See the instructions for use of the SARS-CoV-2 IgG ELISA kit, including the intended use, background and principle of assay, as well as the handling and preparation of reagents, standards and samples to be used with this kit.
For Research Use Only. Not For Use in Clinical or Diagnostic Procedures.
Cat. No. E88-301
Bethyl’s ELISA (E88-301) is for the detection of Human IgG antibodies specific for SARS-CoV-2 virus, specifically the RBD protein, in serum or plasma. This kit contains sufficient components to detect these antibodies in up to 45 samples, if tested in duplicate. It is intended to aid in identifying individuals with an adaptive immune response to SARS-CoV-2, indicating recent or prior infection.
The Bethyl SARS-CoV-2 IgG ELISA is an Enzyme-Linked Immunosorbent Assay (ELISA) intended for semiquantitative detection of IgG antibodies to SARS-CoV-2 in human serum or plasma collected in Potassium EDTA, Sodium Citrate or Lithium Heparin. The Bethyl SARS-CoV-2 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. Understanding the timing, duration and effectiveness of humoral immune responses in individuals previously infected with SARS-CoV-2 will be important for conducting vaccine and epidemiological research. At this time, it is unknown for how long antibodies persist following infection and if the presence of antibodies confers protective immunity.
Results are for the detection of SARS-CoV-2 antibodies. IgG antibodies to SARS-CoV-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.
Bethyl’s ELISA (E88-301) is for the detection of Human IgG antibodies specific for SARS-CoV-2 virus, specifically the RBD protein, in serum or plasma. This kit contains sufficient components to detect these antibodies in up to 45 samples, if tested in duplicate. It is intended to aid in identifying individuals with an adaptive immune response to SARS-CoV-2, indicating recent or prior infection.
The sensitivity of Bethyl SARS-CoV-2 IgG ELISA early after infection is unknown. Negative results do not preclude acute SARS-CoV-2 infection.
False positive results for Bethyl SARS-CoV-2 IgG ELISA may occur due to cross-reactivity from pre-existing antibodies to SARS-CoV-1 or other possible causes.
Coronavirus Disease 2019 (COVID-19) is a systemic disease caused by the novel Coronavirus designated SARS-CoV-2. The initial disease outbreak was first reported in Wuhan, Hubei province, China in December 2019. Within months the World Health Organization declared a global pandemic. COVID-19 can be a serious and life-threatening disease. "Understanding the number of individuals who are exposed and whether they then develop immunity is absolutely key to managing the epidemic." (Ragon Institute of MGH, MIT and Harvard, n.d.)
This kit provides for an indirect ELISA, in which a recombinant receptor binding domain (RBD) of the Spike1 protein of SARS-CoV-2 is coated on the wells of the microtiter plate. Antibodies to SARS-CoV-2 RBD when present in the test sample bind specifically to the RBD protein. After sample binding, unbound proteins and molecules are washed off, and a HRP-conjugated detection antibody is added to the wells to bind to the captured anti-SARS-CoV-2 IgG antibodies. The chromogenic substrate TMB (3,3’,5,5’-tetramethylbenzidine) is then added. This reaction produces a blue product, which turns yellow when the reaction is terminated by addition of dilute sulfuric acid. The absorbance of the yellow product at 450 nm, corrected for plate imperfections by subtracting the absorbance at 570 nm, is proportional to the amount of RBD-specific anti-SARS-CoV-2 IgG present in the sample. Results can be determined by dividing the corrected OD values of the positive control or sample by the corrected OD value of the calibrator. After determining that the values for the Positive Control and Negative Control are valid and acceptable by comparing them to the value for the Calibrator, values for samples are compared to the Calibrator to generate a ratio. Ratios above a cutoff indicate positive samples and values below a cutoff indicate negative samples.
This kit should be refrigerated upon receipt and stored at 2-8 °C. Do not freeze. Expiration date 6 months post receipt of the kit.
Universal precautions using appropriate personal protective equipment should be observed when handling all reagents. Dispose of all reagents and samples appropriately. Avoid eye and skin contact with reagents and samples. Stop solution contains dilute sulfuric acid. In case of eye or skin contact, rinse area with copious amounts of water. Remove and wash any contaminated clothing. Do not ingest. Individual components may contain preservatives.
Use a Plate Template to record the well locations of the Positive Control, Calibrator, Negative Control, and unknown samples.
Using a manual or automated plate washer, perform the following steps.
Do NOT use glass pipette to measure the TMB Substrate Solution. Do NOT cover the plate with aluminum foil or metalized mylar. If the solution is blue before use, DO NOT USE IT!
Note: Wipe the underside of the wells with a lint-free tissue.
Measure the absorbance on an ELISA plate reader set at 450 nm and 570 nm within 30 minutes of stopping the reaction.
If any condition is not met, repeat the assay.
If the condition is not met, discard the values and repeat the assay of the sample.
Samples procured from a commercial source from 34 distinct patients that had tested positive by molecular test (Roche Swab or Abbot RealTime) and tested as reactive by the Siemens ADVIA Centaur Sars-CoV-2 Total assay performed by an independent clinical laboratory were evaluated with the Bethyl SARS-CoV-2 IgG ELISA.
Bethyl SARS-CoV-2 IgG ELISA Results | ||||
---|---|---|---|---|
Days from Symptom Onset | Number of Samples Tested | IgG Positive results | IgG PPA | 95% CI |
0-7 days | 0 | 0 | n/a | |
8-14 days | 0 | 0 | n/a | |
≥15 days | 34 | 34 | 100% | 94%; 100% |
Serum samples collected prior to December 2019 from 161 distinct donors, from two commercial sources were evaluated. One hundred fifty-eight samples (98.1%) were negative and three (1.9%) were not negative for IgG to SARS-CoV-2.
Bethyl SARS-CoV-2 IgG ELISA Results | ||
---|---|---|
Number of Samples Tested | IgG Negative results | IgG NPA (95% CI) |
161 | 158 | 98.1% (96%; 100%) |
Human serum samples previously identified as either Negative, Equivocal, or Positive in the SARS-CoV-2 IgG ELISA were incubated with either diluent alone (Untreated), diluent + goat polyclonal anti-human IgG (Bethyl A80-104) to deplete IgG, or diluent + goat polyclonal anti-human IgM (Bethyl A80-100) to deplete IgM, and then RBD-specific IgG binding was measured in the SARS-CoV-2 IgG assay. All samples were also evaluated in an assay for anti-SARS-CoV-2 IgM binding (Bethyl, in development).
IgG Ratio Range | IgM Ratio Range | % Untreated Ratio (std dev) | ||
---|---|---|---|---|
IgG-Depleted | IgM-Depleted | |||
Neg (n=5) | 0.09 - 0.38 | 0.17 - 0.51 | 21.3 13.1 | 96.4 3.61 |
Equiv (n=1) | 0.83 | 0.72 | 4.1 0.0 | 102.1 0.0 |
Pos (n=10) | 2.45 - 9.84 | 1.86 - 6.40 | 0.6 0.4 | 102.6 2.4 |
IgG Ratio Range | IgM Ratio Range | IgG-Depleted | IgM-Depleted | |||||
---|---|---|---|---|---|---|---|---|
Neg. | Equiv. | Pos. | Neg. | Equiv. | Pos. | |||
Neg (n=5) | 0.09 - 0.38 | 0.17 - 0.51 | 5 | 0 | 0 | 5 | 0 | 0 |
Equiv (n=1) | 0.83 | 0.72 | 1 | 0 | 0 | 0 | 1 | 0 |
Pos (n=10) | 2.45 - 9.84 | 1.86 - 6.40 | 10 | 0 | 0 | 0 | 0 | 10 |
The results demonstrate that depletion of IgG (>99% reduction in IgG assay ratio) from Positive samples converts them to Negatives and that depletion of IgM (>97% reduction in IgM assay ratio, data not shown) from Positive samples has no effect on their disposition in the assay. Similarly, the Negative and Equivocal samples saw no change in assay disposition with IgM depletion, while the Equivocal sample was converted to Negative with depletion of IgG.
These data show that signal in the SARS-CoV-2 IgG ELISA is due specifically to the IgG component of a patient’s immune response to SARS-CoV-2 without significant interference from SARS-CoV-2-specific IgM.
One known negative serum sample and three known positive serum samples were diluted at 1:100 and 1:500 into each of assay diluent, 1:100 serum, 1:100 heparin plasma, 1:100 citrate plasma, and 1:100 EDTA plasma. The blood-based diluents were derived from a single SARS-CoV-2-RBD IgG negative source:
Mean %Serum Assay Ratio (std dev) | ||||
---|---|---|---|---|
Matrix | Serum | Heparin Plasma | Citrate Plasma | EDTA Plasma |
Negative Samples (n=2) | 100.0 | 104.9 (7.9) | 87.3 (3.6) | 92.4 (5.5) |
Positive Samples (n=6) | 100.0 | 100.9 (3.2) | 95.0 (10.1) | 99.9 (4.3) |
Matrix | Diluent | Serum | Heparin Plasma | Citrate Plasma | EDTA Plasma | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Assay Ratio Range | 0.09 - 11.00 | 0.29 - 10.88 | 0.29 - 10.72 | 0.25 - 11.69 | 0.26 - 11.42 | ||||||||||
Assay Outcome | N. | E. | P. | N. | E. | P. | N. | E. | P. | N. | E. | P. | N. | E. | P. |
Neg. Samples (n=2) | 2 | 0 | 0 | 2 | 0 | 0 | 2 | 0 | 0 | 2 | 0 | 0 | 2 | 0 | 0 |
Pos. Samples (n=6) | 0 | 0 | 6 | 0 | 0 | 6 | 0 | 0 | 6 | 0 | 0 | 6 | 0 | 0 | 6 |
Relative to 1:100 SARS-CoV-2-RBD IgG negative serum as diluent, there was no significant perturbation of assay ratio in any of the plasma diluents and no difference in assay outcome.