|Product Includes||Volume||Solution Color|
|ACE2 Protein Coated Microwells||96 tests|
|SARS-CoV-2 Spike RBD Protein, HRP-linked||1 ea||Red (Lyophilized)|
|Sample Diluent A 71637||25 ml|
|HRP Diluent||11 ml||Red|
|ELISA Wash Buffer (20X) 9801||25 ml|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Kit #69486 Positive Control||1 ea|
|ELISA Kit #69486 Negative Control||1 ea|
NOTE: Prepare solutions with deionized/purified water or equivalent. Prepare only as much reagent as needed on the day of the experiment.
NOTE: Equilibrate all materials and prepared reagents to room temperature prior to running the assay.
Human-sourced samples should be handled in accordance with accepted safety practices. Samples (human serum or plasma) should be diluted at least 1:10 (6.5 μL sample + 58.5 μL Sample Diluent A) with Sample Diluent A and can be further serially diluted if the user needs relative quantification. Positive and Negative Controls do NOT need to be diluted after reconstitution. When using the cutoff criteria described below to determine if a sample is positive for anti-SARS-CoV-2 Spike RBD Protein blocking antibodies, samples diluted 1:10 must be compared to wells containing a mixture of Sample Diluent A and RBD-HRP (RBD-HRP only control), while the Positive and Negative Controls are used undiluted. An equation to calculate the percent inhibition is described at the end of this protocol.
NOTE: Sample storage/handling, including heat-inactivation of samples, can potentially affect observed signals. Therefore, it is strongly recommended that in addition to the Positive and Negative Controls included with the kit, the user includes their own negative and positive patient samples as controls when running the assay in order to establish an appropriate cutoff value. In addition to testing human serum/plasma, this kit may be used to assess blocking activity of non-human antibodies and small molecules. In this scenario, the user will have to determine the appropriate dilution/concentration of their samples to use, along with running the proper controls.
Add 100 μL of STOP Solution to each well and shake gently for a few seconds.
NOTE: Initial color change is blue, which changes to yellow upon addition of STOP Solution.
100 - [(OD value of Sample ÷ OD Value of RBD-HRP only control) x 100%]
≥ 20% inhibition- Positive result, SARS-CoV-2 blocking antibody detected.
< 20% inhibition- Negative result, no detectable SARS-CoV-2 blocking antibody.
* Experimental cutoffs were determined by assaying a set of confirmed SARS-CoV-2 positive samples (from donors with positive SARS-CoV-2 diagnosis and seropositive) and uninfected donor serum collected prior to the SARS-CoV-2 pandemic. Researchers can establish or modify this cutoff using additional samples.
posted November 2020
Protocol Id: 2367
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