A combination of monoclonal antibodies for detecting feline coronavirus NP protein and application thereof

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more particularly to a combination of monoclonal antibodies for detecting feline coronavirus NP protein and its application. Background Technology

[0002] Feline coronavirus (FCoV) is a single-stranded positive-sense RNA virus belonging to the Coronaviridae family. Since its discovery, it has become one of the most widely transmitted pathogens in cats. Feline coronavirus has diverse transmission routes and is highly prevalent globally, infecting not only domestic cats but also wild cats and other felines. Infection in cats primarily causes gastrointestinal symptoms such as diarrhea and vomiting; in severe cases, it can lead to dehydration and even death. Some studies also suggest a link to chronic enteritis and certain tumors in cats.

[0003] Based on the different clinical manifestations and pathological changes after infection in cats, feline coronaviruses can be divided into two types: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV infection is usually asymptomatic or only causes mild diarrhea, has low pathogenicity, and is endemic. In contrast, FIPV is the main pathogen of feline infectious peritonitis (FIP), which can cause severe inflammatory responses, rapid disease progression, and high mortality. Studies have shown that FIP ​​is not caused by direct transmission of exogenous viruses, but by mutations in the viral genome during FECV infection. Although the incidence of FIP in cats infected with FECV is relatively low, once the disease develops, the prognosis is poor, making it one of the most important infectious diseases affecting feline health. Furthermore, with the increasing number of pet cats globally and the increasingly frequent pet trade, the spread of feline coronaviruses is expanding, posing a serious threat to the health of cats and other felines, as well as to public health.

[0004] Feline coronavirus particles are mainly composed of structural proteins such as nucleocapsid protein (NP), envelope protein (E), membrane protein (M), and spike protein (S), as well as other non-structural proteins. Among them, the NP protein, as the main structural protein of FCoV, plays a crucial role in viral replication and inducing the body's immunity, and is also the most abundant in viral particles. In addition, compared with the S protein, the NP protein shows significant conservation in coronaviruses, making it very suitable as a target protein for feline coronavirus antigen detection.

[0005] Currently, there are no commercially available feline coronavirus vaccines in China, and treatment for feline coronavirus infection is mostly symptomatic. Detection of feline coronavirus infection primarily relies on nucleic acid testing and etiological testing. Nucleic acid testing has high sensitivity, but it requires sophisticated laboratory conditions and highly skilled personnel, which ordinary veterinary hospitals often lack, thus limiting its widespread clinical application. Etiological testing includes virus isolation and antigen immunological detection. Virus isolation, as the gold standard for infection diagnosis, requires more complex and specialized procedures. In contrast, immunological detection of viral antigens, especially colloidal gold rapid test strips, is simpler to perform and more suitable for routine screening or self-testing. However, existing immunological testing products still have shortcomings in sensitivity and specificity, leading to inaccurate results, which seriously affects the detection, diagnosis, and treatment of feline coronavirus infection.

[0006] Therefore, providing a rapid, convenient, low-cost method suitable for screening and detection is of great practical significance for the prevention and control of feline coronavirus infection. Summary of the Invention

[0007] (a) Technical problems to be solved

[0008] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a monoclonal antibody combination for detecting feline coronavirus NP protein and its application in colloidal gold immunochromatographic detection, which solves the technical problems of low sensitivity, poor specificity, complicated operation, and difficulty in being applied to rapid screening at the grassroots level or on-site by existing feline coronavirus antigen detection reagents.

[0009] (II) Technical Solution

[0010] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0011] A monoclonal antibody ensemble for detecting feline coronavirus NP protein, the monoclonal antibody ensemble comprising monoclonal antibody 3C6 and monoclonal antibody 6H9,

[0012] The heavy chain variable region of the monoclonal antibody 3C6 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively.

[0013] The light chain variable region of the monoclonal antibody 3C6 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

[0014] The heavy chain variable region of the monoclonal antibody 6H9 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively.

[0015] The light chain variable region of the monoclonal antibody 6H9 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

[0016] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 3C6 is shown in SEQ ID NO. 13; the amino acid sequence of the light chain variable region of the monoclonal antibody 3C6 is shown in SEQ ID NO. 14.

[0017] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 6H9 is shown in SEQ ID NO. 15; the amino acid sequence of the light chain variable region of the monoclonal antibody 6H9 is shown in SEQ ID NO. 16.

[0018] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3C6 is shown in SEQ ID NO. 17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3C6 is shown in SEQ ID NO. 18.

[0019] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 6H9 is shown in SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 6H9 is shown in SEQ ID NO. 20.

[0020] Secondly, this application provides the use of the above-mentioned monoclonal antibody combination in the preparation of a tool for detecting feline coronavirus NP protein, or the use of the monoclonal antibody combination in the preparation of a tool for in vitro detection of feline coronavirus NP protein.

[0021] In some embodiments, the tool includes colloidal gold test strips, test strips, reagents, kits, and antibody chips.

[0022] In some embodiments, the colloidal gold test strip or test card uses monoclonal antibody 3C6 as the capture antibody and monoclonal antibody 6H9 as the labeling antibody.

[0023] In some embodiments, the colloidal gold test strip or test card includes a nitrocellulose membrane, a colloidal gold pad, a sample pad, and absorbent paper attached to a backing plate.

[0024] In some embodiments, the nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 3C6, the control line is coated with goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 6H9.

[0025] (III) Beneficial Effects

[0026] This invention provides a monoclonal antibody combination for detecting feline coronavirus NP protein, comprising monoclonal antibody 3C6 and monoclonal antibody 6H9 that specifically recognize NP protein epitopes, with the amino acid sequences of the heavy chain and light chain complementarity-determining regions (CDRs) as shown in SEQ ID NO.1-12, respectively. The antibody pairs can simultaneously recognize the NP proteins of feline enteric coronavirus and feline infectious peritonitis virus, making them widely applicable. 3C6 and 6H9 recognize non-overlapping antigenic epitopes on the NP proteins, and the two can be effectively paired for use in a dual-antibody sandwich immunoassay system, improving the specificity and signal-to-noise ratio of the detection. Experiments have shown that the colloidal gold immunochromatographic test strips / cards developed based on this combination have a detection limit of 1 ng / mL for recombinant FECV-NP and FIPV-NP proteins, exhibiting high sensitivity and no cross-reactivity with common feline pathogens such as feline immunodeficiency virus (FIV), feline panleukopenia virus (FPV), feline herpesvirus (FHV-1), and feline calicivirus (FCV), demonstrating strong specificity. The testing tool is easy to operate, requires no specialized equipment, and the results are visually interpretable, allowing for rapid testing. It is ideal for rapid on-site screening in grassroots settings such as veterinary clinics, farms, or homes. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 Figure showing the SDS-PAGE identification results of purified FECV-NP and FIPV-NP proteins;

[0029] Figure 2 This is a schematic diagram of colloidal gold assembly;

[0030] Figure 3 This is a graph showing the specificity analysis results of the test strip;

[0031] Figure 4 The image shows the sensitivity test results for detecting FECV-NP protein using the test strip.

[0032] Figure 5The image shows the sensitivity test results for the test strip in detecting FIPV-NP protein.

[0033] Figure 6 The image shows the results of antibody purity identification. Detailed Implementation

[0034] The embodiments of this application will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are used to illustrate the principles of this application by way of example, but should not be used to limit the scope of this application. This application can be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0035] The detection method described in this application is not intended for disease diagnosis and treatment.

[0036] Feline coronavirus NP proteins include FECV-NP protein and FIPV-NP protein.

[0037] FECV-NP proteins include native FECV-NP proteins and recombinant FECV-NP proteins.

[0038] FIPV-NP proteins include natural FIPV-NP proteins and recombinant FIPV-NP proteins.

[0039] This invention utilizes hybridoma technology to screen and obtain monoclonal antibody pairs capable of specifically recognizing feline coronavirus NP proteins. These antibody pairs efficiently recognize FECV-NP and FIPV-NP recombinant proteins, exhibiting good specificity and sensitivity. This invention applies these monoclonal antibody pairs to an immunoassay platform, constructing rapid test strips or test cards based on colloidal gold immunochromatography. These test strips demonstrate high sensitivity to both FECV-NP and FIPV-NP recombinant proteins and show no cross-reactivity with other proteins, making them suitable for rapid, on-site detection.

[0040] Techniques, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, they should be considered part of the specification.

[0041] Example 1

[0042] 1. Expression of recombinant NP protein of feline coronavirus

[0043] The N genes for both FECV and FIPV were synthesized and cloned into the pET28a vector for expression.

[0044] The nucleotide sequence of the FECVNP gene is shown in SEQ ID NO.21:

[0045]

[0046] The amino acid sequence is as shown in SEQ ID NO. 22:

[0047] MATQGQRVNWGDEPSKRRGRSNSRGRKNDNIPLSYYNPITLAQGSKFWNVCPRDFVPKGIGNKDQQIGYWNRQDRYRIVKGQRKELPERWFFYFLGTGPRADAKFKDKIDGVFWVARDGAMNKPTTLGTRGTNNESKPLKFDGKIPPQFQLEVNRSRNNSRTGSQSRSVSRNRSQSRGRQQSNGQDNVEDTIIAVLQKLGVSDNQRSRSKSRERSDSKPRDTTPKNANKHTWKKTAGKGDVTNFYGARSASANFGDSDLVANGNAAKCYPQIAECVPSVSSMLFGSQWSAEEAGDQVKVTFTHTYYLPKGDAKTAQFLEQIDAYKRPSQVAKDQRQRKSRSKSVDKKPEELSVTLVEAYTDVFDDTQVEMIDEVTN。

[0048] The nucleotide sequence of the FIPVNP gene is as shown in SEQ ID NO. 23:

[0049]

[0050] The amino acid sequence is shown in SEQ ID NO.24:

[0051] MATQGQRVNWGDEPSKRRGRSNSRGRKNNDIPLSFYNPITLEQGSKFWNLCPRDLVPKGIGNKDQQIGYWNRQIRYRIVKGQRKELAERWFFYF LGTGPHADAKFKDKIDGVFWVARDGAMNKPTTLGTRGTNNESKPLRFDGKIPPQFQLEVNRSRNNSRSGSQSRSVSRNRSQSRGRHHSNNQNNNV EDTIVAVLEKLGVTDKQRSRSKPRERSDSKPRDTTPKNANKHTWKKTAGKGDVTTFYGARSSSANFGDSDLVANGNAAKCYPQIAECVPSVSSIIFGSQWSAEEAGDQVKVTLTHTYYLPKDDAKTSQFLEQIDAYKRPSEVAKDQRQRRSRSKSADKKPEELSVTLVEAYTDVFDDTQVEMIDEVTN.

[0052] Recombinant plasmids pET28a-FECV / NP and pET28a-FIPV / NP were transformed into BL21(DE3) competent cells and induced to express their contents using standard methods. Specifically, the transformed bacteria were plated on LB agar plates (containing 50 μg / mL kanamycin) and incubated overnight at 37°C. Single colonies were picked and inoculated into 5 mL of LB medium (containing 50 μg / mL kanamycin) and incubated overnight at 37°C with shaking at 220 rpm. Then, 1% of the total culture volume was inoculated into LB medium (containing 50 μg / mL kanamycin) and incubated at 37°C with shaking at 220 rpm for approximately 3 hours until OD500 reached. 600 The concentration was 0.6-0.9, and the final concentration was 0.1 mM IPTG. The cells were collected after induction at 30℃ and 200 rpm for 4 hours.

[0053] 2. Purification of recombinant proteins

[0054] Because the expressed recombinant protein carries a histidine tag, it was purified using a protein purification instrument and HisTrap from Suzhou Taidu Biotechnology Co., Ltd. TMPurification was performed using an HP affinity chromatography column. Buffer A consisted of 50 mM PB, 300 mM NaCl, pH 8.0; Buffer B consisted of 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The column was equilibrated with buffer A. The fermented bacterial culture was then centrifuged at 8000 rpm for 10 min. The precipitate was resuspended in buffer A and sonicated in ice water for 30 min, with 5-second sonication intervals. The mixture was then centrifuged at 12000 rpm for 30 min. The supernatant was filtered through a 0.22 μm filter from JetBio and loaded onto the chromatography column. The column was washed with buffer A, followed by gradient elution with buffer B. The collected protein elution peaks were dialyzed overnight at 4°C with buffer A. The purification status was observed by SDS-PAGE electrophoresis. The electrophoresis results of the purified protein are shown below. Figure 1 Protein concentration was determined using an ultra-micro spectrophotometer and stored at -20°C.

[0055] Figure 1 In the diagram, M represents the protein marker; numbers 1, 2, and 3 represent electrophoresis of samples retained during FECV-NP purification; numbers 4, 5, and 6 represent electrophoresis of samples retained during FIPV-NP purification; numbers 1 and 4 represent the loading flow-through buffer; numbers 2 and 5 represent 100 mM imidazole elution buffer; and numbers 3 and 6 represent 500 mM imidazole elution buffer. The results are as follows: Figure 1 As shown, there is a distinct main band near the expected size location (43-55kD), indicating that FECV-NP and FIPV-NP proteins with high purity have been obtained and can be used for subsequent experiments.

[0056] 3. Screening for feline coronavirus NP monoclonal antibodies

[0057] 3.1. Mouse Immunization

[0058] Mice were immunized with two recombinant proteins, FECV-NP and FIPV-NP, using standard methods: FECV-NP and FIPV-NP recombinant proteins were mixed with an equal volume of Freund's complete adjuvant (total volume 200 μL) and injected subcutaneously at multiple sites into 6-week-old female BALB / c mice at a dose of 20 μg / mouse. At weeks 2 and 4, booster immunizations were administered subcutaneously at multiple sites with the same dose mixed with an equal volume of Freund's incomplete adjuvant. At week 6, mice were immunized by direct injection of insulin (5 μg / mouse) into the spleen. Seven days after the final immunization, mouse serum was collected to detect antibody titers. Mice with higher titers were selected for booster immunization with 20 μg of FECV-NP and FIPV-NP recombinant proteins via intraperitoneal pulse. Three days later, the spleens of these mice were harvested for hybridoma cell preparation.

[0059] 3.2 Screening of hybridoma cells

[0060] All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in logarithmic growth phase and then cultured in HAT medium for selection. When the fused cells reached halfway up the well, clones that reacted positively with the recombinant proteins FECV-NP and FIPV-NP were selected using an indirect ELISA method. Since the immunogen is a prokaryotically expressed, His-tagged recombinant protein, background components needed to be screened to identify specific cell lines targeting the feline coronavirus NP protein. Positive cells were cloned to a monoclonal state using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0061] 3.3 Screening of positive clones using indirect ELISA method

[0062] Phylogenetic analysis and sequence alignment of the NP genes of feline enteric coronavirus and feline infectious peritonitis virus revealed a high amino acid similarity of over 95% between the two NP proteins. Therefore, to improve screening efficiency, screening was performed using their respective immunogens and reverse screening antigens. After obtaining positive monoclonal antibodies, cross-reactivity was detected by simultaneously coating both proteins.

[0063] In screening mouse fusion plates immunized with FECV-NP protein, the specific steps were as follows: FECV-NP recombinant protein and other prokaryotically expressed, His-tagged irrelevant recombinant proteins (pET28a-FIV / p24, His tag, patent number: CN120965872 B) were coated into microplates as control antigens. The coating buffer was carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water. The coating concentration was 1 μg / mL, and the plate was incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 150 μL of 3% sucrose + 2% BSA, incubated at 37°C for 2 hours, followed by washing once with PBST wash buffer (PBS containing 0.05% Tween-20, pH 7.4), and then blotted dry. 50 μL of cell culture supernatant was added, and the plate was incubated at 37°C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, blot dry, and add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (Solepro, diluted 5000 times with PBS). Incubate at 37°C for 30 min, wash four more times, blot dry, and add 50 μL / well of TMB chromogenic buffer for incubation at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (Beijing Meikewande, 1001SA) to stop the reaction. Measure the OD using a microplate reader. 450 nm value. Positive cell lines that reacted with the FECV-NP recombinant protein but not with the control recombinant protein (pET28a-FIV / p24) were selected for subsequent experiments.

[0064] When screening mouse fusion plates immunized with FIPV-NP protein, FIPV-NP and pET28a-FIV / p24 were used for detection. Positive cell lines that reacted with the FIPV-NP recombinant protein but not with the control recombinant protein (pET28a-FIV / p24) were selected. The screening results are shown in Table 1.

[0065] Table 1: Screening Results of Monoclonal Antibodies

[0066]

[0067] After the selected hybridoma cell lines were expanded and cultured, 0.2 ml (containing 2.5 × 10⁻⁶ cells) was injected intraperitoneally. 6 Female BALB / c mice (cells) were used to collect ascites fluid approximately 10 days later, when the mice’s abdomens were noticeably swollen.

[0068] 4. Purification and identification of monoclonal antibodies

[0069] Centrifuge the ascites fluid at 12000 rpm for 10 minutes, collect 1 ml of the supernatant, add 4 ml of acetate-sodium acetate buffer (0.06 M, pH 4.5), mix well, and slowly add 10 μl of n-octanoic acid while stirring. After the addition is complete, continue stirring for 30 minutes. Centrifuge at 12000 rpm for 30 minutes at 2–8°C, and collect the supernatant. Filter the supernatant through defatted cotton, and add saturated ammonium sulfate at a final volume ratio of 50% (V / V) while stirring. After the addition is complete, continue stirring for 30 minutes, and let it precipitate overnight at 2–8°C. Centrifuge at 12000 rpm for 30 minutes at 2–8°C, and collect the precipitate. After the precipitate was completely dissolved in binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), it was filtered through a 0.22 μm filter. The filtered sample was then pumped slowly through a peristaltic pump into a Protein L purification column equilibrated with binding buffer. The column was connected to a protein purification instrument, and the sample was washed with binding buffer for 5-10 column volumes until the UV absorption peak leveled off. Elution was then performed with elution buffer (0.1 M glycine, pH 2.7), and the elution peak was collected. The collected sample was adjusted to neutral with 1 M Tris-HCl at pH 9 and placed in a dialysis bag (MW: 8000-14000). Dialysis was performed at 2-8 °C in 20 mM PBS pH 7.4 solution for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 rpm for 5 minutes. The supernatant was the purified monoclonal antibody.

[0070] The purified monoclonal antibodies were diluted in a concentration gradient of 10 μg / ml, 1 μg / ml, 100 ng / ml, and 10 ng / ml. Their binding activity to FECV-NP and FIPV-NP recombinant proteins and the irrelevant antigen FIV / p24 was detected by indirect ELISA. The specific results are shown in Table 2. Table 2 shows that the screened monoclonal antibodies specifically bound to FECV-NP and FIPV-NP proteins and did not react with the irrelevant antigen FIV / p24, indicating that the screened monoclonal antibodies are specific to feline coronavirus NP proteins and can be used for subsequent pairing tests.

[0071] Table 2: Identification results of purified monoclonal antibodies

[0072]

[0073] 5. Colloidal gold pairing of feline coronavirus NP monoclonal antibodies

[0074] Preparation of antibody-colloidal gold labeled complex:

[0075] Antibody labeling: Colloidal gold solution was prepared using the trisodium citrate reduction method. The specific procedure was as follows: 100 mL of 0.01% chloroauric acid solution was heated to boiling, and then 1 mL of 1% trisodium citrate solution was quickly added until the solution turned wine-red. Boiling was continued for 5 minutes until the colloidal gold particles stabilized, and then cooled to room temperature for later use. 1 mL of colloidal gold solution was placed in a centrifuge tube, and 0.2 M potassium carbonate solution was added in gradients of 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, and 6 μL to obtain the optimal pH for efficient antibody-colloidal gold conjugation. The pH environment of the colloidal gold solution was adjusted gradient, and 5 μL was selected as the optimal conjugation level. After mixing, 5 μg of the 21 FCoV-NP monoclonal antibodies to be labeled were added, and the mixture was quickly mixed and incubated at room temperature for 10 min. Then, 10 μL of 10% (w / v) bovine serum albumin (BSA) was added to block non-specific binding sites, and incubation at room temperature was continued for another 10 min. Add 10 μL of 10% (w / v) polyethylene glycol 20000 (PEG20000) to enhance labeling stability. After mixing, centrifuge at 12000 rpm for 10 min and discard the supernatant. Resuspend the lower precipitate in 1 / 10 volume of reconstitution solution (0.05 M Tris + 0.9% NaCl + 0.5% N100 + 1% BSA + 2% Sucrose + 0.01% Proclin 300, pH 8.4) to obtain the antibody-colloidal gold labeled complex. Store at 4°C protected from light for later use.

[0076] 6. Preparation of test strips coated with different monoclonal antibodies against feline coronavirus NP

[0077] The 21 selected feline coronavirus NP monoclonal antibodies were scribed onto nitrocellulose membranes of different sizes (20 mm × 300 mm). Diluted monoclonal antibodies (diluted to 1 mg / mL with 0.01 M PB + 0.5% BSA + 0.2% Tween-20, pH 7.8) were sprayed horizontally in a linear pattern using a scribing instrument, with each line containing 0.8 μL / cm, forming a test line (T line). Goat anti-mouse IgG antibodies, diluted to 1 mg / mL with 0.01 M PBS (pH 7.4), were then sprayed horizontally in a linear pattern at 6 mm intervals, forming a control line (C line).

[0078] 7. Screening of paired monoclonal antibodies

[0079] Nitrocellulose membranes streaked with different monoclonal antibodies against feline coronavirus NP were individually paired with different colloidal gold-labeled monoclonal antibodies. FECV-NP and FIPV-NP recombinant proteins were diluted to 40 ng / mL and then mixed at a 1:1 volume ratio to achieve a final concentration of 20 ng / mL for each NP protein. Simultaneously, FIV / p24 recombinant protein was diluted to 20 ng / mL as a negative antigen for detection. Combinations that showed the deepest color development for the mixed FECV-NP and FIPV-NP protein and did not react with the control protein were selected. Therefore, the optimal pairing for detecting feline coronavirus NP recombinant proteins was determined to be 3C6 streaking and 6H9 gold labeling. Results are shown in Table 3 and Table 3 (continued).

[0080] Table 3: Results of screening paired monoclonal antibodies using feline coronavirus NP recombinant protein

[0081]

[0082] Table 3 (continued): Results of screening paired monoclonal antibodies using feline coronavirus NP recombinant protein

[0083]

[0084] - indicates a negative result, meaning no color develops; + / ++ / +++ indicates a positive result, meaning a color reaction occurs. The more + signs there are, the deeper the color, and the stronger the positive reaction.

[0085] Table 3 shows the screening results of FECV-NP and FIPV-NP recombinant proteins diluted to a final concentration of 20 ng / ml as positive antigens. The detection results for FIV / p24 recombinant protein and blank dilution were all negative, and are not shown in the table. The results show that the combination of 3C6 monoclonal antibody streaking and 6H9 monoclonal antibody labeling resulted in the deepest staining of the mixed FECV-NP and FIPV-NP recombinant protein, making it the optimal pairing. That is, the combination of monoclonal antibody anti-3C6 as the coating antibody and monoclonal antibody 6H9 as the labeling antibody can specifically recognize FECV-NP and FIPV-NP recombinant proteins.

[0086] 8. Preparation and assembly of colloidal gold test strips

[0087] Preparation of gold-labeled pads: Using a 6mm×300mm glass fiber membrane, the prepared colloidal gold-labeled antibody was evenly dropped onto the glass fiber at a rate of 1200ul / strip, allowed to air dry naturally, and then dried at 37℃ for 2 hours for later use.

[0088] See Figure 3 , Figure 3 This diagram illustrates the assembly of the colloidal gold test strip. A 60mm × 300mm PVC backing plate is used for support. Sample pads, colloidal gold pads (also known as gold pads), NC membranes (nitrocellulose membranes), and absorbent pads are attached to the backing plate. The nitrocellulose membrane is coated with two lines: a detection line (T line, monoclonal antibody 3C6 streaking) and a control line (C line, goat anti-mouse IgG). The strips are dried at 37℃ for 12 hours before use. The colloidal gold pads are coated with monoclonal antibody 6H9. The assembled plate is cut into 4mm strips using a strip cutter and wrapped with colloidal gold plastic casings. The sample pads are exposed at the sample application wells of the plastic casings, while the control and detection lines are exposed at the result observation wells. The colloidal gold test strip assembly is now complete.

[0089] 9. Test strip specificity test

[0090] Recombinant protein samples: FECV-NP recombinant protein, FIPV-NP recombinant protein, FIV / p24 recombinant protein (patent number: CN 120965872 B), FPV-VP2 recombinant protein (recombinantly expressed and identified by the applicant as full-length FPV VP2 protein, GenBank# UVI41039.1), FHV1-gD recombinant protein (recombinantly expressed and identified by the applicant as FHV1 gD protein 30aa-329aa, GenBank# YP003331589.1), FCV-VP1 recombinant protein (recombinantly expressed and identified by the applicant as full-length FCV VP1 protein, GenBank# QZA82901.1) were diluted with 0.01M sample dilution buffer. Tris + 2% Sucrose + 0.5% BSA + 0.15% skim milk powder + 0.1% Tween-20 + 0.1% N100 + 0.1% Proclin 300, pH 7.6, diluted to 1 μg / mL for detection. Add 80 μL of the diluted sample to the sample well of the test strip. Simultaneously, add another 80 μL of the diluent to a new test strip as a blank control. Determine the results within 20 minutes. If both the T and C lines show clear red bands, the result is positive; if only the C line shows color, the result is negative; if the C line does not show color, the result is invalid.

[0091] The test strip can effectively detect FECV-NP and FIPV-NP recombinant proteins without cross-reactivity with other recombinant proteins, indicating that the test strip has good specificity.

[0092] 10. Sensitivity test of test strips

[0093] The recombinant proteins FECV-NP and FIPV-NP were diluted to concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.5 ng / mL, and 0.25 ng / mL, respectively, and then detected. Figure 4 and Figure 5 The results showed that the colloidal gold test strip still showed weak color development at a recombinant protein concentration of 1 ng / mL, while the blank dilution, i.e., the sample dilution (0.01M Tris + 2% Sucrose + 0.5% BSA + 0.15% skim milk powder + 0.1% Tween-20 + 0.1% N100 + 0.1% Proclin 300, pH 7.6) (0 ng / mL), did not show color development, indicating that the limit of detection for FCoV-NP recombinant protein on the test strip is 1 ng / mL.

[0094] 11. Identification of paired monoclonal antibodies

[0095] The selected paired monoclonal antibodies were identified using a mouse monoclonal antibody subtype identification kit (Luoyang Baisheng), confirming that all subtypes were IgG1. The results are shown in Table 4. The purity of the purified paired antibodies was assessed using standard SDS-PAGE electrophoresis, and the results are as follows: Figure 6 This indicates that the purity of the obtained antibody is above 95%.

[0096] Table 4. Antibody subtype identification results

[0097]

[0098] This invention utilizes hybridoma technology to successfully prepare a monoclonal antibody capable of efficiently recognizing the NP protein of feline coronavirus, and applies it to the construction of colloidal gold immunochromatographic test strips. Verification showed that the monoclonal antibody exhibited good detection sensitivity and specificity against the NP proteins of feline enteric coronavirus and feline infectious peritonitis virus, meeting the application requirements for rapid detection and on-site screening, and providing an antibody raw material basis for the development of diagnostic reagents for feline coronavirus.

[0099] 12. Gene sequence of monoclonal antibodies

[0100] Total RNA was extracted from paired monoclonal antibody hybridoma cells using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using Random Primers. Universal primers for the variable region of mouse antibodies were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the primers for the third round of PCR. The PCR products were purified by gel extraction and ligated into the pUC19 vector, transformed into TOP10 strain, and single colonies were picked and sequenced after culturing at 37°C for 14 h to obtain the gene sequences of the light and heavy chains of the monoclonal antibodies.

[0101] The 3C6 sequence of the membrane-scraped monoclonal antibody:

[0102] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3C6 is shown in SEQ ID NO.18:

[0103] GACATCCACATGATTCAATCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTAGTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCGATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAAGTACTGATTTACTGGGCATCC ACTGGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTTATTACTGTCACCAATATTATAACTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATAAAACGTACGGTG.

[0104] Light chain variable region amino acid sequence:

[0105] The amino acid sequence of the variable region of the 3C6 light chain of the monoclonal antibody is shown in SEQ ID NO.14:

[0106] DIHMIQSPSSLAVSVGEKVSMSCKSSQSLLYSSDQKNYLAWYQQKPGQSPKVLIYWASTGESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCHQYYNYPWTFGGGTKLEIKRTV.

[0107] Light chain CDR area annotation:

[0108] The amino acid sequence of the complementarity-determining region CDR-L1 of the light chain variable region of monoclonal antibody 3C6 is shown in SEQ ID NO.4:

[0109] CDR-L1: KSSQSLLYSSDQKNYLA;

[0110] The amino acid sequence of the complementarity-determining region CDR-L2 of the light chain variable region of monoclonal antibody 3C6 is shown in SEQ ID NO. 5:

[0111] CDR-L2: WASTGES;

[0112] The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain variable region of monoclonal antibody 3C6 is shown in SEQ ID NO. 6:

[0113] CDR-L3: HQYYNYPWT.

[0114] Heavy chain variable region nucleotide sequence:

[0115] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3C6 is shown in SEQ ID NO.17:

[0116] CAGGTGCAGCTGCAGCAGCCTGGACCTGAGCTGATGAAACCTGGGGCCTCAGTGAAGATATCCTGCAAGACTTCTGGTTACTCATTCACTAATTACTACATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTGATCCTTTCAGTGGTGCTACTACCGAC AACCAGAGGTTCAGGGACAAGGCCACATTGACTGTAGACAGATCTTCCAGCTCAGTCTCCATGCATCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGAGGGAGATGTACGACGGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA.

[0117] The amino acid sequence of the heavy chain variable region of monoclonal antibody 3C6 is shown in SEQ ID NO.13:

[0118] QVQLQQPGPELMKPGASVKISCKTSGYSFTNYYMHWVKQSHGKSLEWIGYIDPFSGATTDNQRFRDKATLTVDRSSSSSVSMHLSSLTSEDSAVYYCAREGDVRRAWFAYWGQGTLVTVSS.

[0119] Heavy chain CDR region annotation:

[0120] The amino acid sequence of the complementarity-determining region CDR-H1 of the heavy chain variable region of monoclonal antibody 3C6 is shown in SEQ ID NO.1:

[0121] CDR-H1: NYYMH;

[0122] The amino acid sequence of the complementarity-determining region CDR-H2 of the heavy chain variable region of monoclonal antibody 3C6 is shown in SEQ ID NO.2:

[0123] CDR-H2: YIDPFSGATTDNQRFRD;

[0124] The amino acid sequence of the complementarity-determining region CDR-H3 of the heavy chain variable region of monoclonal antibody 3C6 is shown in SEQ ID NO.3:

[0125] CDR-H3: EGDVRRAWFAY.

[0126] The 6H9 sequence of the gold monoclonal antibody.

[0127] Light chain variable region nucleotide sequence:

[0128] The nucleotide sequence encoding the variable region of the 6H9 light chain of the monoclonal antibody is shown in SEQ ID NO.20:

[0129] GATGTTGTGGTGACTCAAACTCCAACCACCATGGCTGCATCTCCCGGGGAGAAGATCACTATCACCTGCAGTGCCAGCTCAAGTATAAGTTCCAATTACTTGCATTGGTATCAGCAGAAGGCAGGATTCTCCCCTAAAGTCTTGATTTATAGGACATCCAATCTGGC TTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTTTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACTTATTACTGCCACCAGGGTAGTAGTATAACCATTCACGTTCGGCTCGGGGACCAAGCTGGAAATAAAACGTACGGTG.

[0130] Light chain variable region amino acid sequence:

[0131] The amino acid sequence of the variable region of the light chain of monoclonal antibody 6H9 is shown in SEQ ID NO.16:

[0132] DVVVTQTPTTMAASPGEKITITCSASSSISSNYLHWYQQKAGFSPKVLIYRTSNLASGVPARFSGSGSGTSYFLTIGTMEAEDVATYYCHQGSSIPFTFGSGTKLEIKRTV.

[0133] Light chain CDR area annotation:

[0134] The amino acid sequence of the complementarity-determining region (CDR-L1) of the light chain variable region of monoclonal antibody 6H9 is shown in SEQ ID NO.10:

[0135] CDR-L1: SASSSISSNYLH;

[0136] The amino acid sequence of the complementarity-determining region CDR-L2 of the light chain variable region of monoclonal antibody 6H9 is shown in SEQ ID NO.11:

[0137] CDR-L2: RTSNLAS;

[0138] The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain variable region of monoclonal antibody 6H9 is shown in SEQ ID NO.12:

[0139] CDR-L3: HQGSSIPFT.

[0140] Heavy chain variable region nucleotide sequence:

[0141] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 6H9 is shown in SEQ ID NO.19:

[0142] GAGGTGCAGCTGCAGGAGTCTGGCCCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTAACCAGCTATGGTGTACATTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGCCGGTGGAAACACAAATTATAA TTCGGCTCTCATGTCCAGACTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCAATCCCTACGATAGTAGCTCCCTATGGTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

[0143] Heavy chain variable region amino acid sequence:

[0144] The amino acid sequence of the heavy chain variable region of monoclonal antibody 6H9 is shown in SEQ ID NO.15:

[0145] EVQLQESGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGNTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCANPTIVAPYGMDFWGQGTSVTVSS.

[0146] Heavy chain CDR region annotation:

[0147] The amino acid sequence of the complementarity-determining region (CDR-H1) of the heavy chain variable region of monoclonal antibody 6H9 is shown in SEQ ID NO. 7:

[0148] CDR-H1; SYGVH;

[0149] The amino acid sequence of the complementarity-determining region (CDR-H2) of the heavy chain variable region of monoclonal antibody 6H9 is shown in SEQ ID NO. 8:

[0150] CDR-H2: VIWAGGNTNYNSALMS;

[0151] The amino acid sequence of the complementarity-determining region (CDR-H3) of the heavy chain variable region of monoclonal antibody 6H9 is shown in SEQ ID NO. 9:

[0152] CDR-H3:PTIVAPYGMDF.

[0153] The embodiments of this application have now been described in detail. To avoid obscuring the concept of this application, some details known in the art have not been described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description.

[0154] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this application. In particular, as long as there is no structural conflict, the various technical features mentioned in the embodiments can be combined in any manner.

Claims

1. A monoclonal antibody combination for detecting feline coronavirus NP protein, characterized in that, The monoclonal antibody combination includes monoclonal antibody 3C6 and monoclonal antibody 6H9. The heavy chain variable region of the monoclonal antibody 3C6 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively. The light chain variable region of the monoclonal antibody 3C6 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively. The heavy chain variable region of the monoclonal antibody 6H9 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively. The light chain variable region of the monoclonal antibody 6H9 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

2. The monoclonal antibody combination for detecting feline coronavirus NP protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 3C6 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 3C6 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for detecting feline coronavirus NP protein according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 6H9 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 6H9 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for detecting feline coronavirus NP protein according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3C6 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3C6 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for detecting feline coronavirus NP protein according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 6H9 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 6H9 is shown in SEQ ID NO.

20.

6. The use of the monoclonal antibody combination according to claim 1 in the preparation of a tool for detecting feline coronavirus NP protein.

7. The application according to claim 6, characterized in that, The tools include colloidal gold test strips, test strips, reagents, kits, and antibody chips.

8. The application according to claim 7, characterized in that, The colloidal gold test strip or test card uses monoclonal antibody 3C6 as the capture antibody and monoclonal antibody 6H9 as the labeling antibody.

9. The application according to claim 8, characterized in that, The colloidal gold test strip or test card includes a nitrocellulose membrane, a colloidal gold pad, a sample pad, and absorbent paper attached to a backing plate.

10. The application according to claim 9, characterized in that, The nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 3C6, the control line is coated with goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 6H9.

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