|88005C||1 Kit (96 assays)||
|Product Includes||Volume||Solution Color|
|Spike Multi-Domain Protein Coated Microwells||96 tests|
|Anti-Human IgG, HRP-linked Antibody (ELISA Formulated)||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 #88005 Positive Control||1 ea|
|ELISA Kit #88005 Negative Control||1 ea|
This ELISA Kit Is Intended For Research Use Only. Not For Use in Diagnostic or Clinical Procedures.
NOTE: Prepare solutions with deionized/purified water or equivalent. Prepare only as much reagent as needed on the day of the experiment.
Positive Control: Reconstitute the vial of lyophilized Positive Control with 1.0 mL Sample Diluent A. Mix thoroughly and gently, hold at room temperature for 1 minute and then follow the steps outlined below in the “Test Procedure” section. Positive Controls are recommended to be used immediately after reconstituting, however remaining material may be stored at -80°C (there may be some loss of the Positive Control signal if freeze/thawed). Positive Controls are supplied as a control reagent, not as an absolute quantitation measure.
IMPORTANT: This control is intended to give a positive signal for the RBD and S1 coated microwell strips, and is NOT a positive control for the S1-NTD or S2 microwells.
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 should be diluted at least 1:400 with Sample Diluent A and can be further serially diluted if relative quantification is needed by the user. Positive and Negative Controls do NOT need to be diluted after reconstitution. Refer to the datasheet which shows typical results observed for the Positive Control, Negative Control, serum from uninfected individuals, and serum from SARS-CoV-2 patients. When using the cutoff criteria described below to determine if a sample is positive for anti-CoV-2 Spike Protein antibodies, samples diluted 1:400 must be compared to the undiluted Negative Control.
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.
Add 100 µL of STOP Solution to each well. Shake gently for a few seconds.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
|Positive Criteria||Negative Criteria||Inconclusive Criteria|
|S1-NTD||> 1.7 x Negative Control||< 1.35 x Negative Control||> 1.35 x Negative Control and < 1.7 x Negative Control|
|RBD||> 3.4 x Negative Control||< 2.7 x Negative Control||> 2.7 x Negative Control and < 3.4 x Negative Control|
|S1||> 4 x Negative Control||< 3 x Negative Control||> 3 x Negative Control and < 4 x Negative Control|
|S2||> 4.1 x Negative Control||< 3.1 x Negative Control||> 3.1 x Negative Control and < 4.1 x Negative Control|
Limitations: Experimental cutoffs were determined by assaying a set of SARS-CoV-2 positive samples (from donors with positive SARS-CoV-2 diagnosis) and uninfected donor serum collected prior to the SARS-CoV-2 pandemic.
NOTE: 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 a positive response against each spike domain protein in this IgG Serological ELISA as differences in disease severity, timing of sample collection relative to disease onset, and patient profiles may affect presence and abundance of antibodies against each spike domain in the reference sample.
Researchers can establish or modify this cutoff using additional samples. Positive or negative results from this assay should not be the sole basis for determining the infection status of a sample. A negative result can occur in SARS-CoV-2 patient samples due to:
NOTE: Absorbance 450 nm values should not be directly compared across different spike domain proteins, as they are not absolute values.
posted September 2020
Protocol Id: 2306
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).
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