CN-121698997-B - Monoclonal antibody combination for detecting feline coronavirus NP protein and application thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody combination for detecting feline coronavirus NP protein and application thereof. The combination consists of monoclonal antibodies 3C6 and 6H9, wherein 3C6 is used as a coating antibody, and 6H9 is used for colloidal gold marking, so that efficient and specific sandwich detection of the feline coronavirus NP protein can be realized. The monoclonal antibody combination is applied to an immune detection platform, and a rapid detection test strip or test paper card based on a colloidal gold immune chromatography technology is constructed. The test paper has high sensitivity to FECV-NP and FIPV-NP recombinant proteins, has no cross reaction with other proteins, and is suitable for rapid and on-site detection.

Inventors

• QIN KUN
• LU SHUAI
• LI YUE
• SUN YUTIAN
• ZHONG MING
• SU DU

Assignees

• 北京溯本源和生物科技有限公司

Dates

Publication Date

20260512

Application Date

20260213

Claims (10)

1. 1. A monoclonal antibody combination for detecting the NP protein of feline coronavirus, characterized in that the monoclonal antibody combination comprises monoclonal antibody 3C6 and monoclonal antibody 6H9, The heavy chain variable region of the monoclonal antibody 3C6 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are respectively shown as SEQ ID NO.1-SEQ ID NO. 3; The light chain variable region of the monoclonal antibody 3C6 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are respectively shown in SEQ ID NO.4-SEQ ID NO. 6; The heavy chain variable region of the monoclonal antibody 6H9 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are respectively shown in SEQ ID NO.7-SEQ ID NO. 9; the light chain variable region of the monoclonal antibody 6H9 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are respectively shown in SEQ ID NO.10-SEQ ID NO. 12.
2. 2. The monoclonal antibody combination for detecting the feline coronavirus NP protein according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody 3C6 is shown in SEQ ID No.13, and the amino acid sequence of the light chain variable region of the monoclonal antibody 3C6 is shown in SEQ ID No. 14.
3. 3. The monoclonal antibody combination for detecting the feline coronavirus NP protein according to claim 2, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody 6H9 is shown in SEQ ID No.15, and the amino acid sequence of the light chain variable region of the monoclonal antibody 6H9 is shown in SEQ ID No. 16.
4. 4. The monoclonal antibody combination for detecting feline coronavirus NP protein according to claim 3, wherein the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3C6 is shown in SEQ ID No.17 and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3C6 is shown in SEQ ID No. 18.
5. 5. The monoclonal antibody combination for detecting feline coronavirus NP protein according to claim 4, wherein the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 6H9 is shown in SEQ ID NO.19 and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 6H9 is shown in SEQ ID NO. 20.
6. 6. Use of a monoclonal antibody combination according to claim 1 for the preparation of a tool for detecting feline coronavirus NP protein.
7. 7. The use of claim 6, wherein the means comprises colloidal gold test strips, paper cards, reagents, kits and antibody chips.
8. 8. The use according to claim 7, wherein the colloidal gold test strip or strip card uses monoclonal antibody 3C6 as a capture antibody and monoclonal antibody 6H9 as a labeling antibody.
9. 9. The use of claim 8, wherein the colloidal gold test strip or card comprises a nitrocellulose membrane, a colloidal gold pad, a sample pad, and a bibulous paper attached to a backing plate.
10. 10. The use according to claim 9, wherein the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with monoclonal antibody 3C6, the quality control line is coated with goat anti-mouse IgG, and the colloid Jin Dianbao is coated with monoclonal antibody 6H9.

Description

Monoclonal antibody combination for detecting feline coronavirus NP protein and application thereof Technical Field The invention relates to the field of biological detection, in particular to a monoclonal antibody combination for detecting feline coronavirus NP protein and application thereof. Background Feline coronavirus (Feline Coronavirus, FCoV) belongs to the family coronaviridae, which has been one of the widely transmitted pathogens in the feline group since its discovery. The feline coronavirus transmission routes are various, and the feline coronavirus has a high epidemic situation on the global scale, and can infect other felines such as wild cats. The cat is infected with coronavirus and mainly causes diarrhea, vomiting and other digestive tract symptoms, and dehydration and even death can be caused when the cat is serious, and the cat is also studied and related to chronic enteritis and the occurrence of certain tumors. Cat coronaviruses can be divided into two types, feline enterocoronavirus (FELINE ENTERIC Coronavirus, FECV) and feline infectious peritonitis virus (Feline Infectious Peritonitis Virus, FIPV) according to their clinical manifestations and pathological changes after infection. FECV infections are usually asymptomatic or cause only mild diarrhea, less pathogenic, and endemic. In contrast, FIPV is the primary pathogen of Feline Infectious Peritonitis (FIP), which can lead to severe inflammatory reactions, rapid disease progression and high mortality. Studies have shown that FIP is not directly transmitted by exogenous viruses, but rather is caused by mutations in the viral genome during FECV infection. Although FIP is relatively low in cats infected with FECV, once it is developed, it is a poor prognosis, one of the most important infectious diseases affecting the health of cats. In addition, with the increasing number of global pet cats and the increasing frequency of pet trade, the spread of feline coronaviruses is expanding, and the health and public health of other felines such as cats are a serious threat. The feline coronavirus particle mainly comprises structural proteins such as Nucleocapsid Protein (NP), envelope protein (E), membrane protein (M) and spike protein (S) and other non-structural proteins. The NP protein is used as a main structural protein of FCoV, plays a key role in viral replication and inducing body immunity, and is also the most abundant in virus particles. In addition, compared with the S protein, the NP protein shows remarkable conservation in coronaviruses, and is very suitable for serving as a target protein for cat coronavirus antigen detection. At present, no commercial feline coronavirus vaccine is marketed in China, and most of treatment aiming at feline coronavirus infection is symptomatic treatment, and detection of feline coronavirus infection mainly depends on nucleic acid detection and etiology detection. The nucleic acid detection sensitivity is higher, but the requirements on experimental conditions and the professional level of operators are higher, and the common pet hospitals can not reach the corresponding detection conditions, so that the popularization and application of the method in clinic are limited. Etiology detection comprises virus separation and antigen immunology detection, wherein the virus separation is used as a gold standard for infection diagnosis, and more complex and professional operation is needed, compared with the immunology detection of virus antigens, especially colloidal gold rapid detection test paper, which is simple and convenient to operate, and is more suitable for routine screening or self-detection. However, the existing immunodetection products still have the defects in detection performances such as sensitivity, specificity and the like, so that the result judgment is inaccurate, and the discovery, diagnosis and treatment of the feline coronavirus infection are seriously influenced. Therefore, the method which is rapid, convenient, low in cost and suitable for screening detection is of great practical significance for preventing and treating the feline coronavirus infection. Disclosure of Invention First, the technical problem to be solved In view of the above-mentioned shortcomings and disadvantages of the prior art, the present invention provides a monoclonal antibody combination for detecting feline coronavirus NP protein and its application in colloidal gold immunochromatography detection, which solves the technical problems of low sensitivity, poor specificity, complex operation, and difficulty in adapting to basic layer or rapid screening on site of the existing feline coronavirus antigen detection reagent. (II) technical scheme In order to achieve the above purpose, the main technical scheme adopted by the invention comprises the following steps: a monoclonal antibody combination for detecting feline coronavirus NP protein, comprising monoclonal antibody 3C6 and monoclonal antibody 6H9, The hea