PREPARATION OF INCLUDED REAGENTS
Prepare 1x assay buffer by mixing the 5x assay buffer (20 ml) with 80 ml of distilled water or deionized water. CAUTION!!! If precipitates are observed in the 5x assay buffer bottle, heat the bottle in a 37 ° C water bath until the precipitates disappear. An incomplete solution leads to a high background . The 1x assay buffer can be up to a month at 2-8° C are stored.
Prepare 1xWash buffer by mixing the 10xWash buffer (40ml) with 360ml distilled water or deionized water. CAUTION!!! If precipitates are observed in the 10xWash buffer bottle ,heat the bottle in a 37 ° C batch of water until the precipitate disappears. An incomplete solution leads to a high background. The 1xWash buffer can be stored for up to a month at 2-8 ° C.
2.1x detection antibody solution
Briefly spin down the 100 × Detection Antibody Solution and dilute the desired amount of Antibody 1: 100 with 1 × Assay Buffer, 100 ml 1 well of the 1 × Detection Antibody Solution is required per well.Prepare only as much 1 × detection antibody solution as necessary.
Immediately return the 100 × detection antibody solution to 2-8 ° C after removing the required volume.
Cut-off and positive control preparation Centrifuge briefly before opening the tube. Add 20 µl 1xAssay buffer to each tube and mix thoroughly.
Serum or plasma sample requires a 100-fold dilution in the 1 × assay buffer. A proposed dilution step is to add 10 µl of sample to 990 µl of 1 × assay buffer.
Prior to use, set all reagents at room temperature (20-25 ° C) for at least 30 minutes.
|Add controls and specimen:
Add 100 μL specimen, 100 μL negative control, 100 μL cut-off control and 100 μL positive control to their respective wells. Double testing is recommended.
Note: Use a separate pipette tip for each test to avoid waste contamination. Mix by gently tapping the plate.
Cover the plate and incubate for 1 hour at room temperature, preferably shaking at 600 rpm if there
a shaker is available.
Discard the contents and tap the plate on a clean paper towel to remove the remaining solution in each well. Add 300 µl of 1 × wash buffer to each well and incubate for 1 minute. Discard the 1 × wash buffer and tap the plate on a clean paper towel to remove the remaining wash buffer. Repeat the wash step for a total of 3 washes.
|Step 4||Add HRP conjugated detection solution:
Add 100 µl of 1 × detection solution to each well.
Cover the plate and incubate for 1 hour at room temperature.
Wash each well 4 times as described in step 3.
Add 100 µl of substrate solution to each well and incubate at room temperature for 15 minutes. Protect
Add 100 µl of stop solution to each well and gently tap the plate frame for a few seconds to make one
ensure thorough mixing.
Immediately measure the absorbance of each well at 450 nm. Note: Read the absorbance within 10
minutes after stopping the reaction.
Each microtiter plate should be considered separately when calculating and interpreting the assay results regardless of the number of plates processed simultaneously.
The test results are valid if the quality control criteria are met. It is recommended that each laboratory establish an appropriate quality control system with quality control materials that are similar or identical to the patient specimens to be analyzed.
- The negative control absorbance value should be <0.160 at 450 nm
- The absorbance value of the positive control should be> 0.50 at 450 nm
TYPICAL RESULTS (examples only)
|Cut off check||0.156|
Serum from healthy subjects
|Serum from COVID-19
– Clinical validation study of ImmunoDiagnostics SARS-CoV-2 S1 IgG ELISA was conducted in 2020 in Shenzhen, China. Samples were collected from cases confirmed by COVID-19 with clinical symptoms, laboratory abnormalities, or manifestations of pulmonary imaging. No tests have been performed on samples of latent infections or patients during the incubation period.
- It is highly recommended that each laboratory have its own normal and pathological reference range for it
anti-S1 IgG level. In addition, it is also recommended that each laboratory include its own panel of control samples in the assay.
The cut-off value is calculated by relating the absorbance at 450 nm of each sample to the absorbance of Cut-off Control.
|OD450 from specimen / OD450 from Cut-off Control||Indication|
|≦ 0.9||Negative result|
|≧ 1.1||Positive result|
This cut-off value has been validated in ImmunoDiagnostics, but it is highly recommended that each laboratory establish its own normal and pathological reference range for anti-S1 IgG levels. In addition, it is also recommended that each laboratory include its own panel of control samples in the assay.
INTERPRETATIONS OF THE RESULTS
Negative result (OD450 of sample / OD450 of cut-off control ≦ 0.9):
- The test result is negative when the ratio between the sample absorbance and the cut-off control is equal to or less than 0.9. A negative result indicates that no SARS-CoV-2 S1 antibodies have been detected with ImmunoDiagnostics SARS-CoV-2 S1 IgG ELISA, so no serological indication of COVID-19 currently or in the Positive result (OD450 of sample / OD450 of cut-off control ≧ 1.1):
- The test result is positive when the ratio between the sample absorbance and the cut-off control is equal to or greater than 1.1. Positive result indicates that SARS-CoV-2 S1 antibodies have been detected with ImmunoDiagnostics SARS-CoV-2 S1 IgG ELISA and can be used as serological indications of COVID-19 at the time of testing or in the
Borderline Result (OD450 from specimen / OD450 from cut-off control = 0.9-1.1):
- The test result is considered a cut-off when the ratio of sample absorbance to cut-off control is between 0.9 and 1.1 Samples with cut-off results cannot be interpreted at the time of testing. It is strongly recommended that other clinical and laboratory data be integrated before diagnosis, as the clinical diagnosis should not be established from only one type of test.
|Sensitivity||≥ 97% (n = 80)|
|Specificity||≥99% (n = 314)|
|Intra assay precision|
Clinical validation study of ImmunoDiagnostics SARS-CoV-2 S1 IgG ELISA was conducted in 2020 in Shenzhen, China. Samples were collected from cases confirmed by COVID-19 with clinical symptoms, laboratory abnormalities, or manifestations of pulmonary imaging. No tests have been performed on samples of latent infections or patients during the incubation period. The kit showed
higher rate of positive detection in samples from delayed-onset patients. Therefore, the interpretation of the test results should take into account the sample collection time.
- Positive results should be confirmed by another available method and interpreted in conjunction with the patient’s clinical information.2. Antibodies may not be detectable in the early stages of the disease and in some immunosuppressed individuals. Therefore, negative results obtained with ImmunoDiagnostics SARS-CoV-2 S1 IgG ELISA are only an indication that the sample does not contain a detectable level of antibodies and a negative result should not be considered as conclusive evidence that the individual is not infected with the virus.
- False positive results can occur for a variety of reasons, most of which are related to, but are not limited to, an inadequate wash step. For more information about ImmunoDiagnostics ELISA troubleshooting, contact ImmunoDiagnostics technical support for further
4. The most common assay errors are the use of kits after the expiration date, poor washing procedures, contaminated reagents, improper assay procedures, insufficient aspiration during washing, failure to add samples or reagents, improper operation with laboratory equipment, timing errors, use of highly hemolyzed specimens or specimens containing fibrin, incompletely clumped serum samples.
- The prevalence of the marker affects the predictive values of the
6. This assay cannot be used to test pooled (mixed) serum or plasma. The kit has only been assessed with individual serum or plasma samples.
- ImmunoDiagnostics SARS-CoV-2 S1 IgG ELISA is a qualitative assay and the results cannot be used to measure the antibody concentration