CN111018972A

CN111018972A - anti-mSEB protein antibody, application thereof and kit comprising same

Info

Publication number: CN111018972A
Application number: CN201911370244.8A
Authority: CN
Inventors: 曾浩; 邹全明; 章金勇; 罗萍; 敬海明; 李海波; 夏鑫; 曾妮
Original assignee: Army Medical University; Chengdu Olymvax Biopharmaceuticals Inc
Current assignee: Army Medical University; Chengdu Olymvax Biopharmaceuticals Inc
Priority date: 2019-12-26
Filing date: 2019-12-26
Publication date: 2020-04-17
Anticipated expiration: 2039-12-26
Also published as: CN111018972B; WO2021128949A1

Abstract

The invention belongs to the technical field of biology, and provides an anti-mSEB protein monoclonal antibody and an application thereof, wherein the antibody comprises a heavy chain and a light chain, the heavy chain and the light chain respectively comprise 3 CDR regions, amino acid sequences of the CDR regions of the heavy chain are respectively shown as SEQ ID NO. 1-3 or 7-9, and amino acid sequences of the CDR regions of the light chain are respectively shown as SEQ ID NO. 4-6 or 10-12. The mSEB protein antibody can be specifically combined with mSEB protein, so that the content of the mSEB protein can be effectively detected, and the sensitivity is high.

Description

anti-mSEB protein antibody, application thereof and kit comprising same

Technical Field

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

Background

Staphylococcus aureus enterotoxin (SE) is a superantigenic exotoxin, commonly produced by pathogenic Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus (MRSA). SE can produce biological effects by causing a number of T cells to release large amounts of cytokines, causing clinical symptoms such as toxin shock syndrome. Currently, SE has 9 serotypes, SEA, SEB, SEC, SED and SEE. SEB is highly conserved in amino acid sequence among strains of Staphylococcus aureus. Studies prove that the SEB has the advantage of better humoral response than other antigens, and the prepared human SEB monoclonal antibody can effectively treat the common toxic shock syndrome caused by staphylococcus aureus infection. The mSEB mutant mSEB with three sites (L45R, Y89A and Y94A) is constructed and obtained as one of the antigen components of the recombinant staphylococcus aureus vaccine by the applicant with reference to relevant research documents. In vivo and in vitro experiments prove that: mSEB has no toxicity and superantigen activity of wild SEB any more, and has good safety; meanwhile, the good immunogenicity and immunoprotection are reserved.

The monoclonal antibody aiming at multiple active epitopes of mSEB, which is obtained by taking the recombinant mSEB protein as 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 mSEB antigen content in the recombinant staphylococcus aureus vaccine. No specific mSEB antibodies are currently available.

Disclosure of Invention

For the above reasons, the present invention provides an anti-mSEB protein antibody that can effectively detect the amount of mSEB protein in a sample such as a vaccine to solve the above problems.

In a first aspect, the invention provides an antibody against the mSEB protein, said antibody comprising a heavy chain and a light chain comprising 3 CDR regions, respectively, wherein:

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

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

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

The amino acid sequence of the heavy chain CDR1 region is GYSVTSGYA (SEQ ID NO: 7);

the amino acid sequence of the heavy chain CDR2 region is ISYNGIT (SEQ ID NO: 8);

the amino acid sequence of the heavy chain CDR3 region is AREYRFDGYYYAMDY (SEQ ID NO: 9); and

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

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

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

The amino acid sequence of the light chain CDR1 region is QSLVHSNGNTY (SEQ ID NO: 10);

the amino acid sequence of the light chain CDR2 region is KVS (SEQ ID NO: 11);

the amino acid sequence of the light chain CDR3 region was SRTTHVPFT (SEQ ID NO: 12).

Preferably, the antibody comprises a heavy chain and a light chain, wherein,

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

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

the amino acid sequence of the heavy chain CDR3 region is AREYRFDGYYYAMDY (SEQ ID NO: 9);

the amino acid sequence of the light chain CDR2 region is KVS (SEQ ID NO: 11);

Further preferably, the antibody is antibody 3C7, the heavy and light chain sequences of which are:

heavy chain:

VKLQESGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNFDPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYFCTRSYYGRGGFDCWGQGTTVTVSS(SEQ ID NO:13)；

light chain:

DVLMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRLTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPPTFGGGTKLEI (SEQ ID NO: 14); or

The antibody is antibody 4C7, whose heavy and light chain sequences are:

heavy chain:

GPGLVKPSQSLSLTCTVTGYSVTSGYAWNWIRQFPGNKLEWMGYISYNGITGYNPSLKSRVSLTRDTSKNQFFLQLNSVTTEDTATYYCAREYRFDGYYYAMDYWGQGTPVTVSS(SEQ ID NO:15)；

light chain:

DIVMTQTTLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSRTTHVPFTFGSGTKLEIK(SEQ ID NO:16)。

preferably, the antibody is an IgG antibody.

In a second aspect, the invention provides a kit for detecting mSEB protein content, comprising an antibody of the invention. Preferably, the antibodies are antibodies 3C7 and 4C 7.

Preferably, the kit further comprises human IgG, a washing reagent, a substrate developing solution A (EDTA-Na, citric acid, glycerol, TMB), a substrate developing solution B (sodium acetate, citric acid, sodium citrate,30％H₂O₂) Stop solution (2M H)₂SO₄) And BSA.

Preferably, the kit further comprises an enzyme-linked plate and a cover plate membrane.

Preferably, the kit further comprises a mSEB protein standard.

More preferably, the antibody 3C7 is a detection antibody and the antibody 4C7 is a coating antibody.

In a third aspect, the invention provides a method for detecting mSEB protein content, which comprises carrying out Elisa detection by using an antibody 3C7 and an antibody 4C7 through a double antibody sandwich method.

Preferably, the method comprises:

1) coating the enzyme conjugate plate with antibody 4C 7;

2) reacting the tested sample with an antibody 4C 7;

3) detecting and developing with antibody 3C7, and measuring absorbance at 450 nm;

4) and (4) preparing a standard curve by using the mSEB protein standard, and calculating the mSEB protein content in the detected sample according to the standard curve.

Preferably, the mSEB protein content detected by the method is 0.046875-3 mu g/ml.

In a fourth aspect, the invention provides the use of an anti-mSEB protein antibody of the invention for detecting mSEB protein content.

In the invention, the mSEB protein has a sequence as follows:

MESQPDPKPDELHKSSKFTGLMENMKVLYDDNHVSAINVKSIDQFRYFDLIYSIKDTKLGNYDNVRVEFKNKDLADKYKDKYVDVFGANAYYQCAFSKKTNDINSHQTDKRKTCMYGGVTEHNGNQLDKYRSITVRVFEDGKNLLSFDVQTNKKKVTAQELDYLTRHYLVKNKKLYEFNNSPYETGYIKFIENENSFWYDMMPAPGDKFDQSKYLMMYNDNKMVDSKDVKIEVYLTTKKK (SEQ ID: 17). For specific information on mSEB, including preparation, see CN103694321A, the amino acid sequence of mSEB protein is shown in SEQ ID NO:1 of the patent document. CN103694321A is hereby incorporated by reference in its entirety.

The anti-mSEB protein antibody can be specifically combined with mSEB protein, the content of the mSEB protein can be effectively detected, the sensitivity is high, the lowest limit can reach 0.046875 mu g/mL, the detection range is wide, 0.046875-3 mu g/mL, and the requirement of quantitative detection of vaccines can be met.

Drawings

FIG. 1: screening results of the 3# mouse monoclonal cell strain;

FIG. 2: screening results of 2# mouse monoclonal cell strains;

FIG. 3: and fitting a standard curve to the equation.

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.

The method uses mSEB protein as immunogen to immunize mice, obtains a plurality of mouse monoclonal antibodies for specifically recognizing the mSEB protein through cell fusion and screening, selects an antibody with higher affinity with the mSEB protein to prepare ascites, and respectively performs HRP marking after G protein purification.

After pairing detection and condition optimization, a pairing antibody for detecting the mSEB content is obtained: the coating antibody 4C7 and the detection antibody 3C7, and the detection conditions and the standard curve are determined, the detection sensitivity reaches 0.046875 mu g/mL, the linear range is 0.046875-3 mu g/mL, and the requirement of quantitative detection of the vaccine is met.

Primary reagent

Main instrument

Laboratory animal

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

Preparation of 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，dH₂O500ml。

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 mSEB protein

The immunogens were prepared using mSEB protein (1.4mg/ml) mixed with the adjuvants CFA and AD11.15, respectively, i.e., recombinant protein 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.

An enzyme label plate (1 mu g/mL) is coated with mSEB recombinant protein, 100 mu L of the enzyme label plate is added into each hole, and the reaction is carried out at 4 ℃ overnight; 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, adding 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: NC is negative control, 5% milk-PBS; PC is 2# mouse serum.

As can be seen from the results, the antibody titer of mSEB recombinant protein recognized by the tail blood of mice at day 14 of immunization was only 1:500, and after one additional boosting of the mice, the antibody titer of the tail blood of the mice was evaluated again, and the results are shown in Table 2.

Table 2: evaluation of antibody titer in tail blood of mice after boosting immunization

Note: NC is negative control; PC was serum from first immunized 2# mice.

From the results, the antibody titer of mSEB recombinant protein recognized by 3# mouse tail blood is the highest and reaches 1:5000, and is remarkably improved compared with the last tail blood evaluation. Therefore, the 3# mouse was selected for cell fusion with the myeloma cell SP 2/0. After the fusion, 564 monoclonal hybridoma cells were selected and cultured, and then positive clone screening was performed by using a conventional ELISA, in which the screening criteria was that the OD value was 2.1 times the NC value, and the screening results are shown in fig. 1.

As can be seen from the screening results, no positive clones were obtained. The remaining mice # 1 and # 2 were boosted once and then the tail blood was again evaluated for antibody titer, as shown in table 3.

Table 3: evaluation of antibody titer in tail blood of mice after boosting immunization

Note: NC is negative control.

From the results, the antibody titer of mSEB protein recognized by the tail blood of the 2# mouse is the highest and reaches 1: 10000. Therefore, 2# mice were selected again for cell fusion with the myeloma cell SP 2/0. After the fusion, 376 monoclonal hybridoma cells were selected and cultured, and then screened by ELISA, and the screening results are shown in fig. 2.

From the screening results, 8 positive clones were selected for confirmation experiments in the following procedure: coating mSEB protein 100 ng/well, adding 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 4.

TABLE 4 confirmation of Positive clones

Clone number	1D1	2A10	2A11	2C4	2E4	3C7	3G1	4C7	NC	NC	NC	PC
													OD₄₅₀	999	1.185	0.564	999	999	999	1.282	999	0.059	0.072	0.071	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 4, 8 positive clones were selected for continuing the sensitivity detection experiment: using mSEB separatelyThe histone coating 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 ℃; 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; 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, experimental results are shown in table 5. And performing a specificity detection experiment: HI, MntC and mSEB recombinant proteins are respectively used for coating an enzyme label plate (the concentration is 1 mu g/mL), 100 mu L of the enzyme label plate is added into each hole, and the overnight reaction is carried out 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, adding 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 adding 50 mu L of stop solution, uniformly mixing, and reading OD on an enzyme-linked immunosorbent assay (ELISA) instrument₄₅₀The values, experimental results are shown in table 6.

TABLE 5 results of sensitivity detection

ng/hole	1D1	2A10	2C4	2E4	3C7	3G1	4C7	2A11	NC	NC	NC	PC
													100	999	0.365	2.045	999	0.933	0.457	999	0.355	0.055	0.064	0.125	999
30	0.24	0.069	0.115	2.768	0.085	0.072	0.189	0.052	0.048	0.054	0.047	999
													10	0.061	0.041	0.042	0.635	0.043	0.044	0.054	0.044	0.042	0.042	0.046	0.953
3	0.055	0.051	0.057	0.612	0.052	0.057	0.063	0.065	0.069	0.076	0.068	0.462

Table 6: specific detection results

The results of detection of binding sensitivity and specificity, among which the results of 4 murine monoclonal antibodies are better, 3C7, 4C7, 2E4 and 3G 1.

In the experiment, the information of the HI protein is shown in CN103695508A, and the amino acid sequence of the HI protein is shown in SEQ ID NO. 1 of CN 103695508A; the information of the MntC protein is shown in CN103694323A, and the amino acid sequence of the MntC protein is shown in SEQ ID NO. 1 of CN 103694323A. The contents of both applications are incorporated by reference into the present application.

Example 2: preparation and screening of purified antibodies

1. Preparation of ascites

Ascites was prepared from the above-obtained 4-strain positive clones (3C7, 4C7, 2E4 and 3G 1). Respectively about 10⁷After 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 elution was performed using glycine eluent of pH 2.7, one tube per 1mL of eluent was collected (pre-addedAdding 100 μ L of neutralizing solution containing 1M Tris-HCl, 10mM EDTA, 1.5M NaCl, pH8.0-8.38), collecting 5 tubes; then eluting with glycine eluent with pH of 1.9, collecting one tube (300 μ L of neutralizing solution is added in advance) per 1mL of eluent, and collecting 3 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 by a factor of 1.4; antibody concentration ═ OD₂₈₀/1.4。

The G protein purified murine mAb was evaluated using the indirect ELSIA method and the results are shown in Table 7.

TABLE 7 evaluation of purified murine mAb antibodies

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

As seen from the results in Table 7, the sensitivity of the purified antibodies 3C7 and 4C7 was between 0.005 and 0.05. mu.g/mL, and the sensitivity of 2E4 was higher, between 0.005 and 0.05. mu.g/mL. Whereas 3G1 had too poor affinity.

Example 3: paired detection and screening experiments for antibodies

The binding ability of the labeled antibodies to the mSEB recombinant protein was evaluated by ELISA after HRP labeling of 3C7, 4C7, and 2E4 using sodium periodate oxidation, respectively, and the results are shown in Table 8.

Table 8: evaluation results of HRP-labeled antibody

As can be seen from table 8, the binding capacity of the HRP-labeled antibodies 3C7 and 4C7 to the mSEB protein is not significantly reduced compared with that of the antibody before labeling, and the pairing experiment (see table 9) is performed to select the paired antibody, while the binding capacity of 2E 4-labeled antibodies to the mSEB protein is significantly reduced, and the paired antibody is not used as an enzyme-labeled antibody to perform the pairing experiment.

Table 9: results of pairwise pairing experiments with antibodies

According to the matching results in table 9 and considering the purified antibody yields of 3 murine mabs together, the matching results of murine mab 4C7 as coating antibody and murine mab 3C7 as detection antibody were selected to be optimal.

Through sequencing analysis, the heavy chain sequence of the antibody 3C7 is shown as SEQ ID NO. 13, and the light chain sequence is shown as SEQ ID NO. 14; the heavy chain sequence of the antibody 4C7 is shown as SEQ ID NO. 15, and the light chain sequence is shown as SEQ ID NO. 16.

Example 4: preparation and application of mSEB protein content detection kit

According to the above results, monoclonal antibodies 3C7 and 4C7 were selected as antibody preparation kits, and reagents and articles in the kit may include:

1. antibody 4C7 coated ELISA plates, 8 wells × 12 strips

2. Enzyme conjugate (antibody 3C7), 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 in addition, single-component mSEB protein is used as a standard substance.

Procedure for the preparation of the

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

2. Preparing liquid:

① washing solution (1 × washing solution) 1 bottle of 20 × washing solution is diluted to 1000ml with deionized water and mixed for use.

② enzyme conjugate dilution (3% BSA) BSA (3 g/bag) was dissolved completely in 100ml of 1 × Wash solution prepared ① and mixed well for use.

③ working solution of enzyme conjugate is prepared through diluting the required enzyme conjugate with ② diluted enzyme conjugate, and mixing.

④, diluting the standard sample and the sample to be tested by taking appropriate amount of human IgG, and diluting the human IgG 100 times by using ② diluent for later use.

3. And (3) sample adding, namely taking the antibody 4C7 coated enzyme-linked plate out of the sealed bag, diluting the standard substance and the sample to be detected, adding 100 mu l of sample into each hole, and setting a negative control. 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.

Through a series of optimization operations, a standard curve is finally determined, see table 10 and the double logarithm fitting equation fig. 3.

Table 10: standard curve

As can be seen from the results, the detection sensitivity of the kit reaches 0.046875 mug/mL, and the linear range is 0.046875-3 mug/mL.

Example 5: detection experiment

The detection method for detecting the protein content of the mSEB antigen in 20180903 batches of finished products of the staphylococcus aureus vaccine by using the kit comprises the following steps:

1. sample pretreatment: taking 20 vaccine finished products, sucking 600 mu L of each vaccine finished product into a 1.5mL EP tube after uniformly mixing, centrifuging for 10min at 5000rpm, sucking 300 mu L of supernatant, removing, adding 300 mu L of 2mol/L Na2CO3 solution, uniformly mixing, centrifuging after the solution is clear, and taking a proper amount of supernatant for later use;

2. the samples were tested according to the procedure of example 4;

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 11: mSEB protein content detection result by kit

From the results, the mSEB protein content in 20 samples detected by the kit is greater than 21.375 mu g/mL specified by the standard, and the RSD is 6.90% and less than 10%, which indicates that the kit can be used for quantitative detection of mSEB protein and has good applicability.

Claims

1. An antibody against a mSEB protein, said antibody comprising a heavy chain and a light chain comprising 3 CDR regions, respectively, wherein:

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

the amino acid sequence of the light chain CDR2 region is KVS (SEQ ID NO: 11);

2. The antibody of claim 1, the antibody comprises a heavy chain and a light chain, wherein,

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

the amino acid sequence of the light chain CDR2 region is KVS (SEQ ID NO: 11);

3. The antibody of claim 2, wherein the antibody is antibody 3C7 having the heavy and light chain sequences:

heavy chain:

light chain:

The antibody is antibody 4C7, whose heavy and light chain sequences are:

heavy chain:

light chain:

4. the antibody of any one of claims 1 to 3, wherein the antibody is an IgG antibody.

5. A kit for detecting the amount of mSEB protein, the kit comprising an antibody according to any one of claims 1 to 4.

6. The kit according to claim 5, further comprising human IgG, a washing reagent, a substrate developing solution A, a substrate developing solution B, a stop solution and BSA.

7. The kit of claim 6, wherein the kit further comprises an enzyme conjugate plate, a closure plate membrane and/or a mSEB protein standard.

8. The kit according to any one of claims 5 to 7, wherein the antibodies are antibody 3C7 and antibody 4C7, the antibody 3C7 is a detection antibody, and the antibody 4C7 is a coating antibody.

9. A method for detecting mSEB protein content, which comprises the steps of carrying out Elisa detection by using the antibody of any one of claims 1-4 by using a double antibody sandwich method; preferably, the antibodies are antibody 3C7 and antibody 4C7, the method comprising:

1) coating the enzyme conjugate plate with antibody 4C 7;

2) reacting the tested sample with an antibody 4C 7;

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

10. Use of the antibody of any one of claims 1 to 4 for detecting mSEB protein content.