CN111072775A - anti-MntC protein antibody, application thereof and kit containing same - Google Patents

Abstract

The invention belongs to the technical field of biology, and provides an anti-MntC protein antibody, application thereof and a kit containing the same, 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, and amino acid sequences of the CDR regions of the light chain are respectively shown as SEQ ID NO. 4-6. The anti-MntC protein antibody can be specifically combined with MntC protein, effectively detects the content of the MntC protein, and has high sensitivity and wide detection range.

Description

anti-MntC protein antibody, application thereof and kit containing same

Technical Field

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

Background

The acquisition of manganese ions is related to the metabolism, survival and adhesion of staphylococcus aureus, and is closely related to the virulence of staphylococcus aureus. In addition, manganese ions play an important role in the immune escape of staphylococcus aureus by protecting it from neutrophil attack. Manganese ion transporter C subunit (MntC) is a highly conserved cell surface protein in a staphylococcus aureus manganese ion transporter complex (MntA \ B \ C), consists of 285 amino acids, and has a molecular weight of 32 kD. Research shows that MntC can be used as a vaccine antigen to rapidly mediate effective immune response to prevent the infection of staphylococcus aureus. In addition, the monoclonal antibody against MntC induces strong neutrophil phagocytic activity in mice infected with staphylococcus aureus, and produces immune protection in a passive immune protection model of mice.

The monoclonal antibody aiming at multiple active epitopes of MntC, which is obtained by taking the recombinant MntC 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 content of MntC antigen in recombinant staphylococcus aureus vaccine. However, no specific monoclonal antibody exists at present.

Disclosure of Invention

For the above reasons, the present invention provides a monoclonal antibody against MntC protein, which can effectively detect the content of MntC protein in a sample such as a vaccine, to solve the above problems.

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

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

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

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

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

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

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

Preferably, the antibody is antibody 3C10, the heavy and light chain sequences of which are:

heavy chain:

VKLQESGPSLVKPSQTLSLTCSVTGDSITNGYWNWIRKFPGNKLEYMGYISYSGTTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCSRIRPYRHLGFDYWGQGTTVTVSS(SEQ ID NO:7)；

light chain:

DIVMTQSTASLAVSLGQRATISCKASQSVDYGGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDGATYYCQQSNEDPWTFGGGTKLEIK (SEQ ID NO: 8); or

The antibody is antibody 3F6, the heavy and light chain sequences of which are:

heavy chain:

VQLQQSGPSLVKPSQTLSLTCSVTGDSITNGYWNWIRKFPGNKLEYMGYISYSGTTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCSRIRPYRHLGFDYWGQGTTVTVSS(SEQ ID NO:9)；

light chain:

DIVMTQTPASLAVSLGQRATISCKASQSVDYGGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDGATYYCQQSNEDPWTFGGGTKLEIK(SEQ ID NO:10)。

preferably, the antibody is an IgG antibody.

In a second aspect, the invention provides a kit for detecting the amount of MntC protein, said kit comprising an antibody of the invention. Preferably, the antibodies are antibodies 3C10 and 3F 6.

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 (acetic acid)Sodium, citric acid, 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 MntC protein standard.

More preferably, the antibody 3C10 is a detection antibody and the antibody 3F6 is a coating antibody.

In a third aspect, the invention provides a method for detecting the content of MntC protein, which comprises the step of carrying out Elisa detection by using an antibody 3C10 and an antibody 3F6 through a double antibody sandwich method.

Preferably, the method comprises:

1) coating the enzyme conjugate plate with antibody 3F 6;

2) reacting the tested sample with an antibody 3F 6;

3) detecting and developing with antibody 3C10, and measuring absorbance at 450 nm;

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

Preferably, the content range of MntC protein detected by the method is 0.046875-0.75 mu g/ml.

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

In the invention, the MntC protein sequence is as follows:

GPLGSSSDKSNGKLKVVTTNSILYDMAKNVGGDNVDIHSIVPVGQDPHEYEVKPKDIKKLTDADVILYNGLNLETGNGWFEKALEQAGKSLKDKKVIAVSKDVKPIYLNGEEGNKDKQDPHAWLSLDNGIKYVKTIQQTFIDNDKKHKADYEKQGNKYIAQLEKLNNDSKDKFNDIPKEQRAMITSEGAFKYFSKQYGITPGYIWEINTEKQGTPEQMRQAIEFVKKHKLKHLLVETSVDKKAMESLSEETKKDIFGEVYTDSIGKEGTKGDSYYKMMKSNIETVHGSMK (SEQ ID NO: 11). Specific information of the MntC protein, including preparation and the like, is shown in CN103694323A, and the amino acid sequence of the MntC protein is shown in SEQ ID NO. 1 of the patent document. CN103694323A is hereby incorporated by reference in its entirety.

The anti-MntC protein antibody can be specifically combined with MntC protein, effectively detects the content of the MntC protein, has high sensitivity reaching 0.046875 mu g/mL, has wide detection range of 0.046875-0.75 mu g/mL, and can meet the requirement of quantitative detection of vaccines.

Drawings

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

FIG. 2: screening results of the second batch of monoclonal cell strains;

FIG. 3: and (5) linear fitting results of the standard.

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 MntC protein as immunogen to immunize mice, obtains 2 mouse monoclonal antibodies 3F6 and 3C10 for specifically recognizing the MntC protein through cell fusion and screening, prepares ascites, and respectively carries out HRP marking after G protein purification.

After pairing detection and condition optimization, a pair of paired antibodies (coating antibody 3F6 and detection antibody 3C10) is obtained, and detection conditions and a standard curve are determined, the detection sensitivity reaches 0.046875 mu g/mL, the linear range is 0.046875-0.75 mu g/mL, and the requirement of quantitative detection of the vaccine is met.

Primary reagent

Main instrument

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

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 and screening of mouse monoclonal antibody for specifically recognizing MntC protein

1. Preparation of first mouse monoclonal antibody

The MntC protein was mixed with the adjuvants CFA and AD11.15, respectively, to prepare immunogens, i.e. the protein was first mixed with CFA in a volume ratio of 5: 6 as immunogen A, and the 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.

MntC protein is used for coating an enzyme label plate (1 mu g/mL), 100 mu L of MntC protein 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 1 time with PBS solution, adding 5% cattleReacting the mouse tail blood with milk-PBS solution 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 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.

From the results, the antibody titer of the 2# mouse tail blood recognizing the MntC recombinant protein was the highest and reached 1: 50000. Therefore, 2# mice were selected for cell fusion with myeloma cell SP 2/0. 564 monoclonal hybridoma cells were picked after fusion and cultured in a 96-well plate, and the cell culture supernatants in the 96-well plate were evaluated by the indirect ELISA method described above, and monoclonal cell strains capable of secreting monoclonal antibodies that recognize proteins were selected, and the results are shown in fig. 1.

From the screening results, 17 positive clones were selected for confirmation experiments in the following procedure: coating MntC protein by 100 ng/hole, and respectively adding the supernatant of each clone 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	1B12	1B3	1B7	1B8	1C8	1F1	2C12	3E10	3F6	3G3	NC	PC
													OD450	0.853	999	0.719	1.001	2.599	0.886	0.108	0.691	999	3.101	0.07	999
Clone number	4F2	4H5	4H7	5A12	5B5	5F3	5H9	NC	NC	NC	NC	PC
													OD450	0.651	0.461	1.935	0.316	999	0.877	0.906	0.045	0.047	0.057	0.052	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.

As can be seen from table 2, 16 positive clones, except 2C12, all appeared positive after cell passage. Next, sensitivity detection of cell supernatants was performed on all 16 positive clones: respectively using MntC recombinant protein to coat the ELISA plates (the concentration is sequentially1. mu.g/mL, 0.3. mu.g/mL, 0.1. mu.g/mL and 0.03. mu.g/mL), 100. mu.L per well, and reacting 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; 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 50. mu.L of stop solution (2M H) was added₂SO₄) After mixing, the OD is read on a microplate reader₄₅₀The values, results are shown in Table 3.

Table 3: sensitive detection of first monoclonal cell lines

ng/hole	1B12	1B3	1B7	1B8	1C8	1F1	3E10	3F6	3G3	4F2	NC	PC
													100	0.105	2.601	0.132	0.098	0.327	0.089	0.107	999	0.289	0.081	0.051	999
30	0.056	0.15	0.083	0.051	0.195	0.081	0.101	999	0.244	0.084	0.051	999
													10	0.041	0.047	0.042	0.04	0.054	0.042	0.039	1.961	0.066	0.041	0.044	2.023
3	0.036	0.033	0.034	0.039	0.036	0.033	0.033	0.204	0.036	0.141	0.055	0.324
													ng/hole	4H5	4H7	5A12	5B5	5F3	5H9	NC	NC	NC	NC	NC	PC
100	0.063	0.106	0.044	0.276	0.062	0.062	0.037	0.039	0.039	0.042	0.05	999
													30	0.055	0.048	0.041	0.052	0.043	0.054	0.046	0.046	0.045	0.042	0.049	999
10	0.043	0.044	0.043	0.045	0.05	0.042	0.042	0.041	0.041	0.038	0.042	1.403
													3	0.05	0.036	0.046	0.044	0.044	0.7039	0.044	0.031	0.04	0.044	0.043	0.405

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.

As can be seen from the results in Table 3, only the positive clones 1B3 and 3F6 were able to detect strong recognition signals after cell passage.

2. Preparation of second batch mouse monoclonal antibody

The second monoclonal antibody was prepared according to the method described above. 3 mice were immunized with the immunogen prepared using the MntC protein, and the blood level of the mouse tail at 14 days after immunization was evaluated by the indirect ELISA method described above, and the results are shown in Table 4.

Table 4: 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; PC is the serum of the first immunized 1# mouse.

From the results, the antibody titer of 3# mouse tail blood recognizing MntC protein was highest, reaching 1: 50000. Therefore, the 3# mouse was selected for cell fusion with the myeloma cell SP 2/0. 564 monoclonal hybridoma cells were picked after fusion and cultured, and then screened by ELISA, and the screening results are shown in FIG. 2.

From the screening results, 13 positive clones were selected and subjected to a confirmation test for screening, and the results are shown in table 5.

TABLE 5 confirmation of Positive clones

Clone number	1A9	2E9	2B12	3E7	3C10	4A10	4A8	4B3	4B8	4B9	NC	PC
													OD450	0.369	0.059	0.147	0.288	999	0.341	0.392	0.9	0.424	0.37	0.043	999
Clone number	5A11	5B9	6A10	NC	NC	NC	NC	NC	NC	NC	NC	PC
													OD450	0.271	0.228	0.272	0.043	0.047	0.044	0.044	0.043	0.042	0.052	0.042	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 results of rescreening the positive clones listed in Table 5, 6 positive clones were selected and subjected to the sensitivity detection test, and the results are shown in Table 6.

TABLE 6 sensitivity detection of the second set of monoclonal cell lines

ng/hole	4A10	1A9	3C10	4A8	4B3	4B9	NC	NC	NC	NC	NC	PC
													100	0.091	0.058	2.063	0.063	0.053	0.055	0.055	0.067	0.066	0.061	0.075	999
30	0.056	0.06	0.054	0.055	0.056	0.059	0.062	0.065	0.068	0.072	0.075	0.996
													10	0.061	0.057	0.057	0.055	0.053	0.051	0.053	0.056	0.055	0.069	0.069	0.137
3	0.115	0.066	0.049	0.052	0.065	0.065	0.084	0.076	0.082	0.096	0.11	0.125

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

As can be seen from the results in table 6, the positive clone 3C10 was able to detect a strong recognition signal after cell passage.

Example 2: preparation and screening of purified antibodies

1. Preparation of ascites

Ascites was prepared from the 3-strain positive clones (1B3, 3F6 and 3C10) obtained above. 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 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, pH8.0-8.38), and 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 of Table 7, the sensitivity of the purified antibodies 3F6 and 3C10 was between 0.005 and 0.05. mu.g/mL, while the affinity of 1B3 was too poor, so that 3F6 and 3C10 could be used in pair for detecting the content of MntC protein.

Example 3: antibody pairing detection assay

HRP labeling was performed on 3F6 and 3C10 using sodium periodate oxidation, respectively, and then the binding ability of the labeled antibody to MntC protein was evaluated by ELISA, 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 ability of the HRP-labeled antibody to MntC protein was not significantly reduced compared to the antibody before labeling.

Further pairing experiments were performed on these two antibodies.

Table 9: results of pairwise pairing experiments with antibodies

According to the matching results in table 9, the matching results of the murine monoclonal antibody 3F6 as the coating antibody and the murine monoclonal antibody 3C10 as the detection antibody are optimal, and can be used for detecting the content of MntC protein.

Through sequencing analysis, the heavy chain sequence of the antibody 3C10 is shown as SEQ ID NO. 7, and the light chain sequence is shown as SEQ ID NO. 8; the heavy chain sequence of the antibody 3F6 is shown as SEQ ID NO. 9, and the light chain sequence is shown as SEQ ID NO. 10.

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

Based on the above results, 3C10 and 3F6 were selected as antibody preparation kits, and reagents and articles in the kits may include:

1. antibody 3F6 coated enzyme Linked plate 8 wells × 12 strips

2. Enzyme conjugate (antibody 3C10) 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

In addition, single-component MntC 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 3F6 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, uniformly mixing, and then placing in a dark place at room temperature for developing for 7 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 a table 10 and a double logarithm fitting equation 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 0.046875-0.75 mug/mL.

Example 5: test kit

The detection method for detecting the protein content of the MntC 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, 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 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: result of detecting MntC protein content by kit

From the results, the MntC protein content in 20 samples detected by the kit is greater than 21.375 mu g/mL in the standard specification, and the RSD is 6.01% and less than 10%, which indicates that the kit can be used for quantitative detection of the MntC protein and has good applicability.

Claims (10)

1. An antibody against a MntC 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 GDSITNGY (SEQ ID NO: 1);

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

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

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

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

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

2. The antibody of claim 1, wherein,

the antibody is antibody 3C10, the heavy and light chain sequences of which are:

heavy chain:

VKLQESGPSLVKPSQTLSLTCSVTGDSITNGYWNWIRKFPGNKLEYMGYISYSGTTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCSRIRPYRHLGFDYWGQGTTVTVSS(SEQ ID NO:7)；

light chain:

DIVMTQSTASLAVSLGQRATISCKASQSVDYGGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDGATYYCQQSNEDPWTFGGGTKLEIK (SEQ ID NO: 8); or

The antibody is antibody 3F6, the heavy and light chain sequences of which are:

heavy chain:

VQLQQSGPSLVKPSQTLSLTCSVTGDSITNGYWNWIRKFPGNKLEYMGYISYSGTTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCSRIRPYRHLGFDYWGQGTTVTVSS(SEQ ID NO:9)；

light chain:

DIVMTQTPASLAVSLGQRATISCKASQSVDYGGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDGATYYCQQSNEDPWTFGGGTKLEIK(SEQ ID NO:10)。

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

4. A kit for detecting the content of MntC protein, said kit comprising the antibody according to any one of claims 1 to 3.

5. The kit according to claim 4, further comprising human IgG, a washing reagent, a substrate developing solution A, a substrate developing solution B, a stop solution and BSA.

6. The kit of claim 5, further comprising an enzyme-linked plate, a closure plate membrane, and/or a MntC protein standard.

7. The kit according to any one of claims 4 to 6, wherein the antibodies are antibody 3C10 and antibody 3F6, the antibody 3C10 is a detection antibody, and the antibody 3F6 is a coating antibody.

8. A method for detecting the content of MntC protein comprises the step of carrying out Elisa detection by using an antibody 3C10 and an antibody 3F6 through a double-antibody sandwich method.

9. The method of claim 8, comprising:

1) coating the enzyme conjugate plate with antibody 3F 6;

2) reacting the tested sample with an antibody 3F 6;

3) detecting and developing with antibody 3C10, and measuring absorbance at 450 nm;

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

preferably, the content range of MntC protein detected by the method is 0.046875-0.75 mu g/ml.

10. Use of the anti-MntC protein antibody according to any one of claims 1 to 3 for detecting MntC protein content.

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