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PREPARATION OF SUPPLIED 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 precipitate disappears. An incomplete solution leads to a high background. The 1x assay buffer can be stored at 2-8 ° C for up to a month.

1.1xWash buffer

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.

  1. 1x detection antibody solution

Rotate the 100 × Detection Antibody Solution briefly 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 solutionto 2-8 ° C after removing the required volume.

  1. Cut-off and positive control preparation

 

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Centrifuge briefly before opening the tube. Add 20 µl 1xAssay buffer to each tube and mix thoroughly.

 

SAMPLE PREPARATION

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.

ASSAY PROCEDURES

Prior to use, set all reagents at room temperature (20-25 ° C) for at least 30 minutes.

 

 

 

Step 1

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.

 

Step 2

Incubation:

Cover the plate and incubate for 1 hour at room temperature, preferably shaking at 600 rpm if there

a shaker is available.

 

 

Step 3

Wash:

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.

 

Step 5 Incubation:

Cover the plate and incubate for 1 hour at room temperature.

Step 6 Wash:

Wash each well 4 times as described in step 3.

 

Step 7

 

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Color:

Add 100 µl of substrate solution to each well and incubate at room temperature for 15 minutes. Protect

ht. Step 8

Stop response:

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.

 

Step 9

Measurement:

Immediately measure the absorbance of each well at 450 nm. Note: Read the absorbance within 10

minutes after stopping the reaction.

 

QUALITY CONTROL

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 absorbance value of the negative control should be <0.170 at 450 nm.

  • The absorbance of the positive control should be> 0.500 at 450 nm

TYPICAL RESULTS (examples only)

 

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Samples OD450
 

Negative control

0.068
0.093
Cut off check 0.163
0.168
 

Positive control

1,715
1,693
 

Serum from healthy subjects

0.086
0.072
0.083
Serum from COVID-19

patients

0.927
0.293
1,839

 

  • Clinical validation study of ImmunoDiagnostics SARS-CoV-2 S1RBD IgG ELISA was conducted in Shenzhen in 2020, 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 strongly recommended that each laboratory have its own normal and pathological reference range for it anti-S1RBD IgG level. In addition, it is also recommended that each laboratory include its own panel of control samples in the assay.

CUT-OFF VALUE

The cut-off value is calculated by relating the absorbance of each sample to the absorbance of Cut-off Control at 450 nm.

OD450 from specimen / OD450 from Cut-off Control Indication
≦ 0.9 Negative result
≧ 1.1 Positive result
0.9-1.1 Borderline 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-S1RBD 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 S1RBD antibodies have been detected with ImmunoDiagnostics SARS-CoV-2 S1RBD IgG ELISA, therefore 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 S1RBD antibodies have been detected with ImmunoDiagnostics SARS-CoV-2 S1RBD 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 between sample absorbance and cut-off control is between 0.9 and 1.1. Samples with limit results cannot be interpreted at the time of testing. 
  • It is strongly recommended to integrate other clinical and laboratory data before diagnosis, as the clinical diagnosis should not be established from only one type of test.

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PERFORMANCE CHARACTERISTICS

Sensitivity ≥ 96.25% (n = 80)
Specificity ≥ 99% (n = 296)
Inter-assay precision
Samples CV
1 5.93%
2 3.87%
3 4.52%
Intra assay precision
Samples CV
1 6.66%
2 7.21%
3 5.89%

 

Clinical validation study of ImmunoDiagnostics SARS-CoV-2 S1RBD 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 a higher positive detection rate in samples from delayed onset patients. Therefore, the interpretation of the test results should take into account the sample collection time.

RESTRICTIONS

  1. 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 individuals with immune suppression. Therefore, negative results obtained with ImmunoDiagnostics SARS-CoV-2 S1RBD IgG ELISA are only an indication that the specimen contains no detectable level of antibodies and a negative result should not be considered as conclusive evidence that the individual is not infected with the
  3. 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.
  1. The prevalence of the marker affects the predictive values ​​of the
  2. This assay cannot be used to measure pooled (mixed) serum or plasma. The kit has only been evaluated with individual serum or plasma samples.
  3. Immuno Diagnostics SARS-CoV-2 S1RBD IgG ELISA is a qualitative assay and the results cannot be used to measure the antibody concentration