CN113354727B

CN113354727B - Monoclonal antibody and test strip for simultaneously detecting B/C/E group adenovirus

Info

Publication number: CN113354727B
Application number: CN202110910863.2A
Authority: CN
Inventors: 卢帅; 张兴林; 李月; 陈曼丽; 张东晖; 宋文俊
Original assignee: Beijing Subenyuanhe Biotechnology Co ltd
Current assignee: Beijing Subenyuanhe Biotechnology Co ltd
Priority date: 2021-08-10
Filing date: 2021-08-10
Publication date: 2021-11-02
Anticipated expiration: 2041-08-10
Also published as: CN113354727A

Abstract

The invention provides a group of monoclonal antibodies and test strips for simultaneously detecting B/C/E group adenoviruses, and relates to the technical field of adenovirus detection. The monoclonal antibody specific to the currently popular respiratory adenovirus HAdV-B/C/E group is obtained by screening, and a colloidal gold test strip for detecting the B/C/E group adenovirus is prepared by using the monoclonal antibody, and has a good detection effect, wherein the minimum detection limit of ADV1/2/5/6 can reach 1.0 multiplied by 10³VP/ml; the detection limit of ADV3/4/7 can reach 1.0 multiplied by 10⁴VP/ml; and the good specificity is verified. The invention provides a pair of monoclonal antibody cell strains and a rapid detection method for simultaneously detecting B/C/E group adenovirus.

Description

Monoclonal antibody and test strip for simultaneously detecting B/C/E group adenovirus

Technical Field

The invention belongs to the technical field of adenovirus detection, and particularly relates to a group of monoclonal antibodies and test strips for simultaneously detecting B/C/E group adenovirus.

Background

Adenovirus (Adenovirus) is a non-enveloped icosahedral double-stranded DNA virus, and often invades multiple organs such as respiratory system, digestive system, urinary system, conjunctiva of eye and the like. Adenovirus is prevalent in people all year round, can occur in any age group, and has been subjected to outbreak of adenovirus infection in various countries and regions all over the world, and no specific treatment method is available at present. Adenovirus is an opportunistic pathogen, and approximately 50% of infections are asymptomatic, but still infectious.

Adenoviruses that have been found to infect humans have approximately 69 serotypes, which can be divided into 7 subgroups of A-G. The different serotypes of adenovirus cause differences in tissue tropism for infection, with groups B, C and E often causing respiratory tract infections, group E also infecting the conjunctiva of the eye, and groups a and F primarily infecting the gastrointestinal tract. Adenovirus infection is most common with respiratory symptoms, wherein 3/7 type in group B and 1/2/5/6 type in group C mostly cause acute respiratory infection in children, and 4 type in group E is mostly related to acute respiratory disease epidemic in military training camps. Adenovirus has strong resistance and infectivity, and often causes fulminant epidemics of crowds such as families, kindergartens, primary schools and the like, so that rapid and accurate diagnosis and treatment are necessary for controlling and preventing adenovirus infection and transmission.

Virus culture is currently used as a gold standard for laboratory diagnosis, but is limited by the cell culture cycle and is difficult to apply clinically. Compared with a cell culture method, the fluorescence quantitative PCR has higher sensitivity, but the consumption is expensive, the operation is carried out by professional personnel, and the false positive is easily caused by pollution when a large number of samples are operated, so the field application is limited.

Disclosure of Invention

In view of the above, the present invention aims to provide a group of hybridoma cell groups producing monoclonal antibodies 1H3 and 2a7, monoclonal antibodies, and a preparation method and applications thereof, wherein the operation is simple and convenient, the time consumption is short, the adenovirus infection in a respiratory tract sample can be rapidly detected, the detection accuracy is high, and the method is very suitable for rapid auxiliary diagnosis of respiratory tract adenovirus infection.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a group of monoclonal antibodies for simultaneously detecting B/C/E group adenoviruses, which comprise 1H3 and 2A7, wherein a heavy chain variable region of 1H3 comprises three complementarity determining regions, and 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 1H3 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are respectively shown as SEQ ID NO. 4-SEQ ID NO. 6;

the heavy chain variable region of 2A7 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are respectively shown as SEQ ID NO. 11-SEQ ID NO. 13; the light chain variable region of 2A7 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are respectively shown in SEQ ID NO. 14-SEQ ID NO. 16.

Preferably, the amino acid sequence of the heavy chain variable region of 1H3 is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region of 1H3 is shown in SEQ ID NO. 8.

Preferably, the nucleotide sequence encoding the heavy chain variable region of 1H3 is shown in SEQ ID NO.9, and the nucleotide sequence encoding the light chain variable region of 1H3 is shown in SEQ ID NO. 10.

Preferably, the amino acid sequence of the heavy chain variable region of 2A7 is shown in SEQ ID NO.17, and the amino acid sequence of the light chain variable region of 2A7 is shown in SEQ ID NO. 18.

Preferably, the nucleotide sequence encoding the heavy chain variable region of 2A7 is shown in SEQ ID NO.19, and the nucleotide sequence encoding the light chain variable region of 2A7 is shown in SEQ ID NO. 20.

The invention also provides application of the monoclonal antibody in preparation of a tool for simultaneously detecting B/C/E group adenoviruses.

Preferably, the means comprises a colloidal gold test strip.

The invention also provides a colloidal gold test strip for simultaneously detecting the B/C/E group adenovirus, wherein the colloidal gold test strip takes 1H3 in the monoclonal antibodies as a capture antibody and takes 2A7 in the monoclonal antibodies as a marker antibody.

Preferably, the colloidal gold test strip comprises a cellulose membrane, a gold label pad, a sample pad and a water absorption pad which are sequentially connected to a back plate, and the cellulose membrane is provided with a test line and a quality control line; the detection line is coated with monoclonal antibody 1H3, and the quality control line comprises goat anti-mouse IgG;

the gold-labeled pad was coated with monoclonal antibody 2a 7.

Has the advantages that: the invention obtains the monoclonal antibody specific to the currently popular respiratory adenovirus HAdV-B/C/E group through screening,the colloidal gold test strip for detecting B/C/E group adenovirus is prepared, has good detection effect on adenovirus group B (3/7), group C (1/2/5/6) and group E (4), wherein the minimum detection limit on group C ADV1/2/5/6 can reach 1.0 multiplied by 10³VP/ml; the detection limit of ADV3/4/7 can reach 1.0 multiplied by 10⁴VP/ml; and the test proves that the vaccine has good specificity and does not have cross reaction with common respiratory tract A/B influenza virus, coronavirus and respiratory syncytial virus. The invention provides a pair of monoclonal antibody cell strains and a rapid detection method for simultaneously detecting B/C/E group adenovirus.

Drawings

FIG. 1 is a diagram showing the results of screening positive clones;

FIG. 2 is an SDS-PAGE identification of monoclonal antibody purity;

FIG. 3 is a diagram showing the result of the specificity identification of a monoclonal antibody;

FIG. 4 is a schematic diagram of the assembly of a colloidal gold test strip;

FIG. 5 is a diagram showing the results of a test strip specificity assay;

FIG. 6 is a graph showing the results of the sensitivity analysis of the test strip.

Detailed Description

The amino acid sequence of the heavy chain variable region of the 1H3 is preferably shown as SEQ ID NO.7, and the nucleotide sequence for coding the heavy chain variable region is preferably shown as SEQ ID NO. 9; the amino acid sequence of the light chain variable region of 1H3 is preferably shown as SEQ ID NO.8, and the nucleotide sequence encoding the light chain variable region of 1H3 is preferably shown as SEQ ID NO. 10.

The amino acid sequence of the heavy chain variable region of 2A7 is preferably shown as SEQ ID NO.17, and the nucleotide sequence of the heavy chain variable region of 2A7 is preferably shown as SEQ ID NO. 19; the amino acid sequence of the light chain variable region of 2A7 is preferably shown as SEQ ID NO.18, and the nucleotide sequence encoding the light chain variable region of 2A7 is preferably shown as SEQ ID NO. 20.

The method for producing the monoclonal antibody of the present invention is not particularly limited, and preferably a method for producing a hybridoma cell group using ascites and a hybridoma cell, wherein the method for producing the hybridoma cell group comprises the steps of: (1) immunizing a plurality of female mice for the first time by using group C adenovirus, performing secondary boosting immunization on the female mice by using group B adenovirus, performing tertiary boosting immunization by using group E adenovirus, screening mice with higher titer in serum for group C adenovirus abdominal cavity impact, and extracting spleen cells after 3 days;

(2) fusing the spleen cells and SP2/0 cells to obtain the hybridoma cells.

According to the method, a plurality of female mice are immunized for the first time by using the group C adenovirus, the female mice are subjected to secondary boosting immunization by using the group B adenovirus, the female mice are subjected to tertiary boosting immunization by using the group E adenovirus, the mice with higher titer in serum are screened for group C adenovirus abdominal cavity impact, and spleen cells are extracted after 3 days.

The first immunization of the invention preferably comprises immunizing the female mouse by using a mixed emulsion of the group C adenovirus and Freund's complete adjuvant; the volume ratio of the group C adenovirus to Freund's complete adjuvant is 1: 1. The group C adenovirus of the present invention is preferably group HAdV-C (2), and when the first immunization is performed, the group C adenovirus is preferably diluted to 1X 10⁸After VP/ml, Balb/C female mice are immunized with an equal volume of Freund's complete adjuvant according to a total dose of 200 mu L/mouse for 4-6 weeks.

The invention describesThe boosting immunization preferably comprises immunizing female mice after the first immunization with a mixed emulsion of group B adenovirus and Freund's incomplete adjuvant; the volume ratio of the group B adenovirus to Freund's incomplete adjuvant is 1: 1. The group B adenovirus is preferably diluted to 1 x 10⁸After VP/ml, the above first immunized female mice were immunized with an equal volume of Freund's incomplete adjuvant at a total dose of 200. mu.L/mouse. The group B adenovirus of the present invention is preferably group HAdV-B (7).

The third boosting immunization preferably comprises the step of immunizing a female mouse subjected to the second boosting immunization by using a mixed emulsion of the group E adenovirus and a Freund's incomplete adjuvant; the volume ratio of the group E adenovirus to Freund's incomplete adjuvant is 1: 1. The invention preferably dilutes the group E adenovirus to 1X 10⁸After VP/ml, the female mice after the above booster immunization were immunized with an equal volume of Freund's incomplete adjuvant at a total dose of 200. mu.L/mouse. The group E adenovirus of the present invention is preferably group HAdV-E (4). In the present invention, the time intervals of the primary immunization, the secondary boosting immunization and the tertiary boosting immunization are preferably 2 weeks.

The invention preferably further comprises collecting serum to measure titer after the three boosts, and selecting mice with higher titer in HAdV-C group (2) (1 × 10)⁸VP/ml) 200. mu.L/peritoneal punch, and three days later spleen cells were harvested for fusion. The method for extracting the spleen cells in the present invention is not particularly limited, and any conventional method in the art may be used.

After spleen cells are obtained, the spleen cells and SP2/0 cells are fused to obtain the hybridoma cells. In the present invention, preferably, the spleen cells are fused with myeloma cells SP2/0 by PEG1450 to prepare hybridoma cells.

After obtaining the hybridoma, the invention preferably further comprises diluting the hybridoma to a monoclonal state, measuring the titer of cell culture supernatant, and screening monoclonal antibodies which only specifically react with the C group adenovirus, the B group adenovirus and the E group adenovirus. In the present invention, it is preferable to select a specific monoclonal antibody by an ELISA method, and in the selection, the specific monoclonal antibody is detected on a HAdv2/3/4/6/7 type virus coated plate, and is coated with 200-fold dilution of Hep-2 cell disruption solution (repeated freeze-thaw) and protein amounts corresponding to adenovirus of RSV, A type (A/PR 8)/B type influenza virus (B/Yamagata) and coronavirus HCoV (OC 43, NL 63), and is detected by an indirect ELISA method, and three monoclonal antibodies specifically reacting only with HAdv2/3/4/6/7 from 1H3, 2A7 and 6D8 strains are selected, and are subjected to extensive culture and cryopreservation after limited dilution to a monoclonal state, and can be used for preparing the specific monoclonal antibody.

The present invention preferably utilizes the hybridoma cell group to immunize a mouse in the abdominal cavity to prepare ascites, and more preferably, after the hybridoma cell line selected is expanded and cultured, 0.2mL (about 2X 10) is injected into the abdominal cavity⁶Individual cells) to female Balb/C mice, and after about 10-15 days, when the abdomen of the mice is obviously swollen, using a sterile syringe needle to collect ascites. Preferably, the collected ascites is centrifuged at 12000 r/min for 10min, the upper grease and the lower sediment are removed, and the supernatant is collected; diluting the ascites supernatant with binding buffer at a ratio of 1:10, filtering with a filter membrane, and binding to a Protein G column (equilibrated with binding buffer); washing about 5 column volumes with binding buffer, eluting with elution buffer (0.1M glycine, pH 2.7), and collecting the elution peak; then quickly neutralized with 1M Tris-HC1 (pH 9.0) solution to a dry neutral range; and (3) dialyzing the purified antibody into 1 × PBS after changing the solution for many times to obtain the monoclonal antibody. In the invention, monoclonal antibody subtype identification kit (Proteitech) is used for identifying three antibody subtypes (1H 3, 2A7 and 6D 8), the operation is carried out according to the specification, the result shows that 1H3 is IgG2a, 2A7 and 6D8 are IgG1 subtypes, meanwhile, the paired antibodies are screened by ELISA method, the result shows that OD450nm value of 1H3 coating and 2A7 marking as paired monoclonal antibody detection is highest, negative and positive can be distinguished well, the best paired monoclonal antibodies are determined to be 1H3 and 2A7, and the monoclonal antibodies are used as capture antibodies and marking antibodies of the adenovirus detection test strip.

The kit of the present invention preferably comprises a colloidal gold test strip. The structure and the preparation method of the colloidal gold test strip are not specially limited.

The colloidal gold test strip comprises a cellulose membrane, a gold label pad, a sample pad and a water absorption pad which are sequentially connected to a back plate, wherein a test line and a quality control line are arranged on the cellulose membrane; the detection line is coated with monoclonal antibody 1H3, and the quality control line comprises goat anti-mouse IgG; the gold-labeled pad was coated with monoclonal antibody 2a 7.

The detection line and the quality control line are preferably prepared by diluting the purified antibody monoclonal antibody 1H3 to 1mg/ml by PBS (phosphate buffer solution) with the pH value of 7.4, and coating the purified antibody monoclonal antibody 1H3 on a cellulose membrane by the amount of 1 mu L/cm to be used as a detection line, namely a T line; the goat anti-mouse IgG is diluted to 1mg/mL by using PBS (pH7.4) at the interval of 6mm, and then coated on a cellulose membrane in the amount of 1 muL/cm to be used as a quality control line, namely a C line.

The gold-labeled pad of the invention is preferably coated with a gold-labeled antibody 2A7, and the preparation method of the gold-labeled antibody 2A7 gold label preferably comprises the steps of preparing a 1.0% gold particle solution by a citric acid reduction method; add 5. mu.L of 0.2M K to 1ml of gold solution₂CO₃Adjusting the pH value of the aqueous solution to 7.6, uniformly mixing, adding the purified antibody 2A7 (5 mu g/ml) and 20 mu L of 10% BSA, uniformly mixing, standing for 10min, adding 10 mu L of 10% PEG, standing for 5min, centrifuging at 12000rpm for 5min, removing supernatant, adding a complex solution (0.01M PB +1% BSA +2% sucrose) and re-suspending to obtain the gold-labeled antibody 2A 7. In the invention, the gold-labeled antibody 2A7 is preferably diluted by a diluent, sprayed on glass fiber as a gold-labeled pad in an amount of 1200 mu L/strip, and dried at 40 ℃ for later use.

According to the invention, preferably, as shown in fig. 4, the coated cellulose membrane, gold label pad, sample pad and absorbent pad are adhered to the back plate in the required sequence, cut by a slitter and wrapped by a plastic card shell, so that the sample pad is exposed at the position of the sample adding hole of the plastic card shell, and the quality control line and the detection line are exposed at the position of the result observation hole, thereby assembling the complete detection card.

The following examples are provided to illustrate the group of hybridoma cells producing monoclonal antibodies 1H3 and 2a7, their preparation and use, but should not be construed as limiting the scope of the invention.

The materials used in the invention are all conventional commercial products unless otherwise specified.

1. Virus

The human adenovirus serotype HAdV-B group (3/7), the HAdV-C group (1/2/5/6) and the HAdV-E group (4) are separated and identified by a nucleic acid positive clinical throat swab and propagated and amplified in a Hep2 cell, human coronavirus HCoV (OC 43, NL 63), influenza A virus (PR 8), influenza B virus (Yamagata) and respiratory syncytial virus (RSV-long) are separated, identified and stored by a virus disease prevention and control center in China, and Balb/C mice are purchased from Beijing Huafukang Biotechnology GmbH (SCXK (Beijing) 2019-.

2. Reagent

The conventional chemical reagents such as sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, Tris, surfactant and the like are domestic analytical reagents, Freund's adjuvant and chloroauric acid are Sigma products, goat anti-mouse IgG-HRP is purchased from Beijing Solebao, and TMB color developing solution is produced by Wande of Beijing Meike.

Example 1

1. Preparation of hybridoma cells

1.1 immunization of mice

First diluted to 1X 10 with HAdV-C group (2)⁸VP/ml, immunizing Balb/C female mice for 4-6 weeks; group (7) (1X 10) of HAdV-B for Secondary Booster immunization⁸VP/ml), HAdV-E group for three boosts (4) (1X 10)⁸VP/ml）；

The dosage of the venom is 100 mu L/venom for each immunization, and the venom is mixed with equivalent volume of Freund's complete adjuvant into emulsion for immunization during the first immunization; the two and three boosts were carried out by mixing and emulsifying 100. mu.l/venom with Freund's incomplete adjuvant at an equal volume, and the interval between each immunization was 2 weeks.

Collecting serum after the third immunization to determine titer, and selecting mice with higher titerGroup (2) (1X 10) of HAdV-C⁸VP/ml) 200. mu.l/peritoneal punch (without adjuvant), and three days later spleen cells were harvested for fusion.

1.2 cell fusion and Positive clone screening

The mouse orbit after the peritoneal boosting immunization is killed after blood drawing, spleen cells of the mouse are taken and myeloma cells SP2/0 are fused through PEG1450 to prepare hybridoma cells, the hybridoma cells are detected by using various virus coating plates of an HAdV-B group (3/7), an HAdV-C group (1/2/5/6) and an HAdV-E group (4), and are diluted by 200 times by using Hep-2 cell breaking solution (repeated freeze thawing) and RSV, A type (A/PR 8)/B type influenza virus (B/Yamagata), coronavirus HCoV (OC 43, NL 63) according to 100 times by an indirect ELISA method to screen monoclonal antibodies of 1H3, 2A7 and 6D8 which specifically react with the HAdV-B group (3/7), the HAdV-C group (1/2/5/6) and the HAdV-E group (4), after limited dilution to a monoclonal state, carrying out amplification culture and freezing storage. As a result, as shown in FIG. 1, the obtained monoclonal cell line was used to immunize a mouse with the abdominal cavity, and ascites was prepared.

The ELISA detection method comprises the following steps: the antigen was diluted with a coating buffer (carbonate buffer: 1.59g sodium carbonate and 2.93g sodium bicarbonate to 1L pure water) using a carbonate buffer (pH 9.6) according to the above requirements, added to an ELISA plate at 50. mu.l/well, coated overnight at 4 ℃, blocked with 2% horse serum and 1% sucrose at 37 ℃ for 2h, and spun-dried for use. Adding 50 μ l/well of cell supernatant into coating plate, incubating at 37 deg.C for 35min, spin-drying, washing with 0.5% PBST lotion for four times, beating, adding HRP-labeled goat anti-mouse IgG (solarbio) for 5000 times dilution, incubating at 37 deg.C for 35min, repeating the plate washing step, adding TMB developing solution (Beijing Meike Wander Biotech Co., Ltd.) for 50 μ l/well, incubating at 37 deg.C for 10min, adding 0.5M sulfuric acid to stop reaction, and adding OD wavelength₄₅₀And (6) reading. The measurement results are shown in the figure.

1.3 preparation and purification of ascites

After the selected hybridoma cell line was subjected to amplification culture, 0.2mL (about 2X 10) was injected into the abdominal cavity⁶Individual cell) to a female Balb/C mouse, and collecting ascites by using a sterile syringe needle after about 10 to 15 days when the abdomen of the mouse is obviously swollen.And (4) centrifuging the collected ascites at 12000 r/min for 10min, removing upper-layer grease and lower-layer precipitates, and collecting supernatant. The ascites supernatant was diluted 1:10 with binding buffer, filtered through a filter and then bound to a Protein G column (equilibrated with binding buffer first). The column was washed with binding buffer for about 5 column volumes, eluted with elution buffer (0.1M glycine, pH 2.7) and the peak was collected. Then neutralized rapidly with 1M Tris-HC1 (pH 9.0) solution, allowing to reach a dry neutral range. The purified antibody was dialyzed into 1 XPBS after changing the solution for several times, the concentration was measured and the antibody was dispensed into 1 mL/tube and stored at-20 ℃ or below.

2. Identification of purified monoclonal antibodies

2.1 antibody purity characterization

Preparing SDS-PAGE electrophoresis gel: cleaning and airing the vertical electrophoresis glass plate, vertically placing the vertical electrophoresis glass plate on a glue making frame, preparing a separation glue system according to requirements, adding a gap between the glass plates until the glue surface reaches the required height, and adding absolute ethyl alcohol for sealing; after solidification at room temperature, removing the sealing liquid on the upper layer of the gel, sucking with absorbent paper, preparing the concentrated gel according to the proportion, inserting a proper comb, and solidifying at room temperature.

SDS-PAGE electrophoresis: adding 2 xSDS loading buffer with the same volume into the antibody sample, boiling in boiling water bath for 10min, centrifuging at 12000rpm for 3min, and taking the supernatant to add into the sample adding hole. Diluting 5 Xglycine buffer solution to working concentration, adding the buffer solution into an electrophoresis tank to a proper liquid level height, and slightly pulling out a comb from the coagulated gel. Protein marker and 10. mu.l of the treated protein sample were added to the well. And (3) switching on a power supply, adjusting the voltage to 80V, performing constant-voltage electrophoresis until the gel is separated, and changing the voltage to 120V until bromophenol blue reaches the bottom of the gel.

Dyeing and decoloring: the gel was cut from the glass plate and placed in Coomassie brilliant blue stain and stained on a shaker overnight. And (3) taking out the gel during decolorization, washing with clear water, placing in a decolorization solution, and repeatedly decolorizing on a shaking table until the strips are clear. The results are shown in FIG. 2, and the purity of 3 monoclonal antibodies can reach more than 90%.

2.2 antibody binding and specificity identification

The purified antibody was subjected to ELISA identification by coating adenovirus antigen and control antigen respectively using the above ELISA detection method, and the antibody was diluted to 1. mu.g/ml, as shown in FIG. 3, 1H3, 2A7 and 6D8 were able to specifically recognize HAdv2/3/4/6/7 virus types.

2.3 antibody subtype identification

The subtype of the three antibodies was identified by using a commercial mouse monoclonal antibody subtype identification kit (Proteintech), and the results, which were performed according to the instructions, showed that 1H3 was IgG2a, and 2A7 and 6D8 were IgG1 subtypes, and the results are shown in Table 1.

TABLE 1 monoclonal antibody subtype identification results (OD)₄₅₀）

3. Establishment of adenovirus rapid detection method

3.1 screening of paired antibodies by ELISA method

The three antibodies were each HRP-labeled and stored at-20 ℃ with glycerol 1:1 after labeling. Monoclonal antibodies were diluted to 0.5 μ g/ml with ph7.4 PBS, coated overnight at 4 ℃, washed 1 time with PBST, and then blocked for 2h at 37 ℃ using a blocking solution containing 2% horse serum +1% sucrose. Adding 200-fold diluted HAdV-B group (3/7), HAdV-C group (1/2/5/6) and HAdV-E group (4) viruses, Hep2 cell lysate 200 dilution, RSV virus, A/B type influenza virus and coronavirus, 50 mu l/hole, reacting at 37 ℃ for 35min, and washing the plate for 4 times; adding HRP-labeled enzyme-labeled mouse monoclonal antibody (diluted 1:3000 times), reacting at 37 ℃ for 35min, and washing the plate for 4 times; adding TMB developing solution, developing at 37 deg.C for 10min, adding stop solution, and reading OD₄₅₀The results are shown in table 2, sensitivity detection is performed on various adenovirus type mixtures diluted by different gradients, and it is found that the OD450nm value detected by 1H3 coating and 2A7 labeling as the paired monoclonal antibodies is the highest, so that negative and positive can be well distinguished, and the optimal paired monoclonal antibodies are determined to be 1H3 and 2A7 and are used as capture antibodies and labeled antibodies of the adenovirus detection test strip.

TABLE 2 determination of paired mAbs (OD)₄₅₀）

3.2 preparation of test paper for detecting colloidal gold

Scribing a film: diluting the purified antibody 1H3 to 1mg/ml with PBS (phosphate buffer solution) with the pH value of 7.4, and coating the purified antibody on a cellulose membrane in an amount of 1 mu l/cm to serve as a detection line, namely a T line; the goat anti-mouse secondary antibody is diluted to 1mg/mL by using PBS (pH7.4) at the interval of 6mm, and then coated on a cellulose membrane in an amount of 1 mu l/cm to be used as a quality control line, namely a C line.

Preparing a gold-labeled antibody: preparing a 1.0% gold particle solution by adopting a citric acid reduction method; mu.l of 0.2M K was added to 1ml of gold solution₂CO₃The pH was adjusted to 7.6, after mixing, purified antibody 2A7 (5. mu.g/ml) and 20. mu.l 10% BSA were added, after mixing, the mixture was left to stand for 10min, 10. mu.l 10% PEG was added, after standing for 5min, the mixture was centrifuged at 12000rpm for 5min, the supernatant was discarded and the mixture was resuspended in a reconstituted solution (0.01M PB +1% BSA +2% sucrose).

Preparing a gold label pad: the gold-labeled 2A7 monoclonal antibody was diluted with a diluent (0.01M PB +1% BSA +0.1% Tween-20), sprayed onto glass fibers at 1200. mu.L/strip to form a gold-labeled pad, and dried at 40 ℃ for use.

Assembling the detection test paper: the coated cellulose membrane, gold label pad, sample pad and absorbent pad are stuck on the back plate according to the required sequence, cut by a slitter and wrapped by a plastic card shell, so that the sample pad is exposed at the position of the sample adding hole of the plastic card shell, and the quality control line and the detection line are exposed at the position of the result observation hole, thus assembling the complete detection card (figure 4).

4. Performance detection of test strips

4.1 specific detection

The assembled test paper card is laid flat, HAdV-B group (3/7), HAdV-C group (1/2/5/6), HAdV-E group (4), human coronavirus HCoV (OC 43, NL 63), influenza A virus (FluA-PR 8), influenza B virus (FluB-Yamagata), respiratory syncytial virus (RSV-long) and Hep2 cell lysate samples are diluted by 10 times (90 mul of sample diluent is added into 10 mul, and one is directly added into sample diluent to be used as a negative control, 100 mul of sample diluent is respectively dripped into a sample adding hole of the test paper card, the result is rapidly judged at 15min, the result is displayed after 20min is invalid, the test paper can well detect ADV1/2/3/4/5/6/7 type as shown in figure 5, but has no cross reaction with other samples and has good specificity.

4.2 sensitivity identification

Purified virus cultures from group HAdV-B (3/7), HAdV-C (1/2/5/6), and HAdV-E (4) were selected and diluted to a particle concentration of: 1.0X 10⁷VP/ml, starting with the initial concentration, and then 10 dilutions of the virus sample (0.01M PB +0.1% Tween-20 +0.2% sucrose)³、10⁴Diluting, and mixing. And (3) horizontally placing the assembled test paper card, dripping 100 mu l of samples into the sample adding holes of the test paper card respectively, quickly judging the result after 15min, and displaying the invalid result after 20 min. As shown in FIG. 6, the lowest detection limit of group C ADV1/2/5/6 was 1.0X 10 as determined by dilution of the virus culture purified with ADV1/2/3/4/5/6/7³VP/ml; the detection limit of ADV3/4/7 can reach 1.0 multiplied by 10⁴VP/ml。

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Beijing date origin and Biotechnology Co., Ltd

<120> a group of monoclonal antibodies and test paper for simultaneously detecting B/C/E group adenoviruses

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 1

Gly Tyr Thr Phe Ser Ser Tyr Trp

1 5

<210> 2

<211> 8

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 2

Ile Leu Pro Gly Ser Gly Ser Thr

1 5

<210> 3

<211> 15

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 3

Ser Arg Ala Leu Ser Thr Met Ile Thr Thr Arg Gly Met Asp Cys

1 5 10 15

<210> 4

<211> 10

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 4

Glu Ser Val Asp Asn Tyr Gly Leu Ser Phe

1 5 10

<210> 5

<211> 3

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 5

Ala Ala Ser

1

<210> 6

<211> 9

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 6

Gln Gln Ser Lys Glu Val Pro Trp Thr

1 5

<210> 7

<211> 122

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 7

Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Phe Thr Ala Asp Ile Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys

85 90 95

Ser Arg Ala Leu Ser Thr Met Ile Thr Thr Arg Gly Met Asp Cys Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 8

<211> 111

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 8

Asn Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Gln Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Leu Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 9

<211> 367

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 9

gaggtgcagc tgcaggagtc tggagctgag ctgatgaagc ctggggcctc agtgaagata 60

tcctgcaagg ctactggcta cacattcagt agctattgga tagagtgggt aaagcagagg 120

cctggacatg gccttgagtg gattggagag attttacctg gaagtggtag tactaactac 180

aatgagaagt tcaagggcaa ggccacattc actgcagata tatcctccaa cacagcctat 240

atgcaactca gcagcctgac atctgaggac tctgccatct attactgttc aagagcccta 300

tctactatga ttacgacgag aggtatggac tgctggggtc aaggaacctc agtcaccgtc 360

tcctcag 367

<210> 10

<211> 334

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 10

aacattgtgc tgacccaatc tccagcttct ttggctgtgt ctcaagggca gagggccacc 60

atctcctgca gagccagcga aagtgttgat aattatggcc ttagttttat gaactggttc 120

caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa ccaaggatcc 180

ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat 240

cctatggagg aggatgatac tgcaatgtat ttctgtcagc aaagtaagga ggttccgtgg 300

acgttcggtg gaggcaccaa gctggaaatc aaac 334

<210> 11

<211> 8

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 11

Gly Phe Thr Phe Ser Thr Tyr Gly

1 5

<210> 12

<211> 8

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 12

Ile Ser Ser Gly Gly Ser Tyr Thr

1 5

<210> 13

<211> 18

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 13

Ala Arg Arg Met Ile Tyr Tyr Asp Tyr Asp Ala Lys Ser Trp Gly Leu

1 5 10 15

Asp Tyr

<210> 14

<211> 10

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 14

Gln Ser Leu Asp Tyr Asp Gly Asp Arg Tyr

1 5 10

<210> 15

<211> 3

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 15

Ala Ala Ser

1

<210> 16

<211> 9

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 16

Gln Gln Ser Asn Glu Val Pro Leu Thr

1 5

<210> 17

<211> 125

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 17

Asp Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Met Ile Tyr Tyr Asp Tyr Asp Ala Lys Ser Trp Gly Leu

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120 125

<210> 18

<211> 111

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 18

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Leu Asp Tyr Asp

20 25 30

Gly Asp Arg Tyr Met Asn Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Ser Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile His

65 70 75 80

Pro Val Glu Lys Glu Asp Thr Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 19

<211> 376

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 19

gacgtgaagc tggtggagtc tgggggagac ttagtgaagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggatt cactttcagt acctatggca tgtcctgggt tcgccagact 120

ccagacaaga gactggagtg ggtcgcaact attagtagtg gtggtagtta tacctactat 180

ccagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa taccctgtac 240

ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgc aagacggatg 300

atctactatg attacgacgc aaagtcctgg gggttggatt actggggcca aggcaccact 360

ctcacagtct cctcag 376

<210> 20

<211> 334

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 20

gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60

atctcctgca aggccagcca aagtcttgat tatgatggtg atcgttatat gaactggtac 120

caacagaaag cgggacagcc acccaaactc ctcatctctg ctgcatccaa tctagaatct 180

gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caccatccat 240

cctgtggaga aggaggatac tgcaacctat tactgtcagc aaagtaatga ggttccgctc 300

acgttcggtg ctgggaccaa gctggagctg aaac 334

Claims

1. The monoclonal antibodies comprise 1H3 and 2A7, the heavy chain variable region of 1H3 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 1H3 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are respectively shown as SEQ ID NO. 4-SEQ ID NO. 6;

2. The monoclonal antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region of 1H3 is shown in SEQ ID No.7, and the amino acid sequence of the light chain variable region of 1H3 is shown in SEQ ID No. 8.

3. The monoclonal antibody of claim 1 or 2, wherein the nucleotide sequence encoding the heavy chain variable region of 1H3 is set forth in SEQ ID No.9, and the nucleotide sequence encoding the light chain variable region of 1H3 is set forth in SEQ ID No. 10.

4. The monoclonal antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region of 2A7 is shown in SEQ ID No.17, and the amino acid sequence of the light chain variable region of 2A7 is shown in SEQ ID No. 18.

5. The monoclonal antibody of claim 1 or 4, wherein the nucleotide sequence encoding the heavy chain variable region of 2A7 is set forth in SEQ ID No.19, and the nucleotide sequence encoding the light chain variable region of 2A7 is set forth in SEQ ID No. 20.

6. Use of a monoclonal antibody according to any one of claims 1 to 5 for the preparation of a means for the simultaneous detection of group B/C/E adenoviruses.

7. The use of claim 6, wherein the means comprises a colloidal gold test strip.

8. A colloidal gold test strip for simultaneously detecting B/C/E group adenoviruses, wherein the colloidal gold test strip takes 1H3 in the monoclonal antibody of any one of claims 1-5 as a capture antibody and takes 2A7 in the monoclonal antibody of any one of claims 1-5 as a marker antibody.

9. The colloidal gold test strip of claim 8, wherein the colloidal gold test strip comprises a cellulose membrane, a gold-labeled pad, a sample pad and a water absorption pad, which are sequentially connected to a back plate, and the cellulose membrane is provided with a detection line and a quality control line; the detection line is coated with monoclonal antibody 1H3, and the quality control line comprises goat anti-mouse IgG;

the gold-labeled pad was coated with monoclonal antibody 2a 7.