CN111484552B

CN111484552B - Monoclonal antibody against SpA5 protein, application thereof and kit containing monoclonal antibody

Info

Publication number: CN111484552B
Application number: CN202010575079.6A
Authority: CN
Inventors: 邹全明; 曾浩; 顾江; 鲁东水; 杨峰; 张卫军; 徐丽敏; 程平
Original assignee: Third Military Medical University TMMU; Chengdu Olymvax Biopharmaceuticals Inc
Current assignee: Third Military Medical University TMMU; Chengdu Olymvax Biopharmaceuticals Inc
Priority date: 2019-12-26
Filing date: 2020-06-22
Publication date: 2022-03-04
Anticipated expiration: 2040-06-22
Also published as: CN111072776A; WO2021128946A1; CN111484552A

Abstract

The invention belongs to the technical field of biology, and provides a monoclonal antibody for resisting SpA5 protein, application thereof and a kit comprising the same, wherein the monoclonal antibody for resisting SpA5 protein comprises a heavy chain and a light chain, the heavy chain and the light chain respectively comprise 3 CDR regions, the amino acid sequences of the CDR regions of the heavy chain are respectively shown as SEQ ID NO. 1-3, and the amino acid sequences of the CDR regions of the light chain are respectively shown as SEQ ID NO. 4-6. The antibody can be specifically combined with SpA5 protein, effectively detects the content of SpA5 protein, and has high sensitivity and wide detection range.

Description

Monoclonal antibody against SpA5 protein, application thereof and kit containing monoclonal antibody

Technical Field

The invention belongs to the technical field of biology, and relates to a monoclonal antibody.

Background

Staphylococcus aureus protein a (spa) is present in most pathogenic staphylococcus aureus, but not in staphylococcus epidermidis, staphylococcus saprophyticus. Blast alignment analysis showed that SpA is a highly conserved antigenic component. It has a molecular weight of 55kDa and is distributed on the cell wall surface. In mouse models of septic arthritis, sepsis and skin abscesses of staphylococcus aureus infections, the reduced virulence of SpA-deficient mutant staphylococcus aureus strains is believed to be due to the reduced SpA binding to the IgG-Fc fragment, reducing the anti-host phagocytosis of staphylococcus aureus.

The applicant reports that the five domains of wild type SpA (wSpA) are screened for multiple mutations of the active site by molecular design, and finally determines that the [ E (KKAA) -D (KKAA) -A (KKAA) -B (KKAA) -C (KKAA) -mutant is one of the recombinant staphylococcus aureus vaccine components and is named as SpA 5. The biological activity detection of the system proves that the modified and mutated SpA5 no longer has the superantigen activity of promoting the apoptosis of wild wSpA B cells, and the binding activity of the modified and mutated SpA5 with an IgG-Fc segment is obviously reduced. Animal safety evaluation and a large number of animal immune protection experiments prove that SpA5 has good safety and good immunogenicity, and the BALB/c mouse immune toxicity attack protection rate generated by single-component immunity is more than 30%. Furthermore, no changes in other biological functions were found.

The monoclonal antibody aiming at a plurality of active epitopes of SpA5, which is obtained by taking the recombinant SpA5 protein as an immunogen through immunization, screening and identification, can be applied to the preparation of specific therapeutic antibody medicines for preventing and treating staphylococcus aureus infection through immunization and a detection kit for detecting the SpA5 antigen content in the recombinant staphylococcus aureus vaccine. No specific anti-SpA 5 antibody is currently present.

Disclosure of Invention

For the reasons, the invention provides an antibody against SpA5 protein, which can effectively detect the content of SpA5 protein in vaccines and other preparations containing SpA5 protein so as to solve the problems.

In a first aspect, there is provided a monoclonal antibody against SpA5 protein, said antibody comprising a heavy chain and a light chain, each comprising 3 CDR regions, wherein,

the amino acid sequence of the heavy chain CDR1 region is GYSITSDYG (SEQ ID NO: 1);

the amino acid sequence of the heavy chain CDR2 region is ISYSGST (SEQ ID NO: 2);

the amino acid sequence of the heavy chain CDR3 region is ARGNYYALDN (SEQ ID NO: 3);

the amino acid sequence of the light chain CDR1 region is QSLLYSSNQKNY (SEQ ID NO: 4);

the amino acid sequence of the light chain CDR2 region is WAS (SEQ ID NO: 5);

the amino acid sequence of the light chain CDR3 region is QQYYSYT (SEQ ID NO: 6).

Preferably, the antibody is antibody 5C8, wherein,

the sequence of the heavy chain is:

LQADATSTSRVALGPGLVKPSQSLSLTCTVTGYSITSDYGWNWIRQFPGNKLEWMGYISYSGSTSYNPSLKGRISITRDTSKNQFFLQLNSVTTEDTATYHCARGNYYALDNWGQGTTVTVSS(SEQ ID NO:7)；

the sequence of the light chain is:

DIQLTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYTFGGGTKLEI(SEQ ID NO:8)。

preferably, the antibody is an IgG antibody.

In a second aspect, a method for detecting the content of SpA5 protein is provided, which comprises detecting with the monoclonal antibody of the invention; preferably, detection is by ELISA. Further preferably, the monoclonal antibody of the present invention is used as a coating antibody, and a rabbit polyclonal antibody prepared by a conventional method is used as a detection antibody.

Preferably, the method comprises:

1) coating an enzyme-linked plate with the monoclonal antibody of the invention;

2) reacting a sample to be tested with the monoclonal antibody of the invention;

3) detecting and developing rabbit polyclonal antibody prepared by conventional method, and measuring absorbance at 450 nm;

4) preparing a standard curve by using the SpA5 protein standard, and calculating the content of SpA5 protein in the detected sample according to the standard curve;

preferably, the content of SpA5 protein detected by the method is in the range of 0.015625-0.5 mu g/mL.

In a third aspect, a kit for detecting SpA5 protein is provided, wherein the kit comprises a monoclonal antibody against SpA5 protein of the present invention. Preferably, the kit further comprises a rabbit polyclonal antibody against SpA5 protein and an HRP-labeled goat anti-rabbit igg (fc) secondary antibody.

Further preferably, the kit further comprises human IgG, a washing reagent, a substrate developing solution A (EDTA-Na, citric acid, glycerol, TMB), and a substrate developing solution B (sodium acetate, citric acid, 30% H)₂O₂) Stop solution (2M H)₂SO₄) And BSA, as well as elisa and shrouding membrane and/or SpA5 protein standards.

Preferably, the rabbit polyclonal antibody is prepared using conventional methods.

In a fourth aspect, the invention provides an application of the monoclonal antibody against the SpA5 protein in detecting the content of the SpA5 protein.

In the invention, the contents of the sequence and the preparation method of the SpA5 protein refer to CN103694322A, and the SpA5 protein is SpA5(KKAA) protein of CN103694322A, and the sequence of the SpA5(KKAA) protein is shown as SEQ ID NO:1 of CN 103694322A. The content of CN103694322A is incorporated by reference into this application.

The antibody can be specifically combined with SpA5 protein, effectively detects the content of SpA5 protein, has high sensitivity, reaches 0.015625 mu g/mL at the lowest limit, has a wide detection range of 0.015625-0.5 mu g/mL, and can meet the requirement of quantitative detection of vaccines.

Drawings

FIG. 1: screening results of the monoclonal cell strains;

FIG. 2: fitting an equation graph by using double logarithms.

Detailed Description

The present disclosure will be described below with reference to specific embodiments, but the scope of the present disclosure is not limited thereto.

The reagents and apparatus used in the following examples are conventional in the art and are commercially available, unless otherwise specified; the methods used are all routine experimental methods, and the person skilled in the art can, without any doubt, carry out the protocol and obtain the corresponding results according to the contents of the examples.

Primary reagents and instruments

Primary reagent

The main apparatus is as follows:

name (R)	Manufacturer of the product	Model number
			Liquid transfer device	Eppendorf
Liquid transfer device	Thermo Fisher Scientific	Twelve passages
			Constant temperature incubator	Yongguang medical instrument factory in Beijing	DHP-420 type
Micropore plate constant temperature oscillator	China national Co Ltd	SC60-4
			Plate washing machine	Beijing Tuopau Analyzer Co Ltd	DEM-3 type
Enzyme-linked immunosorbent assay (ELISA) instrument	Thermo Scientific	Multiskan Mk3

Experimental animals:

Balb/C mice: SPF (specific Pathologen free) grade Balb/C mice were purchased from Experimental animals technology, Inc. of Viton, Beijing.

Preparing a buffer solution:

the preparation of the substrate color developing solution A (solution A) is as follows: EDTA-Na 0.2g, citric acid 0.95g, glycerol 50ml, TMB (dissolved in DMSO to 10mg/ml and added) 0.2g, dH₂O 500ml。

The preparation of the substrate color developing solution B (solution B) is as follows: 13.6g of sodium acetate, 1.6g of citric acid and H₂O₂(30％)0.3ml，dH₂O 500ml。

20 × the formula of the lotion is as follows: NaCl 818g, Na₂HPO₄.12H₂O 358g，KCl 205g，H₂And O is metered to 5L, and the pH value is 7.4-7.6.

The formulation of glycine eluent with pH 2.7 is as follows: glycine 1.9g, H₂And O is metered to 500mL, and the pH value is 2.68-2.72.

The formulation of glycine eluent with pH 1.9 is as follows: glycine 1.9g, H₂And O is metered to 500mL, and the pH value is 1.88-1.92.

Example 1: preparation of mouse monoclonal antibody for specifically recognizing SpA5 protein

1. Preparation and screening of monoclonal antibodies

The immunogen was prepared by mixing the SpA5 recombinant protein (1.4mg/ml) with the adjuvants CFA and AD11.15, i.e. the recombinant protein was first mixed with CFA in a volume ratio of 5: 6 as immunogen A, and the recombinant protein is mixed with AD11.15 according to the volume ratio of 1:1 as immunogen B. Immunogen a is the primary and immunogen B is the booster. 3 mice were immunized with muscle, and after immunization, tail blood was collected on day 14 and the tail blood antibody titer was evaluated by an indirect ELISA method.

The SPA5 recombinant protein is used to coat an enzyme label plate (1 mu g/mL), 100 mu L of the protein is added into each hole, and the reaction is carried out overnight at 4 ℃;washing the plate with PBS solution for 3 times, and blocking with 5% milk-PBS at room temperature for 1 hr; then washing the plate with PBS solution for 1 time, adding mouse tail blood diluted with 5% milk-PBS solution, reacting at room temperature for 1 hr; then washing the plate 3 times with PBS solution, drying, adding HRP-labeled secondary goat anti-mouse IgG (Fc) antibody diluted at 1:2000, and reacting at room temperature for 1 hr; after washing the plate 5 times with PBS solution, patting dry, add equal volumes of solution A (EDTA-Na, citric acid, glycerol, TMB (tetramethylbenzidine)) and solution B (sodium acetate, citric acid, 30% H)₂O₂) Reacting for 20min under the conditions of light shielding and room temperature; then 50. mu.L of stop solution (2M H) was added₂SO₄) After mixing, the OD is read on a microplate reader₄₅₀The value is obtained. The results of the indirect ELISA evaluation of the tail blood of the mice at 14 days after immunization are shown in Table 1.

Table 1: evaluation of mouse tail blood antibody titer on day 14 after immunization

Note: OD₄₅₀Is represented by 999>3.5, exceeding the reading range of the microplate reader; NC is negative control, 5% milk-PBS; mice # 3 died.

From the results, the antibody titers of three mouse tails recognizing the SpA5 recombinant protein were all over 1:50000, with the highest antibody titer in the # 1 mouse. Therefore, the 1# mouse was selected for cell fusion with the myeloma cell SP 2/0. 564 monoclonal hybridoma cells were selected after fusion and cultured in a 96-well plate, the cell culture supernatant in the 96-well plate was evaluated by the indirect ELISA method described above, and monoclonal cell strains capable of secreting monoclonal antibodies that recognize proteins were selected, the screening results are shown in fig. 1, in which NC is a negative control, 5% milk-PBS; PC was a positive control, serum # 1.

From the screening results, 26 positive clones were selected for confirmation experiments in the following procedure: SpA5 protein 100 ng/well was coated, and the supernatant 1: dilution 1 was used as primary antibody, and goat anti-mouse secondary antibody was added, and still positive clones were retained, and the results are shown in table 2.

Table 2: confirmation of Positive clones

Clone number	1A11	1C12	1C3	1D11	1F10	1G1	2E8	2B7	2E6	2F6	NC	PC
													OD₄₅₀	3.052	0.092	2.901	999	999	999	1.24	2.068	3.389	1.77	0.062	999
Clone number	2G12	2H4	3A11	3B11	3F8	5F6	3G2	3H10	3H3	4B1	NC	PC
													OD₄₅₀	0.275	999	0.85	0.224	999	2.608	999	0.639	999	3.132	0.054	999
Clone number	5B11	5C10	5C8	5G9	6D10	3G12	NC	NC	NC	NC	NC	PC
													OD₄₅₀	999	0.756	999	1.443	2.051	2.328	0.055	0.057	0.066	0.062	0.062	999

Note: NC is negative control, 5% milk-PBS; PC was positive control, serum # 1; OD₄₅₀A value of 999 denotes OD₄₅₀The value is greater than 3.5.

According to the positive clone rescreening results listed in table 2, 23 positive clones were selected for further sensitivity detection experiments: SpA5 recombinant protein is respectively used for coating an enzyme label plate (the concentration is 1 mug/mL, 0.3 mug/mL, 0.1 mug/mL and 0.03 mug/mL in sequence), 100 mug L is added into each hole, and the overnight reaction is carried out at 4 ℃; wash the plate 3 times with PBS solution and use 5% milk-PBS sealing at room temperature for 1 hr; then washing the plate with PBS solution for 1 time, adding mouse tail blood diluted with 5% milk-PBS solution, reacting at room temperature for 1 hr; then washing the plate 3 times with PBS solution, drying, adding HRP-labeled secondary goat anti-mouse IgG (Fc) antibody diluted at 1:2000, and reacting at room temperature for 1 hr; washing the plate with PBS solution for 5 times, drying, adding equal volume of solution A and solution B, and reacting for 20min at room temperature in a dark place; then adding 50 mu L of stop solution, uniformly mixing, and reading OD on an enzyme-linked immunosorbent assay (ELISA) instrument₄₅₀The values, results are shown in Table 3.

TABLE 3 results of sensitivity detection

ng/hole	1A11	1C3	1D11	1F10	1G1	2E8	2B7	2E6	2F6	2H4	NC	NC
													100	999	999	999	999	999	999	999	999	999	999	0.056	0.052
30	2.016	0.599	2.87	3.324	2.78	0.201	0.511	0.713	0.495	2.29	0.046	0.048
													10	0.085	0.062	0.182	0.202	0.088	0.039	0.042	0.051	0.147	0.083	0.039	0.039
3	0.128	0.432	0.038	0.044	0.04	0.032	0.066	0.033	0.033	0.032	0.034	0.036
													ng/hole	3A11	3F8	5F6	3G2	3H10	3H3	4B1	5B11	5G10	5C8	NC	PC
100	1.586	999	999	999	0.656	999	999	999	1.438	999	0.041	999
													30	0.24	2.895	0.86	3.235	0.096	1.959	1.566	2.305	0.22	2.201	0.045	999
10	0.064	0.159	0.052	0.203	0.077	0.096	0.083	0.124	0.083	0.106	0.045	2.375
													3	0.083	0.052	0.047	0.066	0.065	0.047	0.057	0.052	0.081	0.048	0.113	0.576
ng/hole	5G9	6D10	3G12	NC	NC	NC	NC	NC	NC	NC	NC	PC
													100	999	999	999	0.051	0.046	0.051	0.051	0.061	0.06	0.061	0.054	999
30	1.045	0.734	0.61	0.044	0.041	0.044	0.043	0.043	0.048	0.049	0.048	999
													10	0.069	0.055	0.055	0.058	0.041	0.039	0.037	0.038	0.04	0.039	0.039	2.712
3	0.047	0.037	0.052	0.031	0.033	0.033	0.035	0.03	0.07	0.034	0.035	0.723

2. Preparation and screening of purified antibodies

1) Preparation of ascites

Preparing ascites from 13 mouse monoclonal antibodies obtained by fusing and screening the first part of cells, and respectively preparing about 10 monoclonal antibodies⁷After injecting each cell into the abdominal cavity of 2 Balb/C mice previously injected with IFA adjuvant, about 10 days later, the ascites produced by each positive clone was extracted, and then centrifuged at 12000rpm for 15min at 4 ℃ to collect the supernatant for the next G protein purification.

2) Purification of mouse monoclonal antibodies

Adding 1mL of column material coupled with G protein into an empty column, washing with PBS solution, diluting 2mL of ascites with 8mL of PBS, loading on the column, and loading the flow-through liquid on the column again; then eluting with glycine eluent with pH of 2.7, collecting one tube per 1mL eluent (adding 100 μ L of neutralization solution in advance, wherein the components of the neutralization solution are 1M Tris-HCl, 10mM EDTA, 1.5M NaCl, pH is 8.0-8.38), and collecting 5 tubes; immediately thereafter, elution was carried out using glycine eluent of pH 1.9, and a tube (300. mu.L of neutralizing solution was previously added) was collected for every 1mL of the eluent, followed by co-collection3, tubes; then OD was performed on each tube of eluate separately₂₈₀Read out, OD₂₈₀Mixing the eluents more than 0.5, and measuring OD of the mixed solution again₂₈₀The antibody concentration was calculated according to a factor of 1.4: antibody concentration ═ OD₂₈₀/1.4。

The murine monoclonal antibody purified by the G protein was evaluated by an indirect ELSIA method, and the evaluation method was adjusted to coat 10. mu.g/mL of human IgG, and after blocking with blocking buffer, 2. mu.g/mL of SpA5 protein was added. The method can also realize the specific detection of the mouse monoclonal antibody for identifying the SpA5, and has the advantages that the SpA5 protein capable of being combined on the ELISA plate is combined with the human IgG, so that sites which are combined with antibodies in a non-specific way are necessarily used, and the occurrence of non-specific results caused by incomplete blocking can be effectively avoided. The evaluation results of the purified antibody are shown in Table 4.

Table 4: evaluation results of purified murine monoclonal antibody

Note: OD₄₅₀A value of 999 denotes OD₄₅₀A value greater than 3.5; NC is negative control; PC was the respective cell supernatant.

Based on the results in table 4, 1F10, 1G1, 1D11, 5C8, 3H3, 5C10, and 2H4 were selected and used for the next development of the kit.

Example 2: preparation and application of kit for detecting SpA5 protein by double-antibody sandwich ELISA (enzyme-Linked immunosorbent assay)

The preparation method of the rabbit polyclonal antibody against SpA5 protein comprises the following steps: the recombinant protein SpA5 solution was adjusted to a concentration of 1mg/mL with a diluent (10mM His, 0.9% NaCl, pH 6.0), and emulsified with freund's adjuvant (complete freund's adjuvant for the first immunization, incomplete freund's adjuvant for the enhanced immunization) in a volume ratio of 1:1, respectively. ② the emulsified antigen is injected subcutaneously near the lymph node of the four limbs of the rabbit respectively, each point is 200 μ L, and the total amount is 0.4 mg. The immunization program is 0, 14 and 21 days, and the immunization is carried out 3 times in total. ③ at the 28 th day after the first immunization, the immunized rabbit was subjected to blood sampling by ear vein of 100. mu.L, and the serum was isolated, and the antibody titer corresponding to the recombinant protein SpA5 was detected by indirect ELISA. Fourthly, the antibody titer corresponding to the recombinant protein SpA5 reaches 1: after 219 or more, 100mL of the immunized rabbit was subjected to cardiac blood collection. The collected blood was first placed in an incubator at 37 ℃ for 30min and then left to stand overnight in a refrigerator at 4 ℃. And finally, centrifuging for 15min at 4000g in a low-temperature centrifuge, wherein the collected serum is the hyperimmune serum corresponding to the recombinant protein SpA 5. Fifthly, purifying the rabbit antiserum of the recombinant Protein SpA5 by a 5HiTrap Protein A HP chromatographic column. Sixthly, the BCA method is used for measuring the concentration of the immune globulin of the SpA5-pcAb after purification, and then the SpA5-pcAb is adjusted to be 20 mg/mL. Seventhly, the result of detecting the antibody titer corresponding to the recombinant protein SpA5 by indirect ELISA is 2048000.

The preparation of the kit is completed by using a rabbit polyclonal antibody resisting SpA5 protein, adopting a development scheme that a coating antibody is a mouse monoclonal antibody, a detection antibody is the rabbit polyclonal antibody prepared by the method, and adding a goat anti-rabbit IgG (Fc) secondary antibody marked by HRP for color development.

The reagents and articles in the kit may include:

1. antibody 5C8 coated enzyme Linked plate 8 wells × 12 strips

2. Enzyme conjugate (Rabbit polyclonal antibody) 120. mu.l X1 tube (for 1: 100-fold dilution)

BSA 3 g/bag X1 bag

4. Human IgG 10. mu.g/ml, 120. mu.l X1 tube (for 1: 100-fold dilution)

5.20 Xlotion 50ml X1 bottle

6. Substrate developing solution A (EDTA-Na, citric acid, glycerol, TMB) 7ml × 1 bottle

7. Substrate developing solution B (sodium acetate, citric acid, 30% H)₂O₂) 7ml X1 bottle

8. Stop solution (2M H)₂SO₄) 7ml X1 bottle

9. 2 sheets of sealing plate film

10. 1 part of the specification

And the single component SpA5 protein is used as a standard substance.

Kit handling procedure

1. Balancing: the desired reagents were allowed to equilibrate for 30 minutes at room temperature (18-25 ℃).

2. Preparing liquid:

1 × washing solution: 1 bottle of 20 Xwashing solution is taken, diluted to 1000ml by deionized water and evenly mixed for standby.

Enzyme conjugate diluent (3% BSA): BSA (3 g/bag) was completely dissolved in 100ml of 1. times. washing solution (r) and mixed well for use.

Enzyme conjugate working solution: diluting the required enzyme conjugate with the enzyme conjugate diluent prepared by the second step, and fully mixing for later use.

And fourthly, diluting the standard product and the to-be-detected product: taking a proper amount of human IgG, diluting the human IgG by 100 times by using a diluent of II for later use.

3. Sample application, the enzyme-linked plate coated with the antibody 5C8 was taken out from the sealed bag, and after diluting the standard and the sample to be tested, 100. mu.l of sample was applied to each well, and a negative control was set. The plates were sealed with a sealing plate and incubated at 37 ℃ for 60 minutes.

4. Washing, namely discarding liquid in each hole, filling the micropores with washing liquid, standing for 30 seconds, and then discarding liquid in the holes; repeating for 3 times, and drying the face tissues after the last plate washing is finished.

5. Adding enzyme, adding 100 μ l of the working solution of enzyme conjugate into each well, sealing with sealing plate, and incubating at 37 deg.C for 60 min.

6. Washing, namely discarding liquid in each hole, filling the micropores with washing liquid, standing for 30 seconds, and then discarding liquid in the holes; repeating for 3 times, and drying the face tissues after the last plate washing is finished.

7. And (3) developing, namely adding 50 mu l of substrate developing solution A and 50 mu l of substrate developing solution B into each hole, slightly shaking and uniformly mixing, and then placing in a dark place at room temperature for developing for 10 minutes.

8. For measurement, 50. mu.l of stop solution was added to each well and mixed gently. The absorbance (OD value) of each well was measured by selecting a microplate reader wavelength of 450 nm.

And performing linear fitting on the result of the standard substance by adopting a logX-LogY fitting mode to finally determine a standard curve, which is shown in Table 5 and figure 2.

Table 5: standard curve

From the above results, the detection sensitivity of the kit reaches 0.015625. mu.g/mL, and the linear range is 0.015625-0.5. mu.g/mL.

The sequence of the murine monoclonal antibody 5C8 was determined by sequencing analysis to be:

heavy chain sequence:

the light chain sequence:

further analyzed, the heavy chain comprises 3 CDR regions with amino acid sequences: CDR1 region GYSITSDYG (SEQ ID NO: 1); the CDR2 region is ISYSGST (SEQ ID NO: 2); CDR3 region ARGNYYALDN (SEQ ID NO: 3); the light chain also comprises 3 CDR regions, whose amino acid sequences are: CDR1 region QSLLYSSNQKNY (SEQ ID NO: 4); the CDR2 region is WAS (SEQ ID NO: 5); the CDR3 region is QQYYSYT (SEQ ID NO: 6).

Example 3: sample detection

The kit prepared in example 2 was used to detect the protein content of SpA5 antigen in the finished staphylococcus aureus vaccine 20180903 batches, the detection steps were as follows:

1. sample pretreatment: taking 20 vaccine finished products, mixing each, sucking 600 μ L into 1.5mL EP tube, centrifuging at 5000rpm for 10min, sucking supernatant 300 μ L, removing, adding 2mol/L Na₂CO₃Mixing the solution 300 μ L, centrifuging after the solution is clear, and collecting appropriate amount of supernatant;

2. the samples were tested according to the procedure of example 2;

3. the result of the standard substance is subjected to linear fitting by adopting a logX-LogY fitting mode to obtain a standard curve, the light absorption value (OD value) of the detected sample is substituted into the standard curve to calculate the concentration of the sample, and the detection result is as follows:

table 6: kit detection result of SpA5 protein content

From the results, the SpA5 protein content in 20 samples detected by the kit is greater than 42.75 mu g/mL specified by the standard, and the RSD is 4.73% and less than 10%, which indicates that the kit can be used for quantitative detection of SpA5 protein and has good applicability.

Sequence listing

<110> Chengdu European forest Biotechnology Ltd

ARMY MEDICAL University

<120> monoclonal antibody against SpA5 protein, application thereof and kit containing same

<130> DSP1F201463ZX

<150> CN201911367592X

<151> 2019-12-26

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gly Tyr Ser Ile Thr Ser Asp Tyr Gly

1 5

<210> 2

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Ile Ser Tyr Ser Gly Ser Thr

1 5

<210> 3

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Ala Arg Gly Asn Tyr Tyr Ala Leu Asp Asn

1 5 10

<210> 4

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr

1 5 10

<210> 5

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Trp Ala Ser

1

<210> 6

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gln Gln Tyr Tyr Ser Tyr Thr

1 5

<210> 7

<211> 123

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

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

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Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly

20 25 30

Tyr Ser Ile Thr Ser Asp Tyr Gly Trp Asn Trp Ile Arg Gln Phe Pro

35 40 45

Gly Asn Lys Leu Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr

50 55 60

Ser Tyr Asn Pro Ser Leu Lys Gly Arg Ile Ser Ile Thr Arg Asp Thr

65 70 75 80

Ser Lys Asn Gln Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp

85 90 95

Thr Ala Thr Tyr His Cys Ala Arg Gly Asn Tyr Tyr Ala Leu Asp Asn

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Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 8

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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

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Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

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

100 105 110

Claims

1. A monoclonal antibody against SpA5 protein, the antibody comprising a heavy chain and a light chain, the heavy chain and the light chain respectively comprising 3 CDR regions, wherein,

the amino acid sequence of the light chain CDR2 region is WAS (SEQ ID NO: 5);

the amino acid sequence of the light chain CDR3 region is QQYYSYT (SEQ ID NO: 6);

wherein the amino acid sequence of the SpA5 protein is as follows:

GPLGSAQHDE AKKNAFYQVL NMPNLNADQR NGFIQSLKAA PSQSANVLGE AQKLNDSQAP KADAKKNKFN KDQQSAFYEI LNMPNLNEEQ RNGFIQSLKA APSQSTNVLG EAKKLNESQA PKADNNFNKE KKNAFYEILN MPNLNEEQRN GFIQSLKAAP SQSANLLAEA KKLNESQAPK ADNKFNKEKK NAFYEILHLP NLNEEQRNGF IQSLKAAPSQ SANLLAEAKK LNDAQAPKAD NKFNKEKKNA FYEILHLPNL TEEQRNGFIQ SLKAAPSVSK EILAEAKKLN DAQAPK。

2. the antibody of claim 1, wherein,

the sequence of the antibody heavy chain is:

the sequence of the antibody light chain is:

3. the antibody of claim 1 or 2, wherein the antibody is an IgG antibody.

4. A method for detecting the content of SpA5 protein for non-disease diagnosis purposes, which comprises the steps of detecting by using the monoclonal antibody of any one of claims 1-3 and detecting by an ELISA method, wherein the monoclonal antibody is used as a coating antibody, and a rabbit polyclonal antibody which is prepared by a conventional method and is used for resisting SpA5 protein is used as a detection antibody.

5. The method of claim 4, wherein the method comprises:

1) coating an enzyme-linked plate with the monoclonal antibody according to any one of claims 1 to 3;

2) reacting a sample to be tested with the monoclonal antibody;

4) and preparing a standard curve by using the SpA5 protein standard, and calculating the content of SpA5 protein in the tested sample according to the standard curve.

6. The method of claim 4 or 5, wherein the amount of SpA5 protein detected by the method is in the range of 0.015625-0.5 μ g/mL.

7. A kit for detecting the content of SpA5 protein, which comprises the monoclonal antibody against SpA5 protein as claimed in any one of claims 1-3.

8. The kit of claim 7, further comprising a rabbit polyclonal antibody against SpA5 protein and an HRP-labeled goat anti-rabbit IgG Fc secondary antibody, prepared by conventional methods.

9. The kit according to claim 7 or 8, wherein the kit further comprises human IgG, a washing reagent, a substrate developing solution A, a substrate developing solution B, a stop solution and BSA, an enzyme-linked plate and a plate-sealing membrane, and a SpA5 protein standard; wherein the substrate color developing solution A is prepared by the following steps: EDTA-Na 0.2g, citric acid 0.95g, glycerin 50ml, TMB 0.2g, dH₂O500 ml, and adding TMB after dissolving with DMSO to 10 mg/ml; the preparation of the substrate color developing solution B comprises the following steps: 13.6g of sodium acetate, 1.6g of citric acid and 30% of H₂O₂ 0.3ml，dH₂O 500ml。

10. Use of the monoclonal antibody against SpA5 protein according to any one of claims 1-3 for detecting the content of SpA5 protein for non-disease diagnostic purposes.