CN118126163A

CN118126163A - Respiratory syncytial virus G protein antibody, preparation method and application

Info

Publication number: CN118126163A
Application number: CN202211540006.9A
Authority: CN
Inventors: 赵干
Original assignee: Advaccine Suzhou Biopharmaceutical Co ltd
Current assignee: Advaccine Suzhou Biopharmaceutical Co ltd
Priority date: 2022-12-02
Filing date: 2022-12-02
Publication date: 2024-06-04

Abstract

The invention provides a respiratory syncytial virus G protein antibody, a preparation method and application. The antibody comprises an anti-respiratory syncytial virus G protein antibody 8H6-1 or 5B2-1. The invention also provides an ELISA detection kit, which comprises an anti-respiratory syncytial virus G protease label antibody, an anti-respiratory syncytial virus G protein antibody, a closed system, a coating system, a buffer system and a color development liquid. The kit provided by the invention can be applied to the detection of the G protein antigen of the respiratory syncytial virus, is simple to operate, has a wider detection range, higher sensitivity, can shorten the detection time, and has high accuracy and stable analysis performance.

Description

Respiratory syncytial virus G protein antibody, preparation method and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a respiratory syncytial virus G protein antibody, a preparation method and application thereof.

Background

Respiratory syncytial virus (Respiratory syncytial virus, RSV) can infect the lungs of humans and is the primary causative agent of lower bronchi infections in humans, especially young children. At present, clinical and laboratory researches on the infection mechanism of respiratory syncytial virus and the immune response of human body after infection are gradually perfected, but no approved effective and safe vaccine is yet developed, so that the development of effective respiratory syncytial virus vaccine is urgent.

Chinese patent 201710617822.8 discloses an RSV-PCV combined vaccine and a preparation method thereof, wherein the vaccine takes streptococcus pneumoniae polysaccharide-protein carrier-RSV specific protein as antigen, and has the advantages of simultaneously inducing organisms to generate dual immune responses against RSV and streptococcus pneumoniae, but the preparation method is complex.

RSV is a negative-sense, continuous, single-stranded RNA virus, belonging to the taxonomic family paramyxoviridae, genus pneumovirus. The entire genome of RSV may encode up to 10 viral proteins, including three transmembrane G proteins: an adhesion-acting G protein, a fusion-acting F protein and a small hydrophobin SH protein; two matrix proteins: m protein and M2 protein (including M2.1 and M2.2); three nucleocapsid proteins: n, P and L proteins; there are two additional non-structural proteins: NS1 protein and NS2 protein.

Among the proteins encoded by the respiratory syncytial virus gene, glycosylated proteins (G proteins, glycoprotein) are known as the two "mucin-like" regions in their structure. Similar to cell mucins, G proteins are rich in proline, serine and threonine, the N-terminus is rich in glycosylated side chains, and the secondary structure has a large number of irregular curls. The G protein has a large molecular weight and a very large molecular weight variation range (32,000 kd-90,000 kd) due to the large amount of glycosylation. During viral infection, the G protein, which is the contact protein (ATTACHMENT PROTEIN), acts as a viral anchor by interacting with the epithelial cell surface receptor CX3CR1 via the CX3C motif on its 182-186 amino acid sequence. Experiments prove that compared with wild RSV, the G protein CX3C motif-deleted RSV virus has obviously reduced infection capability and correspondingly reduced lung immune response of infected mice. As one of the major proteins located on the surface of the viral particle, the G protein can produce long-acting neutralizing antibodies. And high affinity G protein antibodies can also be effective in neutralizing RSV viral infection.

In view of the important role and good antigenicity of the G protein in the viral infection process, it is increasingly being studied as a major antigen target for respiratory syncytial virus vaccine development and treatment.

For example, chinese patent 201080042586.8 discloses an immunogenic polypeptide as a vaccine component that corresponds to one or more respiratory syncytial virus G proteins or analogues thereof. The composition of the present invention is useful as a prophylactic and therapeutic agent for the prevention and treatment of respiratory syncytial virus G protein infection and related pulmonary diseases or other diseases.

However, because the respiratory syncytial virus G protein is rich in a 'mucin-like' region, the protein characterization and accurate quantification method of the respiratory syncytial virus G protein are difficult to establish in the development of respiratory syncytial virus vaccine based on the G protein, and in order to solve the problems, the invention provides an ELISA detection kit for quantifying the respiratory syncytial virus G protein and a preparation method thereof.

Disclosure of Invention

The invention provides a respiratory syncytial virus G protein antibody, a preparation method and application. According to the respiratory syncytial virus G protein sequence shown in SEQ ID No.1, the kit is prepared by constructing a vector and expressing a protein to obtain an anti-respiratory syncytial virus G protein antibody and an enzyme-labeled antibody. The ELISA detection kit for quantifying the respiratory syncytial virus G protein can accurately quantify the respiratory syncytial virus G protein. The kit is simple to operate, can shorten the detection time, has high accuracy and stable analysis performance, and has low requirement on G protein concentration. The ELISA detection kit for quantifying the respiratory syncytial virus G protein provided by the invention can play a better auxiliary role in the research of developing respiratory syncytial virus vaccines.

In the invention, when describing the antibody marked by horseradish peroxidase, the suffix "-HRP" can be added directly after the name of the antibody, for example, "8H6-1-HRP" represents the 8H6-1 antibody marked by horseradish peroxidase.

In the present invention, "RSV" is respiratory syncytial virus.

In the present invention, "ELISA" refers to an ELISA assay.

In the present invention, "RSV-rabit" refers to rabbit anti-respiratory syncytial virus antibodies.

In the present invention, "G pr concentration" means G protein concentration.

In the present invention, "RSV-mouse anti-G mAb" refers to a murine anti-respiratory syncytial virus G protein monoclonal antibody.

In one aspect, the invention provides an anti-respiratory syncytial virus G protein antibody, which comprises antibody 8H6-1 or 5B2-1;

The antibody 8H6-1 comprises a heavy chain variable region comprising H-CDR1, H-CDR2 and H-CDR3 and a light chain variable region comprising L-CDR1, L-CDR2 and L-CDR3; wherein the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region shown in SEQ ID NO. 14; the amino acid sequences of the L-CDR1, the L-CDR2 and the L-CDR3 are CDR1, CDR2 and CDR3 of the light chain variable region shown in SEQ ID NO. 15;

The antibody 5B2-1 comprises a heavy chain variable region comprising H-CDR1, H-CDR2 and H-CDR3 and a light chain variable region comprising L-CDR1, L-CDR2 and L-CDR3; wherein the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region shown in SEQ ID NO. 16; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 are CDR1, CDR2 and CDR3 of the light chain variable region shown in SEQ ID NO. 17.

Specifically, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the antibody 8H6-1 are shown as SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 are shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 7; wherein the CDRs are defined by a KABAT definition scheme;

The amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the antibody 5B2-1 are shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively, and the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 are shown as SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13 respectively; wherein the CDRs are defined by a KABAT definition scheme.

Specifically, the heavy chain amino acid sequence of the 8H6-1 is shown as SEQ ID No.18, and the light chain amino acid sequence is shown as SEQ ID No. 19; the heavy chain amino acid sequence of the 5B2-1 is shown as SEQ ID No.20, and the light chain amino acid sequence is shown as SEQ ID No. 21.

On the other hand, the invention provides application of the antibody in preparing ELISA detection kit for quantifying respiratory syncytial virus G protein.

The antibody comprises antibody 8H6-1 and/or antibody 5B2-1.

In yet another aspect, the invention provides an ELISA detection kit for respiratory syncytial virus G protein. The kit is a respiratory syncytial virus G protein double-antibody sandwich ELISA quantitative kit.

Specifically, the kit comprises: one or two of the above anti-respiratory syncytial virus G protein antibodies.

Preferably, the ELISA detection kit comprises an antibody 8H6-1 and an antibody 5B2-1, wherein the antibody 8H6-1 is used as a coating antibody, and the antibody 5B2-1 is used as an enzyme-labeled detection antibody.

Specifically, the coding gene sequence of the respiratory syncytial virus G protein is shown as SEQ ID No. 1; vectors for preparing the respiratory syncytial virus G protein include, but are not limited to pET28a (+); expression systems include, but are not limited to, recombinant E.coli, pichia pastoris, lactic acid bacteria.

Specifically, the anti-respiratory syncytial virus G protein antibodies include, but are not limited to: 0.1-2.5 μg/mL anti-respiratory syncytial virus G protein antibodies 8H6-1 and 5B2-1; 8H6-1 is preferred at a concentration of 2.5. Mu.g/mL.

In some embodiments, rabbit anti-respiratory syncytial virus polyclonal antibodies, goat anti-respiratory syncytial virus polyclonal antibodies may be substituted for the above antibodies as part of a kit.

Specifically, the anti-respiratory syncytial virus G protein antibody is prepared from a subclone hybridoma cell strain 8H 6-1; further specifically, the hybridoma cell strain is obtained by PEG induced fusion of spleen single cell suspension of a mouse after immunization with respiratory syncytial virus G protein and SP2/0 myeloma cells.

Specifically, the anti-respiratory syncytial virus G protease-labeled antibody is a horseradish peroxidase (HRP) enzyme-labeled antibody.

Specifically, the anti-respiratory syncytial virus G protease-labeled antibody comprises but is not limited to 0.5 mug/mL of anti-respiratory syncytial virus G protease-labeled antibody 8H6-1-HRP and 5B2-1-HRP; preferably 5B2-1-HRP at a concentration of 0.5. Mu.g/mL.

Specifically, the anti-respiratory syncytial virus G protease-labeled antibody is prepared by coupling anti-respiratory syncytial virus G protein antibodies 8H6-1 and 5B2-1 with horseradish peroxidase (HRP).

Specifically, the coating system includes, but is not limited to, PBS.

In particular, the closed system includes, but is not limited to, 3% bovine serum albumin, 5% nonfat dry milk, 1% casein. More preferably 3% bovine serum albumin.

Specifically, the buffering system includes, but is not limited to, PBST.

Specifically, the kit further comprises one or more of an enzyme reaction stopping solution, a negative control substance, a positive control substance, a reference standard substance, control serum and sample avidin; the reaction termination liquid includes but is not limited to 2M sulfuric acid.

Specifically, the kit also comprises a solid phase carrier; the solid phase carriers include, but are not limited to, one or more of a microtiter plate, a bead, and a cuvette; preferably a microtiter plate.

In still another aspect, the invention also provides a preparation method of the ELISA detection kit for quantifying respiratory syncytial virus G protein, which comprises the following steps:

(1) Preparation of recombinant respiratory syncytial virus G protein: the nucleotide sequence shown in SEQ ID No.1 is utilized to construct a respiratory syncytial virus G protein expression vector and expressed in an expression system.

(2) Preparation of anti-respiratory syncytial virus G protein antibodies: the recombinant respiratory syncytial virus G protein obtained in the step (1) is used for immunizing a mouse, and the spleen single cell suspension of the immunized mouse and SP2/0 myeloma cells are subjected to PEG-induced fusion. The fusion cells after screening express and purify to obtain the anti-respiratory syncytial virus G protein antibody.

(3) Preparing a G protein horseradish peroxidase-labeled antibody: and (3) coupling the anti-respiratory syncytial virus G protein antibody obtained in the step (2) with horseradish peroxidase (HRP) to obtain the G protein horseradish peroxidase-labeled antibody.

(4) Preparation of respiratory syncytial virus G protein double antibody sandwich ELISA quantitative kit closed system: 1 XPBS was formulated.

(5) Respiratory syncytial virus G protein double antibody sandwich ELISA quantitative kit coating system preparation: 3% bovine serum albumin was prepared.

(6) Preparation of respiratory syncytial virus G protein double antibody sandwich ELISA quantitative kit buffer system: 1 XPBST was formulated.

(7) Preparing a reaction stopping solution: concentrated sulfuric acid (109 mL) was taken and diluted with water to 1000mL to prepare 2M sulfuric acid.

In yet another aspect, the invention also provides an application of the ELISA detection kit for quantifying the respiratory syncytial virus G protein.

In particular, the applications include, but are not limited to, quantitative detection of respiratory syncytial virus G protein.

Further specifically, the applications include, but are not limited to, the development of respiratory syncytial virus vaccines.

Compared with the prior art, the invention has the following positive and beneficial effects:

(1) The invention provides an anti-respiratory syncytial virus G protein antibody, which has better affinity and binding activity with respiratory syncytial virus G protein.

(2) The ELISA detection kit for quantitative respiratory syncytial virus G protein prepared by the antibody can accurately quantify respiratory syncytial virus G protein, has simple operation, can shorten detection time, has high accuracy and stable analysis performance, has a detection range of 3.063745-600 ng/mL of a standard curve, has sensitivity of 3.063745ng/mL, has a minimum detection line of 2.490093ng/mL, and has low requirement on G protein concentration.

(3) The ELISA detection kit for quantifying the respiratory syncytial virus G protein provided by the invention can play a better auxiliary role in the research of developing respiratory syncytial virus vaccines.

(4) The kit provided by the application has the advantages of wider detection range, higher sensitivity, lower minimum detection limit and smaller batch-to-batch difference, and can completely fill up ELISA kits which are not used for quantitative detection of RSV glycoprotein in the market.

Drawings

FIG. 1 is a graph showing the results of Western Blot hybridization of recombinant respiratory syncytial virus G protein with goat anti-RSV antibody.

FIG. 2 shows the results of whole G protein and degraded G protein polyacrylamide gel electrophoresis, wherein Lane1 is blank; lane2, marker; lane3, 1.5 μg intact G protein; lane4, 2.5 μg intact G protein; lane5, 3.5 μg intact G protein; lane6, 4.5 μg intact G protein; lane7, blank; lane8, 5 μg degraded G protein (48% -7); lane9, marker; lane10, blank.

FIG. 3 is a graph of a standard test for a kit.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The following basic experimental examples, examples and experimental examples were carried out under conventional conditions unless otherwise specified. Materials, reagents and the like used in the following basic experimental examples, examples and experimental examples are commercially available unless otherwise specified.

The purchase information of the used products is as follows:

vector pET28a (+) is a product of Novagen, cat# 69864-3.

Coli BL21 (DE 3) is a product of Tiangen Biochemical technology (Beijing) Co., ltd., catalog number CB105-02.

1640 Medium is available from Michen technologies Inc. under the product number CM10040.

Goat anti-RSV polyclonal antisera is product of Meridian company, usa, catalog No. B656860G.

TMB color development was purchased from Shanghai Biyun biotechnology Co., ltd., cat# P0209.

EXAMPLE 1 preparation and validation of recombinant respiratory syncytial Virus G protein

(1) Recombinant G protein production

The method of expressing recombinant G protein of respiratory syncytial virus by recombinant colibacillus and separating and purifying the same belongs to the technology known in the industry and is only needed by adopting a common method. The coding gene sequence of the recombinant G protein of the respiratory syncytial virus is shown as SEQ ID No. 1.

(2) Antigen validation

Respiratory syncytial virus recombinant G proteins were separated on a 12% SDS-PAGE gel, and then G proteins were transferred onto PVDF membrane using a Bio-Rad transfer membrane system. The membranes were immersed in 5% nonfat dry milk in PBST, blocked at 37℃for 2h, and the membranes were washed 3 times with PBST. Membranes were immersed in 2000-fold dilution of goat anti-RSV polyclonal antiserum (product of Meridian, USA) or 6000-fold dilution of mouse anti-His-Tag (Abmart, inc.) with 3% skimmed milk powder in PBST, and incubated at 37℃for 1 hour, and the membranes were washed 3 times with PBST. Membranes were immersed in HRP bovine anti-goat IgG or HRP sheep anti-mouse IgG (Santa Cruz Co., ltd.) diluted 1000-fold with 3% nonfat milk powder in PBST, incubated for 1 hour at 37℃and washed 3 times with PBST. Referring to the instructions for use, the immunocomplexes were detected using the chemiluminescence (ECL) method. The target protein of induced expression is verified by Western Blot hybridization through a goat anti-RSV antibody.

The verification result is shown in fig. 1, and the result shows that the soluble expressed recombinant G protein has good antigenicity and can be recognized by an anti-RSV antibody.

EXAMPLE 2 preparation of mouse anti-respiratory syncytial Virus G protein antibody

1. Immunogen preparation

The recombinant G protein prepared in the example 1 is respectively mixed with an equal volume of adjuvant and emulsified uniformly to prepare the recombinant respiratory syncytial virus G protein vaccine for immunizing mice in a water-in-oil state so as to prepare the immunized mice, wherein the adjuvant used for the first immunization is Freund's complete adjuvant, and the second and subsequent immunization are Freund's incomplete adjuvant.

2. Immunization strategies

5 BALB/c mice were immunized subcutaneously with recombinant respiratory syncytial virus G protein vaccine, three times per mouse, 0.1mg per mouse for the first time, followed by 0.05mg each time. Each immunization interval was 2 weeks.

One week after three immunizations, the orbit was bled to isolate serum and stored at-80 ℃.

The antibody titer is detected by an indirect ELISA method, and the specific detection method is as follows:

(1) The reaction plate is coated with antigen.

The whole experiment adopts a 96-well reaction plate special for ELISA. Protein antigen was diluted to a concentration of 2. Mu.g/mL with alkaline coating solution, and 100. Mu.L/well was added to the reaction plate, which was placed in a refrigerator at 4℃and incubated overnight.

(2) The reaction plate was non-specifically blocked.

The coating liquid in the reaction plate is sucked, PBST is used for washing for 3 times, and the reaction plate is dried. 5% nonfat dry milk (PBST formulation) was added to the reaction plate at 100. Mu.L/well and incubated for 2 hours at 37 ℃.

(3) And (5) incubating antisera.

The blocked milk powder solution in the reaction plate was sucked off, washed 3 times with PBST, and the reaction plate was spin-dried. 2% nonfat dry milk (PBST formulation) was added to the corresponding wells in the reaction plate at 100. Mu.L/well and incubated for 1.5 hours at 37 ℃.

(4) And (5) incubating the secondary antibody.

The primary antibody in the reaction plate was blotted off and washed 6 times with PBST. The secondary antibody with horseradish peroxidase was diluted with 2% nonfat milk powder according to the experimental set gradient, then 100 μl/well was added to the corresponding reaction plate and incubated for 1 hour at 37 ℃.

(5) The system is developed.

The secondary antibodies in the reaction plate were blotted off and washed 6 times with PBST. TMB (3, 3', 5' -tetramethylbenzidine) color development solution 100. Mu.L/well was added to the reaction plate and incubated at room temperature for 10-15 minutes in the absence of light.

(6) The reaction was terminated.

To the developed reaction plate was added 50. Mu.L/well of 2M sulfuric acid, and the reaction was terminated.

(7) And reading by an enzyme label instrument.

The OD value of the terminated reaction plate was measured at 450nm and 620 nm. The OD ₄₅₀ minus the OD ₆₂₀ is the final result. The OD value of the experimental well was considered positive when it was twice that of the control well;

the serum antibody titers of each mouse are shown in table 1:

TABLE 1 serum antibody titers of mice

Mouse numbering	Valency of
		G-001	1：8000
G-002	1：1000
		G-003	1：16000
G-004	1：16000
		G-005	1：1000

3. Cell fusion

Two weeks after three immunizations, mice with the highest antiserum titers were given intraperitoneal injections of 0.05mg of sterile respiratory syncytial virus G protein stock, and cell fusion was performed three days later. The method comprises the steps of sacrificing the neck of a mouse, soaking the mouse in 75% ethanol solution for 30 minutes for sterilization, opening the abdominal cavity in a sterile super clean bench, taking out the spleen aseptically, grinding the spleen into single-cell suspension, obtaining spleen single-cell suspension after passing through a 200-mesh sterile copper mesh, adding SP2/0 myeloma cells, and carrying out cell fusion under the action of PEG.

4. Fusion screening.

The fused cells are plated on a 96-well cell culture plate, cultured by HAT culture solution, changed for three days, and then cultured by HT culture solution. After 10 days of culture, cell culture supernatant is removed, indirect ELISA is carried out to detect the anti-respiratory syncytial virus G protein antibody titer (the method is the same as G protein antibody detection), and positive holes with high antibody titer are taken for subcloning.

5. Subcloning and monocloning.

Positive wells were subcloned using limiting dilution and antibody titers were detected after 10 days. Subcloning of positive clones was continued by limiting dilution until the subclones obtained were positive, and finally 2 subclone hybridoma cell lines of highest titer were selected and stored (table 2).

TABLE 2 highest titer 2 subcloned hybridoma cell lines

6. And (5) performing expansion culture.

And (3) performing expansion culture on each positive monoclonal hybridoma cell strain, and performing cryopreservation after expansion.

7. Antibody preparation and purification.

The method for preparing and purifying the anti-respiratory syncytial virus G protein monoclonal antibody generated by the 2 hybridoma cell strains comprises the following steps:

(1) And (5) preparing ascites.

The mice were injected with mineral oil intraperitoneally, one week later, with hybridoma cell lines intraperitoneally, 5X 10 ⁶ hybridoma cells per mouse, and ascites were collected after 10-14 days.

(2) And (5) purifying the monoclonal antibody.

The collected ascites is centrifuged, the supernatant is collected and saturated ammonium sulfate solution is added dropwise to the supernatant under stirring until the saturation of ammonium sulfate reaches 50%, and stirring is continued for 30 minutes. Then 12000rpm room temperature centrifugation for 30 minutes, collecting centrifugal precipitate and re-suspending in PBS buffer, under stirring conditions, dropwise adding saturated ammonium sulfate solution, until the ammonium sulfate saturation reaches 33%, and continuing stirring for 30 minutes. Centrifuge at 12000rpm for 30 min at room temperature, collect pellet and re-suspend in PBS buffer and dialyze overnight. And carrying out affinity chromatography on the dialyzed ascites by using a G protein purification medium to obtain a purified monoclonal antibody pure product of the anti-respiratory syncytial virus G protein.

(3) And (5) potency measurement.

The titers of the individual monoclonal antibodies were detected by indirect ELISA methods (supra). The results are shown in Table 3.

TABLE 3 titers of monoclonal anti-RSV G protein antibodies after purification

(4) Sending the purified anti-respiratory syncytial virus G protein antibodies 5B2-1 and 8H6-1 to a gold Style company for sequencing, wherein the heavy chain amino acid sequence of the 8H6-1 is shown as SEQ ID No.18, and the light chain amino acid sequence is shown as SEQ ID No. 19; the heavy chain amino acid of 5B2-1 is shown as SEQ ID No.20, and the light chain amino acid sequence is shown as SEQ ID No. 21.

Example 3 ELISA monoclonal antibody preparation and antibody pair screening

1. And (3) preparing the horseradish peroxidase-labeled monoclonal antibody.

The purified anti-respiratory syncytial virus G protein monoclonal antibody is coupled with horseradish peroxidase (HRP) and specifically comprises the following steps:

2mg of horseradish peroxidase was dissolved in sterile ultrapure water, and the mixture was added to a NaIO ₄ solution to react for 30 minutes at room temperature. And (3) dialyzing 2mg of anti-respiratory syncytial virus G protein monoclonal antibody with a coupling buffer solution overnight, adding the antibody into a horseradish peroxidase reaction solution, reacting for 2 hours at room temperature, and dialyzing to obtain the coupled horseradish peroxidase ELISA monoclonal antibody. The titers of each monoclonal anti-RSV G protein antibody after HRP conjugation are shown in table 4.

TABLE 4 monoclonal anti-RSV G protein antibody titers after HRP conjugation

2. Antibody pairing screening

ELISA procedure:

(1) Coating: antigen affinity purified antibodies (5B 2-1, 8H 6-1), goat anti-respiratory syncytial virus polyclonal antibody, human anti-respiratory syncytial virus G protein monoclonal antibody, mouse anti-respiratory syncytial virus G protein monoclonal antibody, rabbit anti-respiratory syncytial virus G protein polyclonal antibody were diluted with 1 XPBS to 2.5, 0.5, 0.1 μg/mL, respectively. Adding 100 mu L of the mixture into a 96-well ELISA plate, making 2 compound wells for each concentration, and standing at 2-8 ℃ overnight.

(2) 3% Bovine serum albumin blocking: the coating was removed, washed 3 times with PBST, 3% bovine serum albumin was prepared with PBST, 100. Mu.L was added to each well, and incubated for 1h at 37 ℃.

(3) The blocking solution was discarded, the plates were washed 3 times with PBST, the solution was removed by pipetting, 100. Mu.L of the diluted standard solution or the test solution was added and incubated in a wet box at 37℃for 1 hour. 2 duplicate wells per sample.

G protein (no degradation) and G protein (degradation) dilution (dilution with 1% bovine serum albumin-PBST): 600, 200, 66.7ng/mL, respectively.

(4) The test solution was discarded, the plate was washed 6 times with PBST, and 100. Mu.L of 1% bovine serum albumin-PBST diluted 0.5. Mu.g/mL horseradish peroxidase-labeled antibodies 8H6-1 and 5B2-1 were added, respectively, and incubated at 37℃for 1 hour.

(5) The antibody was discarded, the plate was washed 6 times with PBST, 100. Mu.L of TMB developing solution was added, and the color was developed at room temperature for 10-15 minutes (blue color was formed after the development).

(6) The color development was terminated by adding 50. Mu.L of 2M sulfuric acid (the color turned to bright yellow after termination of the color development). The reading was detected with a microplate reader (OD 450nm/620 nm) within 15 min.

TABLE 5 antibody pair screening results

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As shown in Table 5, the primary screening was carried out according to the criteria (A, standard curve R ² value is greater than 0.9500; B,0 concentration well OD value is less than 0.1000; C, highest concentration well OD value is high), 8H6-1 (2.5. Mu.g/mL) and 5B2-1-HRP (0.5. Mu.g/mL) detection antibody pairs, 8H6-1 (0.5. Mu.g/mL) and 5B2-1-HRP (0.5. Mu.g/mL) detection antibody pairs, RSV-rabit (2.5. Mu.g/mL) and 5B2-1 (0.5. Mu.g/mL) detection antibody pairs, RSV-rabit (0.5. Mu.g/mL) and 5B2-1 (0.5. Mu.g/mL) detection antibody pairs all meet A, B standard, and at the highest concentration well (600 ng/mL) OD value reaches 1.5 or more, which is significantly superior to other antibody pairs, and can be used as ELISA raw materials for the preferred reagent kit.

8H6-1 (2.5. Mu.g/mL) coated with 5B2-1-HRP (0.5. Mu.g/mL) detection antibody pair with OD 1.5071,8H6-1 (0.5. Mu.g/mL) coated with 5B2-1-HRP (0.5. Mu.g/mL) detection antibody pair with OD 1.537,8H6-1 (2.5. Mu.g/mL) coated with 5B2-1-HRP (0.5. Mu.g/mL) at a G protein concentration of 600ng/mL, and less than 8H6-1 (0.5. Mu.g/mL) detection antibody pair with 5B2-1-HRP (0.5. Mu.g/mL) at a G protein concentration of 600 ng/mL. However, the OD value of the 8H6-1 (2.5. Mu.g/mL) coating and 5B2-1-HRP (0.5. Mu.g/mL) detection antibody pair was 1.0619 at a G protein concentration of 200ng/mL, and the difference from the OD value of the G protein coating and 5B2-1-HRP (0.5. Mu.g/mL) detection antibody pair was smaller, and the linearity was better than that of the 8H6-1 (0.5. Mu.g/mL) coating and 5B2-1-HRP detection antibody pair.

Although the RSV-rabit (2.5. Mu.g/mL) coated with 5B2-1 (0.5. Mu.g/mL) detection antibody pair, the RSV-rabit (0.5. Mu.g/mL) coated with 5B2-1 (0.5. Mu.g/mL) detection antibody pair had an OD value higher than 8H6-1 (2.5. Mu.g/mL) coated with 5B2-1-HRP (0.5. Mu.g/mL) detection antibody pair at a G protein concentration of 600ng/mL, polyclonal antibody batches were not generally selected as the starting material for the quantitative detection kit due to the inconsistency between the polyclonal antibody batches.

Therefore, the 8H6-1 (2.5. Mu.g/mL) antibody coating and 5B2-1-HRP (0.5. Mu.g/mL) detection monoclonal antibody pair were finally selected as optimal conditions for the next kit development.

3. The complete glycoprotein and the degraded glycoprotein identification antibody pairs were validated.

ELISA procedure:

(1) Coating

Antigen affinity purification antibody 8H6-1 was diluted to 2.5. Mu.g/mL with 1 XPBS. Adding 100 mu L of the mixture into a 96-well ELISA plate, making two compound wells for each concentration, and standing at 2-8 ℃ overnight.

(2) Blocking with 3% bsa: the coating was discarded, washed 3 times with PBST, 3% BSA was prepared with PBST, 100. Mu.L of each well was added, and incubated at 37℃for 1h.

G protein (no degradation) and G protein (degradation) dilution (dilution with 1% bsa-PBST): 1200, 600, 300, 150, 75, 37.5, 18.75, 0ng/mL, respectively.

(4) The test solution was discarded, the plate was washed 6 times with PBST, and 1.0, 0.5, 0.25. Mu.g/mL horseradish peroxidase-labeled antibody 5B2-1 (5B 2-1-HRP) diluted with 100. Mu.L of 1% BSA-PBST was added, respectively, and incubated at 37℃for 1 hour.

(6) After 50. Mu.L of 2M H ₂SO₄ was added, the color development was terminated (the color turned to bright yellow after the termination of the color development). Detecting the readings (OD 450nm/620 nm) with an enzyme label instrument within 15 min;

the results of the antibody pair suitability confirmation were confirmed.

The results of the detection are shown in FIG. 2 and Table 6, wherein the 8H6-1 (2.5. Mu.g/mL) antibody coating and the 5B2-1-HRP (1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL) detection of monoclonal antibodies both detect intact glycoprotein and degraded glycoprotein. Of these, 0.5. Mu.g/mL of 5B2-1-HRP was the most effective for degradation and was used in subsequent experiments.

TABLE 6 identification of antibodies to intact glycoprotein and degraded glycoprotein pair confirmation results

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Example 4 screening of a coating System of a double antibody sandwich ELISA quantitative kit for RSV glycoprotein

ELISA procedure:

(1) Coating: the ascites fluid was purified by protein A column and 8H6-1 was diluted to 2.5. Mu.g/mL with 1 XPBS and coating buffer. Adding 100 mu L of the mixture into a 96-well ELISA plate, and making three compound wells at each concentration, wherein the temperature is 4 ℃ overnight; wherein the coating buffer concentration was 0.05M and pH 9.6, consisting of 1.59g Na ₂CO₃ and 2.93g NaHCO ₃, distilled water was added to 1000mL.

(3) The blocking solution was discarded, the plates were washed 3 times with PBST, the solution was removed by pipetting, 100. Mu.L of the diluted standard solution or the test solution was added and incubated in a wet box at 37℃for 1 hour. Three wells per sample.

Protein G (no degradation) dilution (dilution with 1% bsa): 1200, 600, 300, 150, 75, 37.5, 18.75, 0ng/mL, respectively.

(4) The test solution was discarded, the plate was washed 6 times with PBST, 100. Mu.L of diluted 0.5. Mu.g/mL horseradish peroxidase-labeled antibody 5B2-1 was added, and incubated in a wet box at 37℃for 1 hour.

(6) After 50. Mu.L of 2M H ₂SO₄ was added, the color development was terminated (the color turned to bright yellow after the termination of the color development). The reading was detected with a microplate reader (OD 450nm/620 nm) within 15 min.

As shown in Table 7, it is clear from Table 7 that the 8H6-1 (2.5. Mu.g/mL) antibody coating and 5B2-1-HRP (0.5. Mu.g/mL) mab detection pair showed better performance in the PBS coating system than in the coating liquid coating system (overall small RSD value).

TABLE 7 screening results of RSV glycoprotein double antibody sandwich ELISA quantitative kit coating system

Example 5 screening of RSV glycoprotein double antibody sandwich ELISA quantitative kit blocking system

ELISA procedure:

(1) Coating: the ascites fluid was purified by protein A column and 8H6-1 was diluted to 2.5. Mu.g/mL with 1 XPBS and coating buffer. Adding 100 mu L of the mixture into a 96-well ELISA plate, and making 2 compound wells at each concentration at 4 ℃ overnight; wherein the coating buffer concentration was 0.05M and pH 9.6, consisting of 1.59g Na ₂CO₃ and 2.93g NaHCO ₃, distilled water was added to 1000mL.

(2) 3% Bsa,5% nonfat milk powder, 1% casein blocking: the coating solution was discarded, washed 3 times with PBST, 3% BSA,5% skim milk powder and 1% casein were prepared with PBST, 100. Mu.L was added to each well, and incubated at 37℃for 1 hour.

(3) The blocking solution was discarded, the plates were washed 3 times with PBST, the solution was removed by pipetting, 100. Mu.L of the diluted standard solution or the test solution was added and incubated in a wet box at 37℃for 1 hour. Two wells per sample.

(4) The test solution was discarded, the plate was washed 6 times with PBST, 100. Mu.L of diluted 0.5ug/mL horseradish peroxidase-labeled antibody 5B2-1 was added, and incubated in a wet box at 37℃for 1 hour.

After 50. Mu.L of 2M H ₂SO₄ was added, the color development was terminated (the color turned to bright yellow after the termination of the color development). The reading was detected with a microplate reader (OD 450nm/620 nm) within 15 min.

The results are shown in Table 8:

(1) 8H6-1 (2.5. Mu.g/mL) antibody coating and 5B2-1-HRP (0.5. Mu.g/mL) detection monoclonal antibody pairs in PBS coating system and coating liquid coating system, different closed systems (3% bovine serum albumin, 5% skim milk powder, 1% casein) are suitable for detecting glycoprotein;

(2) The 8H6-1 (2.5. Mu.g/mL) antibody coating and 5B2-1-HRP (0.5. Mu.g/mL) detection mab pair showed overall excellent performance in the PBS coating system, the 3% bovine serum albumin blocking system.

Table 8 screening results of RSV glycoprotein diabody sandwich ELISA quantitative kit blocking system

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Example 6 screening of respiratory syncytial Virus glycoprotein double antibody Sandwich ELISA quantitative kit buffer System

ELISA procedure

(2) 3% Bsa block, 5% nonfat dry milk, 1% casein: the coating was discarded, washed 3 times with PBST or PBS, 3% BSA,5% skim milk powder and 1% casein were prepared with PBST, 100. Mu.L was added to each well, and incubated for 1h at 37 ℃.

(3) The blocking solution was discarded, the plates were washed 3 times with PBST or PBS, the solution was then removed, 100. Mu.L of the diluted standard solution or the test solution was added, and incubated in a wet box at 37℃for 1 hour. Two wells per sample.

(4) The test solution was discarded, the plates were washed 6 times with PBST or PBS, respectively, 100. Mu.L of diluted 0.5. Mu.g/mL 5B2-1-HRP was added, and incubated in a wet box at 37℃for 1 hour.

(5) The antibody was discarded, and the mixture was washed 6 times with PBST or PBS, 100. Mu.L of TMB developing solution was added, and the mixture was developed at room temperature for 10 to 15 minutes (blue color was formed after the development).

The test results are shown in Table 9 below:

The 8H6-1 (2.5. Mu.g/mL) antibody coating and 5B2-1-HRP (0.5. Mu.g/mL) detection monoclonal antibody are excellent in overall glycoprotein detection by the PBST buffer system.

Table 9 screening results of glycoprotein double antibody sandwich ELISA quantitative kit buffer System

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Example 7 an ELISA quantitative detection kit for respiratory syncytial virus glycoprotein antigen the kit comprises the following components: 0.5 μg/mL anti-respiratory syncytial virus glycoprotein ELISA monoclonal antibody 5B2-1-HRP;2.5 μg/mL of anti-respiratory syncytial virus glycoprotein monoclonal antibody 8H6-1;

a 1x PBS coating system;

A 3% bovine serum albumin blocking system;

1 XPBST buffer system;

TMB color development liquid;

2M sulfuric acid.

Example 8 preparation method of respiratory syncytial Virus glycoprotein antigen ELISA quantitative detection kit

(1) Anti-respiratory syncytial virus glycoprotein monoclonal antibody 8H6-1 and anti-respiratory syncytial virus glycoprotein ELISA monoclonal antibody 5B2-1-HRP were prepared according to the method of examples 1-3, and diluted to 2.5 μg/mL and 0.5 μg/mL, respectively, with PBS.

(2) A1 XPBS coating system, a 3% bovine serum albumin, and a 1 XPBST buffer system were each formulated.

(3) 2M sulfuric acid was prepared.

Example 9 precision test of respiratory syncytial Virus glycoprotein double antibody sandwich ELISA quantitative kit

Batch-to-batch difference: the same operator uses a unified method to detect differences in different time periods (time period intervals are greater than 24 hours); or detecting the difference by using a unified method in the same time period of different operators;

intra-batch difference: the same operator runs the same experiment (two ELISA plates are detected simultaneously) at the same time and uses a unified method to detect differences;

ELISA experiments:

(1) Coating: the ascites fluid was purified by protein A column and 8H6-1 was diluted to 2.5. Mu.g/mL with 1 XPBS and coating buffer. Adding 100 mu L of the mixture into a 96-well ELISA plate, and making 6 compound wells at each concentration overnight at 4 ℃; wherein the coating buffer concentration was 0.05M and pH 9.6, consisting of 1.59g Na ₂CO₃ and 2.93g NaHCO ₃, distilled water was added to 1000mL.

(2) Blocking with 3% bsa: the coating was discarded, washed 3 times with PBST, 3% BSA was prepared with PBST, 100. Mu.L of each well was added, and incubated at 37℃for 1 hour.

(3) The blocking solution was discarded, the plates were washed 3 times with PBST, the solution was removed by pipetting, 100. Mu.L of the diluted standard solution or the test solution was added and incubated in a wet box at 37℃for 1 hour. Six duplicate wells per sample.

(4) Protein G (no degradation) dilution (dilution with 1% bsa): 300, 150, 75, 37.5, 18.75, 9.375, 4.6875, 0ng/mL, respectively.

(5) The assay solution was discarded, the plate was washed 6 times with PBST, 100. Mu.L of diluted 0.5. Mu.g/mL 5B2-1-HRP was added and incubated in a wet box at 37℃for 1 hour.

(6) The antibody was discarded, and 100. Mu.L of TMB-developed solution was added thereto, and the mixture was developed at room temperature for 10 to 15 minutes (blue after development).

(7) After 50. Mu.L of 2M H ₂SO₄ was added, the color development was terminated (the color turned to bright yellow after the termination of the color development). Detection of the reading (OD 450nm/620 nm) with an ELISA reader within 15min

Analysis of results (tables 10 to 11): 8H6-1 (2.5. Mu.g/mL) antibody coating and 5B2-1-HRP (0.5. Mu.g/mL) detection of monoclonal antibody pairs in a1 XPBS coating system, a 3% bovine serum albumin closed system, a PBST buffer system, and TMB chromogenic solution detection of glycoprotein with batch-to-batch differences of less than 25%.

TABLE 10 intra-lot difference results

TABLE 11 results of batch-to-batch differences

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Example 10 respiratory syncytial Virus glycoprotein double antibody sandwich ELISA quantitative kit detection range, sensitivity and minimum detection line detection

ELISA procedure:

(1) Coating: the ascites fluid was purified by protein A column and 8H6-1 was diluted to 2.5. Mu.g/mL with 1 XPBS and coating buffer. Adding 100 mu L of the mixture into a 96-well ELISA plate, and making 2 compound wells at each concentration at 4 ℃ overnight; wherein the coating buffer concentration was 0.05M and pH 9.6, consisting of 1.59g Na2CO3 and 2.93g NaHCO ₃, distilled water was added to 1000mL.

Protein G (no degradation) dilution (dilution with 1% bsa): 2400, 1200, 600, 300, 150, 75, 37.5, 18.75, 9.375, 4.6875, 2.34375, 0ng/mL, respectively.

(4) The assay solution was discarded, the plate was washed 6 times with PBST, 100. Mu.L of diluted 0.5. Mu.g/mL 5B2-1-HRP was added and incubated in a wet box at 37℃for 1 hour.

(5) The antibody was discarded, and 100. Mu.L of TMB-developed solution was added thereto, and the mixture was developed at room temperature for 10 to 15 minutes (blue after development).

(6) After 50. Mu.L of 2M H ₂SO₄ was added, the color development was terminated (the color turned to bright yellow after the termination of the color development). The reading is detected with a microplate reader (450/620) within 15 min.

As shown in FIG. 3, the 8H6-1 (2.5. Mu.g/mL) antibody coating and 5B2-1-HRP (0.5. Mu.g/mL) detection monoclonal antibody pairs were combined in a PBS coating system, a 3% bovine serum albumin closed system, a PBST buffer system, a TMB chromogenic solution detection glycoprotein system, and a standard curve detection range of 3.063745-600ng/mL; the sensitivity is 3.063745ng/mL; the lowest detection line was 2.490093ng/mL.

The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. An anti-respiratory syncytial virus G protein antibody, characterized in that: comprising antibody 8H6-1 or 5B2-1;

2. The anti-respiratory syncytial virus G protein antibody according to claim 1, wherein: the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the antibody 8H6-1 are shown as SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively, and the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 are shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 respectively;

the amino acid sequences of the H-CDR1, the H-CDR2 and the H-CDR3 of the antibody 5B2-1 are shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively, and the amino acid sequences of the L-CDR1, the L-CDR2 and the L-CDR3 are shown as SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13 respectively.

3. The anti-respiratory syncytial virus G protein antibody according to claim 1 or 2, characterized in that: the heavy chain amino acid sequence of the 8H6-1 is shown as SEQ ID No.18, and the light chain amino acid sequence is shown as SEQ ID No. 19; the heavy chain amino acid sequence of the 5B2-1 is shown as SEQ ID No.20, and the light chain amino acid sequence is shown as SEQ ID No. 21.

4. The use of an anti-respiratory syncytial virus G protein antibody according to claim 1 for the preparation of an ELISA detection kit for quantifying respiratory syncytial virus G protein.

5. An ELISA detection kit for respiratory syncytial virus G protein, which is characterized in that: the ELISA detection kit comprises one or two of the anti-respiratory syncytial virus G protein antibodies as defined in claim 1.

6. The ELISA detection kit of claim 5 wherein: the ELISA detection kit comprises an antibody 8H6-1 and an antibody 5B2-1, wherein the antibody 8H6-1 is used as a coating antibody, and the antibody 5B2-1 is used as an enzyme-labeled detection antibody.

7. The ELISA detection kit of claim 5 wherein: the concentration of the anti-respiratory syncytial virus G protein antibody is 0.1-2.5 mug/mL; preferably, the concentration of the anti-respiratory syncytial virus G protein antibody is 0.1-0.5 μg/mL.

8. The ELISA detection kit of claim 5 wherein: the ELISA detection kit also comprises a coating system, a closed system, a buffer system and a color development liquid; the coating system is PBS buffer solution; the closed system is 3% bovine serum albumin, 5% skim milk powder or 1% casein; the buffer system is PBST buffer solution.

9. A method for preparing the ELISA detection kit as claimed in claim 5, wherein: the preparation method comprises the following steps:

(1) Preparing recombinant respiratory syncytial virus G protein;

(2) Preparing the anti-respiratory syncytial virus G protein antibody of claim 1;

(3) Preparing a respiratory syncytial virus G protein horseradish peroxidase-labeled antibody;

(4) The respiratory syncytial virus G protein double antibody sandwich ELISA quantitative kit is prepared by a closed system: the closed system is PBS buffer solution;

(5) The respiratory syncytial virus G protein double-antibody sandwich ELISA quantitative kit coating system is prepared by: the coating system is 3% bovine serum albumin;

(6) The buffer system of the respiratory syncytial virus G protein double-antibody sandwich ELISA quantitative kit is prepared: the buffer system is PBST buffer solution;

(7) Preparing a reaction stopping solution: the reaction stopping solution is 2M sulfuric acid.

10. The method of manufacturing according to claim 9, wherein: the step (1) is to construct a pET28a (+) recombinant vector by using a nucleotide sequence shown in SEQ ID No.1, and introduce the recombinant vector into a recombinant escherichia coli expression system to obtain recombinant respiratory syncytial virus G protein.