Figure 1. Patient Testing: Patient samples were tested using the SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit #69486. Serum/plasma samples were heat-inactivated (56°C for 30 min) and diluted 1:10 prior to running the assay, as described in the protocol. Percent inhibition of the SARS-CoV-2 spike RBD-ACE2 interaction (% Inhibition) is plotted for each serum/plasma sample, with values corresponding to samples obtained from donors who had been diagnosed with SARS-CoV-2 (diagnosed positive n=11*) on the left, and values corresponding to samples from presumed uninfected donors collected prior to the SARS-CoV-2 outbreak (presumed negative n=9) on the right. Samples are considered positive or negative for SARS-CoV-2 blocking antibodies based on the 20% inhibition cutoff criteria (dotted line) described in the “Data Analysis” section of the kit protocol.
*Positive reference samples were from patient donors with a positive SARS-CoV-2 diagnosis. However, a positive SARS-CoV-2 diagnosis will not always correlate with the presence of blocking antibodies, as differences in disease severity, timing of sample collection relative to disease onset, and patient profiles may affect presence and abundance of antibodies in the reference sample that block the interaction between SARS-CoV-2 spike RBD and ACE2.
Note: We are continuing to test more samples as available. For the most up-to-date set of data, always refer to the product page for #69486 on the website.
Intra-Assay Precision: Three different serum samples were each tested in 16 replicates using a single assay kit of SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit #69486. Intra-assay CV (%) was calculated for each sample, and each replicate was correctly identified as being positive or negative when compared to the RBD-HRP only control using the cutoff criteria described in the attached protocol.
Inter-Assay Precision: Five different assay kits from one lot of material were tested using 3 different serum samples run in duplicate wells and Positive and Negative Controls also run in duplicate wells. Inter-assay CV (%) was calculated for each sample, and each assay kit correctly identified the samples as being positive or negative when compared to the RBD-HRP only control using the cutoff criteria described in the attached protocol.
|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||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|
The SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit allows for the detection of antibodies (or small molecules) that block the interaction between the host receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and ACE2. In this assay, SARS-CoV-2 spike RBD protein linked to HRP (RBD-HRP) is pre-incubated with the sample and controls, allowing blocking antibodies present in the sample to bind to the RBD-HRP. These pre-incubated mixtures are then added to microwell plates coated with ACE2 protein. The RBD-HRP is captured by ACE2 in the well to varying degrees depending on the blocking activity present in each sample. The wells are then washed to remove unbound material. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is inversely proportional to the ability of the sample to block the interaction between SARS-CoV-2 spike RBD and ACE2 proteins. For example, if no blocking activity is present in the sample, the RBD-HRP will be able to bind to ACE2 on the microwell plates, resulting in high signal. Conversely, samples with high blocking activity will prevent the RBD-HRP from binding to ACE2 on the microwell plates, resulting in low signal. This kit is designed to detect blocking antibodies present in human serum/plasma samples. However, this kit may also be used to assess the blocking activity of non-human antibodies and small molecules. In the latter scenarios (for non-human antibodies and small molecules), the user will have to determine the appropriate dilution/concentration of their samples to use, along with running the proper controls.
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
The SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit detects endogenous levels of antibodies that block the interaction between the SARS-CoV-2 spike RBD (318-541) protein and human ACE2 (18-615). This kit is designed to detect blocking antibodies present in human serum/plasma, however, it may also be used to assess blocking activity of non-human antibodies and small molecules.
The cause of the COVID-19 pandemic is a novel and highly pathogenic coronavirus, termed SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2). SARS-CoV-2 is a member of the Coronaviridae family of viruses (1). The genome of SARS-CoV-2 is similar to other coronaviruses, and is comprised of four key structural proteins: S, the spike protein, E, the envelope protein, M, the membrane protein, and N, the nucleocapsid protein (2). Coronavirus spike proteins are class I fusion proteins and harbor an ectodomain, a transmembrane domain, and an intracellular tail (3,4). The highly glycosylated ectodomain projects from the viral envelope surface and facilitates attachment and fusion with the host cell plasma membrane. The ectodomain can be further subdivided into host receptor-binding domain (RBD) (S1) and membrane-fusion (S2) subunits, which are produced upon proteolysis by host proteases at S1/S2 and S2’ sites. S1 and S2 subunits remain associated after cleavage and assemble into crown-like homotrimers (2,4). In humans, both SARS-CoV and SARS-CoV-2 spike proteins utilize the angiotensin-converting enzyme 2 (ACE2) protein as a receptor for cellular entry (5-7). Spike protein subunits represent a key antigenic feature of coronavirus virions, and therefore represent an important target of vaccines, novel therapeutic antibodies, and small-molecule inhibitors (8,9).
Explore pathways + proteins related to this product.
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