Cat. No.: 3CLP-100 (for 100T)

Cat. No.: 3CLP-500 (for 500T)

Description

Coronavirus Mpro/3CLpro Activity Fluorometric Assay Kit is a reagent kit used for rapid and highly sensitive detection of the main protease (Mpro), also known as 3C-like protease (3CLpro), activity in coronaviruses using fluorescence-based methods.

Towards the end of 2019, a pneumonia outbreak caused by a novel coronavirus emerged, rapidly escalating into a global pandemic from early 2020 onwards, leading to a significant rise in infection cases and global attention. This virus was named 2019-nCoV by the World Health Organization (WHO) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses. The disease caused by the novel coronavirus was officially termed Coronavirus Disease 2019 (COVID-19) by WHO, commonly referred to as "COVID-19" or "novel coronavirus pneumonia."

The novel coronavirus (2019-nCoV) belongs to the single-stranded RNA virus group and shares a high degree of homology with SARS-CoV and MERS-CoV. After infecting host cells, the viral RNA's ORF1a/b is translated to produce two polyproteins (pp1a and pp1ab) with the help of host cell machinery. These polyproteins undergo intramolecular cleavage by the main protease (Mpro) and papain-like proteases to generate multiple non-structural proteins. Mpro is also known as 3C-like protease (3CLpro) due to its similarity to the 3C protease of picornaviruses in terms of cleavage site specificity. The non-structural proteins derived from polyprotein processing are involved in the production of viral subgenomic RNAs and four structural proteins (envelope/E protein, membrane/M protein, spike/S protein, and nucleocapsid/N protein), facilitating the replication and release of progeny viruses. Given the pivotal role of Mpro protease in the virus lifecycle and its absence of a human homologue, it becomes an ideal target for antiviral drug development.

With only 12 amino acids different between 2019-nCoV Mpro/3CLpro and SARS-CoV Mpro/3CLpro, and a homology of over 96%, the structures of these two proteases are essentially identical. The positive control 2019-nCoV Mpro/3CLpro used in this kit is purified using our proprietary protein expression technology platform, ensuring the expressed recombinant 2019-nCoV Mpro/3CLpro protein has no additional labels or extra amino acids, maintaining complete sequence identity with the Mpro/3CLpro amino acid sequence of the 2019-nCoV virus. This facilitates the establishment of a detection system and the comparison of enzyme activity.

The Coronavirus Mpro/3CLpro Activity Fluorometric Assay Kit employs the fluorescence resonance energy transfer (FRET) technique. MCA serves as the fluorescence donor (Donor), and Dnp acts as the fluorescence acceptor (Acceptor) or quencher. The absorption spectra of these two fluorescence groups partially overlap, and when they are at an appropriate distance (generally 7-10 nm), fluorescence energy transfers from the donor to the acceptor, leading to a decrease in donor fluorescence intensity. MCA and Dnp are connected to both ends of the natural substrate (Substrate) of the Mpro/3CLpro protease, namely MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2 (P9731). When the protease does not cleave the substrate, the two groups are close enough for fluorescence resonance energy transfer to occur, resulting in the quenching of MCA fluorescence by Dnp, leading to no detectable fluorescence. After the substrate is cleaved by the protease, the peptide's two ends separate, and the two groups move apart. MCA fluorescence is no longer quenched by Dnp, allowing MCA fluorescence to be detected. This fluorescence-based detection method enables the sensitive measurement of Mpro/3CLpro protease activity. MCA has a maximum excitation wavelength of 325 nm and a maximum emission wavelength of 393 nm.

This kit provides the MCA standard solution, and the MCA standard exhibits a good linear relationship within the range of 0.2-50 μM. By establishing a standard curve, the activity of the Mpro/3CLpro protease in the samples can be calculated.

This kit enables the measurement of the enzymatic activity of coronavirus Mpro/3CLpro from various sample types, including the novel coronavirus, cells or animal tissues containing the novel coronavirus, cell or tissue lysates expressing Mpro/3CLpro, or purified Mpro/3CLpro enzyme.

For testing in a 96-well plate format, the small package of this kit allows for 100 tests, and the medium package of this kit allows for 500 tests.

Storage

Store at -20°C, valid for one year. Substrate and MCA Standard (10mM) should be stored away from light.

Precaution

• Different batches of this product may have varying quantities of Substrate provided and used. Please ensure to follow the instructions accordingly.
• Assay Buffer, Substrate, and MCA Standard (10mM) should be completely thawed and equilibrated to room temperature before use; otherwise, it may affect the detection results. The use of nCoV Mpro/3CLpro (1mg/ml) should be conducted on ice. After use, each reagent should be stored immediately under the conditions specified in the assay kit.
• Please ensure that the sample pH is between 7 and 8, either before adding the sample or after the reaction, as deviations may impact signal values and stability of the results.
• The term "sample lysate" refers to the dilution of lysate used for both the lysis and dilution of the test sample, i.e., dilution of virus lysate or lysate of cells/tissues containing the virus. This solution may introduce interference during testing; it is recommended to use the provided Assay Buffer from this kit as a solvent for diluting the test samples. For reagents with smaller volumes, it is advised to briefly centrifuge them to ensure even distribution before use. Frozen reagents must be completely thawed and mixed well before use.
• When performing the assay, using 96-Well Black Opaque Plates is recommended.
• This product is intended solely for scientific research by professionals and must not be used for clinical diagnosis or treatment, food, or drugs, and should not be stored in regular residential settings.
• For your safety and health, please wear appropriate laboratory attire and disposable gloves during operation.