. The color development is stopped by adding acid that halts development, and the color density of each well is measured using a spectrophotometric microplate reader.
Overview of Test Procedure
|Prepare diluted patient samples, suggested 1:100 dilution in Sample Dilution Buffer (SDB)||1st Dilution: |
10 µL sample + 90 µL SDB;
Duplicate sample: (25 µL of 1:10 solution + 225 µL of SDB to achieve 250 µL at 1:100 dilution)
OR singlet sample: (12 µL of 1:10 solution + 108 µL of SDB to achieve 120 µL at 1:100 dilution)
|Add Sample Dilution Buffer to Calibrator Strips||100 µL/well|
|Add Assay Controls 1 and 2, add diluted Samples||100 µL/well|
|Incubate||1 hour, at room temperature (without shaking)|
|Wash plate with 1X Wash Buffer||300 µL/well, 5 times|
|Add 1X Enzyme Conjugate||100 µL/well|
|Incubate||1 hour, at room temperature, protect from light (without shaking)|
|Wash plate with 1X Wash Buffer||300 µL/well, 5 times|
|Wash plate with Deionized Water||300 µL/well, once only|
|Add TMB Substrate||100 µL/well|
|Incubate||25 minutes or longer at room temperature, protect from light|
|Add Stop Solution||100 µL/well|
|Measure absorbance at 450 nm||Calculate results|
|Validation Status||The AntiCoV-ID™ IgG ELISA has been fully validated within Akston’s Quality System, passing all technical and clinical validation parameters. |
The assay was validated in both single well and duplicate well sample analysis format, and this validation data was included in Akston’s EUA package to the FDA.
|Analytical Sensitivity||The limit of detection is 0.0125 µg/mL.|
|Analytical Specificity||Cross reactivity to other coronaviruses has been shown to be minimal likely due to the low sequence homology between the SARS-CoV-2 spike protein RBD region and other coronavirus spike protein sequences. However, there could be some degree of cross-reactivity to antibodies present in serum or plasma from patients who were exposed to the original SARS-CoV virus that occurred ~15 years ago.|
|Spike Recovery||Serum from 30 normal serum donors was spiked with a recombinant anti-SARS-CoV-2 antibody and recovery was calculated vs. un-spiked serum run in parallel. Recovery ranged between 90% and 107% across all samples tested.|
|Dilutional Linearity||Serum from several COVID-19 positive donors was serially diluted and assayed from 1:100 to 1:10,800 to determine dilutional linearity. Samples diluted from 1:100 to at least 1:1,200 and up to 1:3,600 for high concentration samples showed linearity, indicating the capability of the assay for detecting a broad range of antibody titers in patient samples without any matrix interference.|
|Stability||Assay plates were incubated for up to 3 days at 50°C (equivalent to ~6 weeks stability at 2-8°C) for accelerated stability testing. Incubation at 50°C had no effect on assayed values for antibody controls or negative serum background. Additional stability studies are ongoing in order to further measure the kit’s shelf life.|
|Clinical Sensitivity and Specificity||N=34 of 35 (Sensitivity/PPA = 97.1%) serum samples from COVID-19 patients were above the positive/negative cutoff values and N=78 of 80 (Specificity/NPA = 97.5%) normal donors were below the assay cutoff value.|
The assay cutoff value was set during validation studies conducted within Akston’s Quality System with a library of normal serum samples. Clear differences were seen between the normal serum samples and the Covid-19 positive samples, even when testing a single dilution level (1:100), indicating their antibody titer levels. The test results showed Positive Predictive Value (PPV) of 94.4% and Negative Predictive Value (NPV) of 98.7%.
|Minimal Cross Reactivity||Serum collected from patients with known IgG titers against the following non-coronaviruses was tested on the assay: Mumps (N=8), Measles (N=8), Epstein-Barr virus (EBV; N=8), Cytomegalovirus (CMV; N=8), Varicella zoster virus (VZV; N=8), influenza virus (N=10). None of the samples from any of these donors demonstrated any cross-reactivity on the AntiCoV-ID™ IgG ELISA.|
An analysis on serum samples collected from N=105 patients with known IgG titers against non-SARS coronaviruses (229E, HKU1, NL63 and OC43) and other viruses was conducted at a Tier 1, US-based university hospital system, and the AntiCoV-ID™ IgG ELISA showed essentially no cross-reactivity to any of the samples and demonstrated a significantly lower false positive rate (<1% false positive) than an EUA approved qualitative IgG ELISA kit (5% false positive rate).
The Akston Biosciences AntiCoV-ID™ ELISA is a method for the quantitative and qualitative determination of anti-SARS-CoV-2 spike protein receptor binding domain (RBD) IgG antibodies in human serum, citrate plasma, or EDTA plasma.
The AntiCoV-ID™ IgG ELISA is intended for use as an aid in identifying individuals with an adaptive immune response to SARS-CoV-2, indicating recent or prior infection. At this time, it is unknown for how long antibodies persist following infection and if the presence of antibodies confers protective immunity. Testing is limited to laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. 263a, to perform high complexity tests. Results are for the detection of SARS CoV-2 antibodies. IgG antibodies to SARSCoV-2 are generally detectable in blood several days after initial infection, although the duration of time antibodies are present post-infection is not well characterized. Individuals may have detectable virus present for several weeks following seroconversion. Laboratories within the United States and its territories are required to report all positive results to the appropriate public health authorities. Negative results do not preclude acute SARS-CoV-2 infection.
If acute infection is suspected, direct testing for SARS-CoV-2 is necessary. False positive results for AntiCoV-ID™ IgG ELISA may occur due to cross-reactivity from pre-existing antibodies or other possible causes.
Detailed Intended Use information can be found in the Instructions For Use.
In accordance with FDA guidance:
These kits are provided for use only by laboratories certified under CLIA to perform high complexity testing, they are not to be used for at-home testing. The kits are sold in the United States under Section IV.D of the Policy for Diagnostic Tests for Coronavirus Disease-2019 during the Public Health Emergency.
- This test has not been reviewed by the FDA.
- Negative results do not preclude acute SARS-CoV-2 infection. If acute infection is suspected, direct testing for SARS-CoV-2 is necessary.
- Results from antibody testing should not be used to diagnose or exclude acute SARS-CoV-2 infection.
- Positive results may be due to past or present infection with non-SARS-CoV-2 coronavirus strains, such as coronavirus HKU1, NL63, OC43, or 229E.
- Not for screening of donated blood.