SARS-CoV-2 Nucleocapsid Protein Binding Kit (For rabbit antibody)

Background

SARS-CoV-2 Nucleocapsid protein (NP) is one of the core components of SARS-CoV-2 virus. It forms a complex with viral genomic RNA in a helical symmetrical structure and plays a key role in the process of virus replication and assembly. Since NP is abundantly expressed during infection, it can be used as an important diagnostic marker for COVID-19 and also can be used as a potential drug target or developing vaccines.

Assay Principle

The SARS-CoV-2 Nucleocapsid protein (NP) Binding kit is a TR-FRET based assay, that is designed to detect binding status of NP to the test antibody. Terbium-labeled anti-Tag1 antibody serves as fluorescence donor, that binds to the His-Tagged NP. If a test rabbit antibody binds to NP, fluorescence- labeled anti-rabbit antibody (fluorescence acceptor) will be brought in close proximity with the fluorescence donor. Excitation of Terbium (340 nm) generates fluorescence resonance energy transfer (FRET) to the fluorescence-labeled acceptor, which consequently fluoresces at 665 nm (figure below). Thus, the test antibody binding to NP can be quantitively measured by calculation of the fluorescent ratio of 665 nm/620 nm.

Application

High throughput screening of antibodies that bind to NP.

Instrument Required

Aurora Biolabs LLC, San Diego, CA 92121, USA; www.aurorabiolabs.com; SARS-CoV-2 Nucleocapsid Protein Binding Kit (for rabbit antibody) A HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.

Kit Components

Materials Not Supplied

• Microplate reader, HTRF® certified microplate reader
• Customer Test anti-NP-rabbit antibody (to be tested antibody)
• 0.5 M DTT
• Adjustable micro-pipettor
• Sterile Tips

Assay Protocol

1. Step 1. Prepare SARS-CoV-2 Nucleocapsid protein Dilute SARS-CoV-2 Nucleocapsid protein (NP) 1,500-fold with 1X DTT-containing assay buffer. For example: 1 µl of NP + 1,499 µl of 1X DTT-containing assay buffer. Add 5 µl of diluted NP protein to each well.
2. Step 2. Prepare Antibody solution
3. Step 3. Prepare dye solution Dilute Terbium-labeled anti-Tag1 Ab and fluorescence-labeled anti-rabbit antibody 1:200 in 1X DTT- containing assay buffer. For example: 1 µl of Terbium-labeled anti-Tag1 Ab + 1 µl of fluorescence- labeled anti-rabbit antibody + 198 µl of 1X DTT-containing assay buffer. Add 10 µl of this dye mixture to each well.
4. Step 4. Incubate the reaction at room temperature for 1 hour.
5. Step 5. Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:
6. Step 6. Excitation wavelength at 340 nm and emission at 620 nm.
7. Step 7. Excitation wavelength at 340 nm and emission at 665 nm.

Data Analysis

Calculate sample signal. Calculate percentage activity Aurora Biolabs LLC, San Diego, CA 92121, USA; www. SARS-CoV-2 Nucleocapsid Protein Binding Kit (for rabbit antibody) In the absence of the compound (positive control), the sample signal (P) is defined as 100% activity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% activity. The percent activity in the presence of each compound is calculated according to the following equation: % activity = (S-N)/(P-N) X100, where S= the sample signal in the presence of the compound.

Assay Validation