A protein detection method and its protein detection kit

Abstract

The present invention discloses a monoclonal antibody that specifically binds to rotavirus VP8 protein. The monoclonal antibody includes monoclonal antibody 1 and monoclonal antibody 2. The sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 1 are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively. The sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 2 are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively. The present invention also discloses a rotavirus detection test strip, and the test strip includes a P4 test strip and a P8 test strip. The present invention screened 2 monoclonal antibodies. Among them, monoclonal antibody 1 can only specifically bind to the VP8 protein of P8 rotavirus, and monoclonal antibody 2 can only specifically bind to the VP8 protein of P4 rotavirus. On this basis, a test strip for detecting P4 rotavirus and P8 rotavirus was established. The test strip of the present invention has good specificity, sensitivity and clinical applicability, providing a good tool for the rapid clinical detection and typing detection of rotavirus.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a biomarker for cancer diagnosis and treatment and its application. Background Art

[0002] Rotavirus is one of the main pathogens causing infantile diarrhea. It mainly infects small intestinal epithelial cells, resulting in cell damage and diarrhea. Rotavirus prevails in summer, autumn and winter every year. The infection route is fecal-oral route. The clinical manifestation is acute gastroenteritis, presenting as osmotic diarrhea. The course of disease is generally 6-7 days, with fever lasting for 1-2 days, vomiting for 2-3 days, and diarrhea for 5 days. Severe cases may show dehydration symptoms.

[0003] Rotavirus (RV for short) is a double-stranded ribonucleic acid virus and belongs to the family Reoviridae. It is the single main cause of diarrhea in infants and young children. Almost every child around the world about five years old has been infected with rotavirus at least once. However, after each infection, the body's immunity gradually increases, and the impact of subsequent infections will be reduced. Therefore, adults are rarely affected by it. There are a total of seven types of rotavirus, numbered A, B, C, D, E, F, and G in English letters. Among them, type A is the most common one, and more than 90% of human rotavirus infection cases are caused by this type.

[0004] Rotavirus is divided into G-type serotype and P-type serotype according to VP7 and VP4 proteins. 35 G-types and 50 P-types have been identified so far. VP7 and VP4 constitute the structural proteins of the rotavirus capsid, which can induce the body to produce neutralizing antibodies and play an important role in the body's immunity. VP7 is a glycosylated protein that determines the G serotype. VP4 (VP8 is the main antigen fragment after VP4 hydrolysis) is a protease-sensitive protein that determines the P serotype. So far, 12 G-types and 15 P-types have been found in group A to be related to human diarrhea. G-types and P-types can be combined independently and freely. Epidemiological investigations in different regions of the world show that G1P1A[8], G2P1B[4], G3P1A[8], G4P1A[8], G9P1A[8] are the most common dominant epidemic strains of human rotavirus. Among them, P4 and P8 are the most common. Due to a unique characteristic of rotavirus, namely "heterotypic cross-protection", there is a widespread heterotypic cross-protective immunity between different G serotypes (G1, G2, G3, G4, etc.) and P serotypes (P1, P4, P8).

[0005] The detection of rotavirus is extremely important for disease surveillance and epidemic control. At present, the detection techniques of rotavirus are summarized from three aspects: etiology, immunology, and gene detection. For example, colloidal gold, PCR, and PAGE are used for the qualitative detection of rotavirus in collected samples. The BiolineTM Rotavirus Test Card uses immunochromatography to detect group A rotavirus in fecal samples. In addition, multiplex real-time quantitative PCR (qPCR) is used for the rapid genotyping and quantitative analysis of the VP7 gene of G1-G4 rotavirus. Each specimen needs to be subjected to rotavirus antigen detection and genotyping. However, at present, the genotyping detection of rotavirus in the market mainly focuses on sequencing and comparison, which is time-consuming and laborious. Therefore, it is very necessary to develop a new rotavirus detection method. Summary of the Invention

[0006] In order to make up for the deficiencies of the prior art, one of the objectives of the present invention is to provide a method for detecting rotavirus; another objective of the present invention is to provide an application of the method for detecting rotavirus of the present invention in rotavirus genotyping detection.

[0007] Therefore, on the one hand, the present invention discloses a monoclonal antibody, and the monoclonal antibody is a monoclonal antibody that specifically binds to the VP8 protein of rotavirus; the monoclonal antibody that specifically binds to the VP8 protein of rotavirus includes monoclonal antibody 1 and monoclonal antibody 2; among them, monoclonal antibody 1 specifically binds to the VP8 protein of P8 type rotavirus, and the sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 1 are shown in SEQ ID NO.3 and SEQ ID NO.4; monoclonal antibody 2 specifically binds to the VP8 protein of P4 type rotavirus, and the sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 2 are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0008] Preferably, the amino acid sequence of the monoclonal antibody 1 of the present invention that specifically binds to the VP8 protein of P8 type rotavirus is vdpvdr.

[0009] Preferably, the amino acid sequence of the monoclonal antibody 2 of the present invention that specifically binds to the VP8 protein of P4 type rotavirus is iklgglgy.

[0010] On the other hand, the present invention also discloses a rotavirus detection test strip, and the test strip includes a P4 test strip and a P8 test strip; among them, the P4 test strip is prepared from the monoclonal antibody 2, the rabbit polyclonal antibody against the VP8 protein of rotavirus, and the supporting reagents; the P8 test strip is prepared from the monoclonal antibody 1, the rabbit polyclonal antibody against the VP8 protein of rotavirus, and the supporting reagents.

[0011] On the other hand, the present invention also discloses an application of the monoclonal antibody 1 and monoclonal antibody 2 in the preparation of a rotavirus diagnostic reagent.

[0012] Based on the previous work, the inventors compared the sequences of VP8 proteins of P4-type rotavirus and P8-type rotavirus and conducted in-depth research, screening 2 specific monoclonal antibodies. Among them, monoclonal antibody 1 can only specifically bind to the VP8 protein of P8-type rotavirus, and monoclonal antibody 2 can only specifically bind to the VP8 protein of P4-type rotavirus. On this basis, a test strip for detecting P4-type rotavirus and P8-type rotavirus was established. The test strip of the present invention has good specificity, sensitivity and clinical applicability, providing a good tool for the rapid clinical detection and typing detection of rotavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Purification results of P4-type VP8 protein. Among them, 1 is Marker, 2 is the expression supernatant before loading the column, 3 is the flow-through, 4 is washing solution A1, 5 is washing solution A2, 6 is washing solution A3, 7 is washing solution A4, 8 is elution solution B1, 9 is elution solution B2, 10 is elution solution B3.

[0014] Figure 2 Purification results of P8-type VP8 protein. Among them, 1 is Marker, 2 is before induction, 3 is after induction, 4 is the supernatant after cell disruption, 5 is the flow-through, 6 is washing solution A1, 7 is washing solution A2, 8 is elution solution B1, 9 is elution solution B2, 10 is elution solution B3.

[0015] Figure 3 Results of amino acid sequence alignment of VP8 proteins of P4-type and P8-type.

[0016] Figure 4 Western blot detection results of two monoclonal antibodies. Among them, A is the detection result of monoclonal antibody 1, 1 is Marker, 2 is P4-VP8 protein, 3 is P8-VP8 protein. Among them, B is the detection result of monoclonal antibody 2, 1 is Marker, 4 is P8-VP8 protein, 5 is P4-VP8 protein.

[0017] Figure 5 Schematic diagram of test strip assembly.

[0018] Figure 6 Specificity detection results of the test strip.

[0019] Figure 7 Sensitivity detection results of the test strip. From left to right, they are 2 ng / mL, 4 ng / mL, 8 ng / mL, 16 ng / mL respectively. DETAILED DESCRIPTION OF THE INVENTION

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. The terms used in the description of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0021] Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0022] Example 1: Preparation of VP8 proteins of different P-type (P4, P8) rotaviruses

[0023] The amino acid sequences of the VP8 proteins of P4 type and P8 type (as shown in SEQ ID NO.1 and SEQ ID NO.2) were entrusted to Genewiz (Suzhou) Inc. for codon optimization and construction of prokaryotic expression vectors. The vector was selected as pET-30a(+), the restriction enzyme sites added were BamH I and ECoRI, and the tag was 6His tag. The constructed vectors were transformed, plasmids were extracted, etc. to construct expression strains, which were stored at -80 °C for standby. The specific protein preparation process is as follows:

[0024] (1) Take 10 mL of LB medium, add 100 μL of the expressed bacterial strain liquid, and 20 μL of Kana (kanamycin), and shake the bacteria overnight at 37 °C and 220 rpm. (2) Inoculate 10 mL of the overnight-cultured bacterial liquid into 1 L of LB medium (containing 2 mL of Kana), and culture at 37 °C and 220 rpm for about 5 h. Then add the inducer IPTG (1 mM), and culture at 16 °C and 200 - 220 rpm for 12 h. (3) Transfer 1 L of the bacterial liquid into two 500 mL high-speed centrifuge cups, balance them on the balance, and then centrifuge at 4000 rpm for 40 min. After centrifugation, discard the supernatant as much as possible and collect the precipitate. (4) Add 50 mL of 1×PBS buffer to each, resuspend, centrifuge at 4 °C and 12000 rpm for 10 min, and discard the supernatant. (5) Repeat step (4). (6) Add 50 mL of His Buffer A buffer and 0.5% Triton X-114 (can be replaced with 1×PBS) to the precipitate, resuspend, and transfer the bacterial liquid in the centrifuge cup to two 50 mL centrifuge tubes for ultrasonic disruption (90 ml is used to resuspend 1 L of bacteria). (7) Place it on ice for ultrasonic disruption. The disruption program is to ultrasonicate for 3 s and pause for 5 s, and the total ultrasonic time is 15 min until the bacterial liquid changes from turbid to relatively clear (similar to the appearance of limestone dissolved in water, depending on the vector, strain, and tag specifically, some can be clarified). Ultrasonic disrupt the bacteria in the other tube with the same program. (8) After completion, centrifuge at 4 °C and 12000 rpm for 15 min, collect the supernatant and combine them. (9) Filter the supernatant through a 0.45 μm filter membrane and store it at 4 °C for later use. (10) When treating the bacterial liquid, the nickel column can be treated simultaneously. Open the lid at the bottom of the nickel column to let the original liquid in the nickel column flow down, add His Buffer B buffer with a concentration of 500 mM, place it on ice and shake on an oscillator for 10 min and then wash, and the oscillator speed is 60 rpm. (11) Add His Buffer A, place it on ice and shake on an oscillator for 30 min and then wash to remove the remaining miscellaneous proteins. Repeat more than three times. (12) Load the supernatant filtered through a 0.45 μm filter membrane onto the column, place it in an ice box, and place the ice box on a shaker to bind overnight. (13) Wash with His Buffer A and 0.5% Triton X-114 four times. Each time, let the buffer flow down after binding for 8 h as samples A1, A2, A3, A4, and store them in a -20 °C refrigerator. Elute with 25 mM HisBuffer A, each time binding for 15 min, and wash four times. (14) Elute with 500 mM His Buffer B, each time binding 15 ml for 15 min, collect the flowing liquid separately and label it as B1, B2, B3, B4 and save samples for running SDS-PAGE gel electrophoresis. Dialyze the eluted samples with freshly prepared 1*PBS overnight to calculate and reduce the imidazole concentration to below 10 mM. (15) After use, the nickel column should be bound with 500 mM His Buffer B for 15 min.(16) Wash three times with His Buffer A for 5 minutes each time. (17) Fill the column with His Buffer A and store the column in a 4°C refrigerator. If not used for a long time, place it in 20% ethanol. (18) Dialyze. Place B1 in a treated dialysis bag, clamp the dialysis bag, put it into a beaker filled with 1×PBS, and also place the rotor in the beaker. Let the rotor rotate to drive the dialysis bag to rotate gently overnight. (19) Take out the swollen dialysis bag, dry it, apply sucrose on the surface of the dialysis bag to suck out the solvent, so as to achieve the concentration effect. After 2 - 3 hours, take out the liquid in the dialysis bag, add it to a 10KDa ultrafiltration tube for ultrafiltration at 4°C, 3000 rpm for 15 minutes, repeat twice for quantification. (20) The protein concentration can be directly measured with an instrument for measuring protein concentration, or the BCA kit can be used to measure the protein concentration. Generally, concentrate to a concentration of 1 mg / ml. If the concentration is higher than this, dilute it with 1×PBS. If the concentration is lower than this, continue ultrafiltration. Store the aliquoted and quantified protein at -80°C.

[0025] The results are as Figure 1 and Figure 2 shown. The SDS-PAGE purity of the purified P4 and P8 type VP8 proteins is above 90%, and the concentration detected using the BCA kit is above 1 mg / ml.

[0026] Example 2: Preparation of Monoclonal Antibodies Against Different P-Type (P4, P8) Rotavirus VP8 Proteins

[0027] Perform amino acid sequence alignment analysis on the VP8 proteins of P4 type and P8 type respectively, and find that the homology of these two proteins is very high, reaching 250 / 289 (86.5%), as Figure 3 shown. Therefore, in order to prepare monoclonal antibodies that can specifically bind to P4 type and P8 type VP8 proteins respectively, in this study, antigen polypeptides were prepared in the form of artificially synthesized polypeptides for the preparation of monoclonal antibodies. The antigen polypeptides are respectively:

[0028] P8-VP8 (antigen polypeptide 1): vdpvdr;

[0029] P4-VP8 (antigen polypeptide 2): vsqtnr;

[0030] P4-VP8 (antigen polypeptide 3): iklgglgy.

[0031] Couple the antigen polypeptide with casein (OVA) to prepare the antibody peptide. Immunize female BALB / c mice aged 6 - 8 weeks, with a total of three immunizations, using the subcutaneous multiple-point injection method. The antigen immunization dosage for each mouse each time is 25 μg. For the primary immunization, it is emulsified with an equal volume of Freund's complete adjuvant; for the second immunization, it is emulsified with an equal volume of Freund's incomplete adjuvant; for the third immunization, it is mixed with an equal volume of normal saline and injected intraperitoneally.

[0032] Seven days after the second immunization, collect blood and measure the antibody titer by the indirect method (using the 2 proteins prepared in the examples for coating detection respectively) to determine whether antibodies against the antigen are generated. Compare the mouse titers, and finally select the mice with higher antibody titers for cell fusion. Three days before fusion, boost the immunization directly with the antigen once, with the same dose as before. Through detection, in the immunization group with antigen polypeptide 1, 1 mouse was screened out with an antibody titer of 1:3000 against the P8-VP8 protein and 1:200 against the P4-VP8 protein; in the immunization group with antigen polypeptide 2, no mouse that could only bind to the P4-VP8 protein was screened out; in the immunization group with antigen polypeptide 3, 1 mouse was screened out with an antibody titer of 1:10000 against the P4-VP8 protein and 1:50 against the P8-VP8 protein.

[0033] Take the spleens of the above 2 screened mice respectively, fuse the spleen cells with myeloma cells, and screen for hybridoma cells. After screening and detection, one good hybridoma cell strain was screened out from each mouse. Prepare mouse ascites and monoclonal antibodies from these 2 hybridoma cell strains as follows:

[0034] (1) Take 6-week-old healthy female mice, sensitize them by intraperitoneal injection of 0.5 ml of liquid paraffin. One to two weeks later, inject 1 - 2×10 6 hybridoma cells into each mouse intraperitoneally. Observe the mouse status. After 7 - 10 days, when the mouse abdomen is significantly enlarged, collect the ascites. The centrifuged supernatant is the ascites, which is aliquoted and labeled for standby. (2) Centrifuge the ascites for 15 min (4000 rpm, room temperature), take the supernatant, and purify it using a Protein G column. First, pass 5 ml of ultrapure water through the column, and then equilibrate the purification column with 5 ml of 0.4 M PB buffer (pH 7.0); pass the antibody through the column, and during the process, it is required to pass the column slowly to make the antibody protein better bind to the binding site; continue to equilibrate the purification column with 10 ml of 0.4 M PB buffer (pH 7.0); elute the antibody on the binding site with 5 ml of 0.1 M glycine-hydrochloric acid buffer (pH 2.7), and add 1 M Tris-HCl (pH 8.0) to neutralize glycine to keep the pH neutral for antibody preservation. Determine the concentration of the purified antibody by the BCA method, aliquot it, and store it at -80 °C for standby.

[0035] The Western blot was performed on the two prepared monoclonal antibodies. Monoclonal antibody 1 prepared from antigen polypeptide 1 could only bind to the P8-VP8 protein and not to the P4-VP8 protein, while monoclonal antibody 2 prepared from antigen polypeptide 3 could only bind to the P4-VP8 protein and not to the P8-VP8 protein. Specifically, as shown in Figure 4 shown.

[0036] By examining and analyzing the sequences of the two monoclonal antibodies, the amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 1 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively, and the amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 2 are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0037] Example 3: Preparation of test strips for detecting different P-type (P4, P8) rotaviruses

[0038] The P4-VP8 protein and P8-VP8 protein prepared in Example 1 were mixed in equal mass ratio, emulsified with Freund's complete adjuvant to prepare a vaccine, and the protein content in the vaccine was 100 μg / ml. Two healthy New Zealand white rabbits at 6 weeks old (about 2 Kg) were selected. Each rabbit was immunized subcutaneously at 4 sites on the back, 0.25 ml at each site, and boosted every 2 weeks for a total of 4 immunizations. Cardiac blood was collected 2 weeks after the 4th immunization. The serum was centrifuged for 15 min (4000 rpm, room temperature), and the supernatant was taken for purification of polyclonal antibodies using a ProteinG column, as shown in Example 2 specifically. The purified rabbit polyclonal antibodies were aliquoted and stored at -80 °C for later use.

[0039] The rabbit polyclonal antibodies, monoclonal antibodies, and proteins prepared above were used to prepare test strips for detecting rotaviruses. The specific method is as follows:

[0040] (1) Preparation of nitrocellulose membrane: The nitrocellulose membrane was attached to the corresponding position of the PVC bottom plate. Monoclonal antibody 1 and monoclonal antibody 2 were respectively diluted to 1 mg / mL with coating buffer (PBS buffer containing 5% sucrose). The scribing position and height of the scribing machine were adjusted, and the scribing was the T line, that is, the test line. The T line was close to the gold conjugate pad end. With the quality control line (C line) solution (goat anti-rabbit IgG secondary antibody containing 1 mg / mL), the scribing position and height of the scribing machine were adjusted, and the scribing was the C line, that is, the quality control line. The C line was close to the absorbent pad, and the distance between the two lines was 5 - 8 mm. It was dried in an oven at 37 °C for 15 hours, sealed in an aluminum foil bag, and stored at room temperature for later use.

[0041] (2) Preparation of gold conjugate pad

[0042] Colloidal gold preparation: Heat the 1% chloroauric acid aqueous solution, add the 1% trisodium citrate aqueous solution after stirring evenly, and boil. Keep it for use after cooling. Add rabbit polyclonal antibody (add 5 μg antibody to 1 mL gold water) and react for 10 minutes, then add 1% blocking agent (PBST solution containing 1% BSA) to block for 30 minutes. Then centrifuge to discard the supernatant, add the reconstitution solution, and shake evenly.

[0043] Gold spraying: Spray the resuspended colloidal gold solution onto the glass cellulose membrane at a rate of 2 μL / cm using a gold spraying machine, dry it at 37°C for 16 - 20 hours, seal it with an aluminum foil bag, and store it at room temperature for later use.

[0044] (3) Treatment of sample pad: Evenly apply the sample pad treatment solution (PBS buffer containing 1% casein) on the glass cellulose membrane, dry it at 37°C for 16 - 20 hours, seal it with an aluminum foil bag, and store it at 2 - 30°C for later use.

[0045] (4) Assembly of test strip: As Figure 5 shown, cut the sample pad, gold label pad, and absorbent pad into corresponding widths and paste them in sequence at the corresponding positions on the PVC bottom plate with the nitrocellulose membrane already adhered, making the gold label pad and absorbent pad in partial contact with the nitrocellulose membrane respectively, and the sample pad in partial contact with the gold label pad to make a large plate.

[0046] (5) Packaging: Cut the large plate into test strips 2 - 3 mm wide with a strip cutter, install the outer shell, seal and package it with an aluminum foil bag, containing one test strip, one pipette, and one pack of desiccant, and store it at room temperature in the dark for later use.

[0047] (6) Usage and determination

[0048] ① Restore the test card and the sample solution to be tested to room temperature before use.

[0049] ② Take out the test card from the original packaging bag, and use it as soon as possible within one hour after opening.

[0050] ③ Place the test card flat, use a dropper to suck the sample solution to be tested (usually an anal swab), and vertically drop 2 - 3 drops into the sample addition hole, and start timing after adding the sample.

[0051] ④ The result should be read within 3 - 5 minutes, and the judgment at other times is invalid. Judge the result according to the schematic diagram, and the judgment criteria are as follows:

[0052] Negative (-): There is no band at the test T line, and there is a band at the quality control C line, indicating that the sample does not contain the corresponding type of rotavirus or its concentration is lower than the detection limit.

[0053] Positive (+): There is a band at the test T line, and there is also a band at the quality control C line, indicating that the sample contains the corresponding type of rotavirus.

[0054] Invalid: There is no band on the quality control C line, indicating that the operation process is incorrect or the test strip has expired.

[0055] Example 4: Detection of test strips for different P-type (P4, P8) rotaviruses

[0056] The prepared test strips were used to detect samples clinically identified as positive and negative, and the results are as follows:

[0057] (1) Specificity test: The test strips were used to detect anal sub-samples (which may contain other viruses such as influenza virus) that were PCR-identified as rotavirus-free. The results showed that all samples were negative, indicating that the test strips had good specificity. As shown in Table 1 and Figure 6 shown.

[0058] Table 1 Specificity test results

[0059]

[0060] Note: The test line of the P4 test strip is monoclonal antibody 2, and the test line of the P8 test strip is monoclonal antibody 1.

[0061] (2) Sensitivity test: The prepared test strips were used to detect different concentrations of P4-VP8 protein and P8-VP8 protein respectively. The results showed that the lower detection limit of both test strips was 4 ng / ml. As shown in Table 2 and Figure 7 shown.

[0062] Table 2 Specificity test results

[0063]

[0064] (3) Conformance test: The prepared test strips were used to detect samples that were PCR-identified as positive or negative and typed. The results showed that the coincidence rate of this test strip with the PCR detection result was 100%, as shown in Table 3.

[0065] Table 3 Conformance test results

[0066]

[0067] Note: The test line of the P4 test strip is monoclonal antibody 2, and the test line of the P8 test strip is monoclonal antibody 1.

[0068] In addition, the test strip of the present invention can also be used as a combined test strip, that is, one test strip can achieve the detection of 2 types of rotaviruses simultaneously.

[0069] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A monoclonal antibody composition, characterized in that: The monoclonal antibody composition comprises monoclonal antibody 1 and monoclonal antibody 2; wherein monoclonal antibody 1 specifically binds to VP8 protein of rotavirus P8, and the sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 1 are shown in SEQ ID NO.3 and SEQ ID NO.4; Monoclonal antibody 2 specifically binds to P4 type rotavirus VP8 protein. The sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 2 are shown in SEQ ID NO.5 and SEQ ID NO.

6.

2. The monoclonal antibody composition according to claim 1, characterized in that The amino acid sequence of the monoclonal antibody 1 that specifically binds to the VP8 protein of the P8 type rotavirus is vdpvdr.

3. The monoclonal antibody composition according to claim 1, characterized in that The amino acid sequence of the monoclonal antibody 2 that specifically binds to the P4 type rotavirus VP8 protein is iklgglgy.

4. A rotavirus test strip, characterized in that: The test strips include P4 test strips and P8 test strips; wherein the P4 test strips are prepared from the monoclonal antibody 2 described in claim 1, a rabbit polyclonal antibody against rotavirus VP8 protein, and matching reagents; and the P8 test strips are prepared from the monoclonal antibody 1 described in claim 1, a rabbit polyclonal antibody against rotavirus VP8 protein, and matching reagents.

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