CN109957006B

CN109957006B - Metabolin polypeptide artificial antigen, antibody and application

Info

Publication number: CN109957006B
Application number: CN201711407757.2A
Authority: CN
Inventors: 任培根; 张键; 姚振宇; 李健; 滕斌; 刘畅; 于向纺
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2017-12-22
Filing date: 2017-12-22
Publication date: 2021-04-20
Anticipated expiration: 2037-12-22
Also published as: CN109957006A

Abstract

The invention relates to the field of antibodies, in particular to a metabolite polypeptide artificial antigen, an antibody and application. A metabolite polypeptide artificial antigen is obtained by coupling polypeptide and carrier protein activated by amine compounds; the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1 is shown. The artificial antigen of the metabolite polypeptide provided by the invention contains cysteine, and is obtained through experiments, the polypeptide is coupled with activated macromolecular protein KLH to form a complete antigen with immunogenicity, and an immune system in a mouse body can be stimulated to generate an anti-metabolite monoclonal antibody.

Description

Metabolin polypeptide artificial antigen, antibody and application

Technical Field

The invention relates to the field of antibodies, in particular to a metabolite polypeptide artificial antigen, an antibody and application.

Background

The metabolin is a conservative sequence in a mouse osteocalcin protein molecule, and the polypeptide has antigenicity after sequence analysis. Osteocalcin is also called bone gamma-carboxyglutamic acid containing protein (BGP). The protein accumulates only after the peak bone mineralization. The content of the protein in the bone can be reduced by using the vitamin K antagonist, but the content of proline in the protein is not influenced, and the mechanical strength of the bone is not influenced. Osteocalcin is secreted from osteoblasts, and the content thereof varies with age, and the state of osteoblasts can be known from the serum osteocalcin content, which is of great significance for determining osteoporosis and the like.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first aim of the invention is to provide a metabolite polypeptide artificial antigen, the polypeptide contains cysteine, the polypeptide is obtained by experiment, the polypeptide is coupled with activated macromolecular protein KLH to form complete antigen with immunogenicity, and the complete antigen can stimulate the immune system in a mouse to produce anti-metabolite monoclonal antibodies.

The second object of the present invention is to provide an antibody against the above antigen, which can be used for detecting a polypeptide molecule related to a metabolite in mouse serum.

The third purpose of the invention is to provide a kit containing the antibody, which facilitates the detection of polypeptide molecules related to the metabolin.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a metabolite polypeptide artificial antigen is obtained by coupling polypeptide and carrier protein activated by amine compounds; the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1 is shown.

The artificial antigen of the metabolite polypeptide provided by the invention contains cysteine, and is obtained through experiments, the polypeptide is coupled with activated macromolecular protein KLH to form a complete antigen with immunogenicity, and an immune system in a mouse body can be stimulated to generate an anti-metabolite monoclonal antibody.

Further, the carrier protein is apocrin.

Further, the amine compound is maleimide.

The artificial antigen of the metabolite polypeptide provided by the invention can be prepared by the following method:

as shown in SEQ ID NO: dissolving the metabolin polypeptide shown in the step 1 to obtain a polypeptide solution with the metabolin polypeptide concentration of 6-9 mg/ml;

dissolving the activated carrier protein to obtain a carrier protein solution with the activated carrier protein concentration of 4-6 mg/ml;

mixing and coupling the polypeptide solution and the carrier protein solution in a volume ratio of 1: 2-3;

and separating and purifying the coupled product to obtain the artificial antigen of the metabolite polypeptide.

The preparation method of the artificial antigen of the metabolite polypeptide provided by the invention is simple and easy to implement, and the coupling efficiency is high and is more than 90%.

Further, the dissolving solvent of the metabolite polypeptide is 20 plus or minus 1mM sodium phosphate buffer solution, which also contains 230 plus or minus 5mM NaCl, 2 plus or minus 0.2mM EDTA, 80 plus or minus 2mM sucrose, and pH6.5-6.7.

Further, the solvent for dissolving the activated carrier protein is 20 + -1 mM sodium phosphate buffer solution, which also contains 100 + -5 mM EDTA, 80 + -2 mM sucrose, pH 6.5-6.7.

Further, the coupling conditions were either 2 hours at room temperature or overnight at 2-8 ℃ with stirring.

The invention also provides an antibody prepared from the artificial antigen of the metabolite polypeptide.

Further, the antibody is a monoclonal antibody.

Further, the subtype of the monoclonal antibody is G2 a.

Further, the titer of the monoclonal antibody was 1: 4000.

The invention also provides the application of the antibody in detecting the polypeptide fragment of the metabolin existing in serum or tissues.

The invention also provides a kit containing the antibody.

Further, the kit is an ELASA kit.

The ELISA kit provides a specific serological index for diagnosing and identifying adult bone and related diseases so as to improve the accuracy of diagnosis.

Furthermore, the kit also comprises any one or more of an enzyme label plate, a positive and negative reference substance, a secondary antibody solution, a developing solution, a reaction stopping solution, a concentrated washing solution, a sample diluent, a plate sealing film, a tin foil bag, a product instruction and a product quality inspection report.

Compared with the prior art, the invention has the beneficial effects that:

(1) the artificial antigen of the metabolite polypeptide provided by the invention contains cysteine, and is obtained through experiments, the polypeptide is coupled with activated macromolecular protein KLH to form a complete antigen with immunogenicity, and an immune system in a mouse body can be stimulated to generate an anti-metabolite monoclonal antibody.

(2) The monoclonal antibody provided by the invention can be used for detecting polypeptide molecules related to the metabolin in mouse serum, and has good specificity and sensitivity.

(3) The invention provides a kit containing the antibody, which facilitates the detection of polypeptide molecules related to the metabolin.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a schematic representation of the structure of maleimide-activated KLH of example 1 of the present invention;

FIG. 2 is a line graph showing the titer results of monoclonal antibody No. 21 in example 3 of the present invention;

FIG. 3 is a graph showing the results of recognition specificity of a monoclonal antibody (21#) to a metabolite polypeptide in example 4 of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

For better understanding of the present invention, the preparation process of monoclonal antibodies is described as a general method unless otherwise specified. In the embodiment, the reagent materials are all conventional biochemical reagents unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged. All phosphate buffers used in the examples were 0.01M PBS buffer at pH 7.4. The carbonate buffers used in the examples were all 0.05M sodium carbonate buffer at pH 9.6. Bovine serum albumin is abbreviated as BSA, and spoonful hemocyanin is abbreviated as KLH.

Example 1

A. Conjugation of maleimide-activated KLH to Polypeptides

1. Opening a tube of maleimide activated KLH (as shown in FIG. 1) and adding 1ml of 20mM sodium phosphate buffer (230mM NaCl, 2mM EDTA, 80mM sucrose, pH 6.6) by reconstituting the maleimide activated KLH to give a 5mg/ml solution ready for use;

2. 20mM sodium phosphate buffer (100mM EDTA, 80mM sucrose, pH 6.6) was prepared as a coupling buffer;

3. 4mg of polypeptide was dissolved in 0.5ml of coupling buffer to give a polypeptide solution, 50. mu.l of the polypeptide solution was left for determination of coupling efficiency (total cys) and stored at 2-8 ℃.

4. Immediately mixing 0.5ml of polypeptide solution and 1ml of maleic amide activated KLH solution in a reaction tube, stirring, and keeping the mixture at room temperature (15-25 ℃) for 2 hours or at 2-8 ℃ overnight (8-15 hours) to obtain polypeptide coupled with KLH, which is named as NH 2-KLH;

5. 100 μ l of the coupling reaction was retained for determination of coupling efficiency (free cys).

B. Isolation of KLH conjugates

1. Adding 0.01% sodium azide into 1L PBS, and storing for a long time;

2. fixing the Sephadex G-25M gel column on a proper beaker;

3. taking off the cover of the gel column, cutting the bottom end of the gel column to allow excessive liquid to flow out, and prohibiting the liquid in the column from flowing dry;

4. equilibrating the gel column with 30ml PBS, and storing to 2-8 ℃ after covering the upper and lower covers if the gel column is not used immediately;

5. adding the reacted liquid into the gel column, and collecting the liquid flowing out from the lower end;

6. washing with 10ml PBS, collecting eluate, collecting 0.5-1.0 ml each time, and measuring absorbance at 280 nm;

7. collecting the protein-containing eluate, and freezing at-20 deg.C.

Standard curve for C cysteine

DTNB assay solution (0.1 mM): prepared by adding 10mMDTNB standard solution with 1 volume and 0.25M Tris buffer solution with 99 volumes, and prepared on site;

2. dissolving 32mg of L-cysteine hydrochloride monohydrate in 1ml of double distilled water, and performing gradient dilution in a range of 0.4-0.04 mg/ml;

3. adding 50 mul of pre-diluted cysteine standard solution into a test tube, and adding 50 mul of double distilled water into a blank tube;

4. adding 0.1ml of double distilled water and 0.75ml of pH8.0DTNB buffer solution into all the tubes, immediately adding 0.1ml of DTNB reagent solution to make the total volume be 1ml, and uniformly mixing;

5. the absorbance values were measured at 412nm and a standard curve was generated and repeated three times.

Determination of the coupling efficiency

1. Add 50. mu.l of polypeptide solution (total cys) to 0.1ml of double distilled water;

2. the following 50 μ l solution was added to the test tube: DTNB buffer (blank), diluted polypeptide sample (total cys, KLH binding), polypeptide-KLH (free cys, KLH binding), diluted polypeptide sample (total cys, BSA binding), polypeptide-BSA (free cys, BSA binding);

3. adding 0.1ml of double distilled water and 0.75ml of DTNB buffer solution with the pH value of 8.0 into all the test tubes, immediately adding 0.1ml of DTNB reagent solution with the final volume of 1ml, and uniformly mixing;

4. measuring the absorbance value at 412nm, and if the absorbance value is greater than 1.4, diluting the sample and repeating the test;

5. calculating the formula: coupling efficiency (%) (Total cys-free cys)/Total cys Gamma 100%

The coupling efficiency obtained was above 90%.

Example 2

Preparation of polypeptide metabolite monoclonal antibody

BALB/c female mice were purchased from the center of laboratory animals in Guangdong province.

A animal immunization

The NH2-KLH immunogen prepared in example 1 was used to immunize BALB/c female mice, the primary immunization was performed in an amount of 60. mu.g protein/mouse, and 4 SPF Balb/c female mice, numbered 1-4, were immunized subcutaneously, boosted every 2 weeks, as follows:

the first subcutaneous booster immunization, the immunization amount is 30 mug protein/mouse;

a second boost subcutaneously with an immune dose of 30 μ g protein/mouse;

a third subcutaneous booster, the immune amount is 30 mug protein/mouse;

the fourth boost was performed subcutaneously with an amount of 30. mu.g protein/mouse.

Blood was collected from each mouse orbit and serum titer was measured.

And (3) detecting the immune titer:

the method comprises the following steps: coating with NH2-BSA 2. mu.g/ml at 4 deg.C overnight; 2% of milk, sealing for 2h at 37 ℃; serum was diluted in a 2-fold gradient starting at 200-fold, with a blank control (blank) in PBS and a negative control (negative) at 200-fold dilution in negative serum. The titer is the dilution corresponding to the minimum OD reading greater than the maximum OD/2.

The titer results are shown in table 1.

TABLE 1 potency results

As can be seen from the measured data, the potency of the mouse No. 4 is excellent, and therefore, the impact 4# mouse was selected for the cell fusion experiment.

B cell fusion assay

Taking the mouse spleen cell and SP2/0 cell, fusing by PEG method. The fused cells were cultured in semi-solid medium (containing HAT) for selection.

1. Experimental equipment

Sterilized surgical instruments: three scissors, three tweezers, a cell sieve, an inner core of an injector and a plate. Wet box, 2 500ml beakers, 250 ml centrifuge tubes, 3 15ml centrifuge tubes.

2. Experimental reagent

IMDM medium; IMDM complete medium (15% serum); 2.2% methylcellulose, manufacturer SIGMA, cat #: M0262-100G; newborn bovine serum: 10 ml; PEG1500, manufacturer Roche, cat #: 78364, respectively; HAT: manufacturer Sigma, cat #: h0262-10 VL.

3. Procedure of fusion experiment

a) Well conditioned sp2/0 cells were gently blown off the flask wall and aspirated into a 50ml centrifuge tube.

b) 50 μ g of NH2-KLH immunogen was used, and #4 mice were impacted intraperitoneally, sacrificed by cervical dislocation, and soaked in 75% ethanol for 5 min.

c) A small amount of serum-free IMDM was poured into the dish, and the cell sieve and plunger were placed in the dish. The spleen of the mouse was removed with scissors and forceps and placed on the cell sieve. The spleen was gently crushed sufficiently with the inner core of the syringe, and the crushed cells were aspirated into a centrifuge tube filled with sp2/0 and centrifuged at 1500r/min for 5 min.

d) The thymus of the mouse was removed with scissors and forceps and ground. The milled thymocytes are put into a 15ml centrifuge tube, 1ml HAT is added, and the obtained product is put into an incubator for standby.

e) And (3) pouring off the supernatant of the centrifuged cells, gently and uniformly blowing the cells by serum-free IMDM, and centrifuging at 1500r/min for 5 min.

f) The centrifuged cell supernatant was discarded as much as possible. Beating the bottom of the centrifuge tube to suspend the cells sufficiently, putting the centrifuge tube into warm water at 37 ℃, slowly adding 1ml of PEG within 1 minute, and standing in warm water for 1min after adding. Then 2ml of serum free IMDM was added slowly over 2min followed by 8ml of serum free IMDM over 2 min. Centrifuging at 1000r/min for 5 min.

g) The supernatant was decanted, 10ml of serum was added, the cells were carefully blown down and poured into the previously prepared thymocytes. Then 25ml of sterilized semi-solid medium was added and mixed well. Then poured into 30 cell culture dishes uniformly. The cell culture dish is put into a wet box and then put into an incubator for culture.

C selected clone

10X 93 cell monoclonals were picked and cultured in 96-well cell culture plates (previously plated with thymocytes, 100. mu.l/well).

D monoclonal cell screening

(1) Experimental reagent

Coating liquid: sodium carbonate-sodium bicarbonate buffer, pH 9.6;

sealing liquid: 2% milk in PBS wash;

color development liquid: 1% solution A + 10% solution B (solution A: 1% TMB in DMSO; solution B: 0.1% H)₂O₂in citrate buffer);

stopping liquid: 2M sulfuric acid;

secondary antibody: goat anti-mouse IgG/HRP

(2) Experimental procedure

1) Diluting "NH 2-BSA" with coating solution to a final concentration of 2. mu.g/ml, 100. mu.l/well, 4 deg.C overnight; then washed 3 times with washing solution.

2) Sealing with 2% milk sealing solution, 200 μ l/hole, incubating at 37 deg.C for 2 h; then washed 3 times with washing solution.

3) Adding primary antibody (cell culture supernatant), negative control (SP2/0 culture supernatant), blank control (PBS), and positive control (positive serum PBS 200 times diluted), all 100 μ l/well, incubating at 37 deg.C for 1 h; then washed 3 times with washing solution.

4) Adding PBS (pH7.4) to dilute 20000 times of secondary antibody, 100 μ l/hole, incubating at 37 deg.C for 1 h; after removal, the plate was washed 3 times with a washing solution.

5) Developing with 100 mul/hole developing liquid for about 5 min.

6) Stop with 50. mu.l stop buffer per well.

7) Absorbance was measured at both wavelengths (450, 630) and the stored data recorded as shown in table 2. In Table 2, the different plates are labeled NO 1-10.

TABLE 2 OD values determined for different plates

And (3) coating a plate by using 'NH 2-BSA', adopting an ELISA method for the selected clones, and performing primary screening on 36 positive hybridoma cell strains except a positive control group with OD values larger than 0.15.

E secondary screening of monoclonal cells

And (3) coating the 36 positive cell strains with NH2-BSA again, and performing secondary screening by adopting an ELISA method to obtain 17 positive hybridoma cell strains.

F monoclonal cell subset identification

The method comprises the following steps:

1. experimental reagent

Coating antibody: southern Biotech; sealing liquid: 2% BSA + 3% sucrose in PBS;

color developing solution 0.2ml of solution A + 10. mu.l of 30% H₂O₂in 10ml of solution B (solution A: 15mg/ml ABTS in H)₂O; and B, liquid B: citrate buffer, ph 4.0); each type was a subclass of secondary antibodies.

2. Experimental procedure

a) The coated antibody was diluted to 0.5. mu.g/ml with 100mM PBS (pH7.4), 0.1ml per well, 4 ℃ overnight.

b) PBS-T washing 2 times, adding 200. mu.l of blocking solution to each well, and incubating at 37 ℃ for 2 h.

c) PBS-T was washed 3 times, 100. mu.l of hybridoma supernatant was added to each well, and incubated at 37 ℃ for 1 h.

d) PBS-T wash 3 times, with blocking solution 1:1000 (κ, λ) or 1: 0.1ml of HRP-labeled antibody diluted 2000 (others) per well was added to the appropriate wells, respectively, and incubated at 37 ℃ for 1 h.

e) PBS-T washing 3 times; add 50. mu.l substrate solution to each well, measure absorbance at dual wavelengths (450, 630) over 10-20min, and record the stored data.

And (4) carrying out subclass identification on the 17 screened positive cell strains to finally obtain 14 IgG positive hybridoma cell strains. Specifically, the results are shown in tables 3 and 4.

TABLE 317 subtype data determined for positive cell lines

Positive hybridoma cell strain of IgG of table 414 strain

Example 3

Monoclonal antibody titer determination-ELISA method

1. Antigen coating: diluting the polypeptide antigen to the concentration of 2 mu g/ml, adding the diluted polypeptide antigen into a 96-well plate, wherein each well is 100 mu l, and the polypeptide antigen is wrapped in a wet box at 4 ℃ overnight;

2. washing: washing the coated ELISA plate with PBS buffer solution containing 0.05% Tween-20 at a concentration of 200 μ l/well for 3 times and 5 min/time;

3. and (3) sealing: blocking a 96-well plate by using 1% BSA solution, blocking the plate at 250 mu l/well in a wet box at 37 ℃ for 2 hours;

4. washing: washing method as above;

5. primary antibody incubation: diluting rabbit serum polyclonal antibody with PBS buffer solution according to the ratio of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000 and 1:64000, setting blank control and negative serum control, 100 μ l/hole, and incubating at 37 deg.C for 1 h;

6. washing: washing method as above;

7. and (3) secondary antibody incubation: diluting an HRP-labeled goat anti-mouse IgG antibody with PBS (phosphate buffer solution) according to a ratio of 1:5000, incubating the diluted antibody at 100 mu l/hole in a wet box at 37 ℃ for 1 h;

8. washing: washing method as above;

9. adding a substrate: adding TMB into a 96-well plate according to 100 mul/well, and reacting for 20min in a 37 ℃ wet box in a dark place;

10. and (3) terminating the reaction: with 2M H₂SO₄Adding a 96-well plate according to 50 mu l/well to terminate the reaction, and measuring the absorbance value by using an enzyme-labeling instrument at the wavelength of 450nm, wherein OD positive/OD negative is more than 2.1, which is a serum titer determination standard.

11. The titer results of the monoclonal antibody numbered 21 obtained by the screening are shown in FIG. 2.

The antibody titer was obtained as 1: 4000.

example 4

Western blot method for identifying specificity of monoclonal antibody

1. Preparation of protein samples:

the silanized tubes were autoclaved, added with a mixed solution of 0.1% BSA and 0.9% NaCl and soaked overnight, the next day the solution was aspirated and the tubes allowed to air dry, and polypeptides were added to the air dried tubes and frozen. The tips were also soaked overnight with a mixed solution of 0.1% BSA and 0.9% NaCl the next day with ddH₂And (4) washing with water for 3 times, then autoclaving and drying for later use.

2.SDS-PAGE

And selecting a proper glass plate to wipe clean for later use. The prepared separation gel is added after the edge sealing is carried out by 1.5 percent agar, a space required by the concentrated gel is reserved, and 1ml of deionized water is added to cover the gel to isolate the gel from air, so as to facilitate the condensation of the separation gel. After the gel was polymerized, the cover water was decanted, the top of the gel was rinsed several times with deionized water to remove unpolymerized acrylamide, and the remaining liquid on the top of the gel was blotted with filter paper. Preparing concentrated gel, carefully and quickly injecting the concentrated gel into the upper layer of the separation gel, immediately inserting a clean comb which cannot generate bubbles, and standing for coagulation.

After the polymerization of the concentrated gel was completed, the electrophoresis buffer was added and the comb was carefully pulled out. The prepared cell total protein samples are loaded in sequence. The sample wells without loading were loaded with 1 Xloading buffer. And (3) performing electrophoresis from the negative electrode to the positive electrode at a constant voltage, wherein the voltage of the bromophenol blue in the concentrated gel is 80V, when the bromophenol blue enters the separation gel, the voltage is increased to 120V, the electrophoresis is continued until the bromophenol blue reaches the bottom of the separation gel, the power supply is turned off, and the gel is detached.

3.Western-blot

1) Cutting a PVDF membrane and a plurality of pieces of filter paper which have the same size as the gel, soaking the filter paper and the gel after electrophoresis in an electrotransfer solution, soaking the PVDF membrane in methanol for 10 seconds, washing the PVDF membrane with deionized water, and then soaking the PVDF membrane in the electrotransfer solution;

2) opening a plastic bracket of the protein electrophoresis apparatus, placing a piece of sponge on each side, placing the sponge in the order of filter paper-gel-PVDF membrane-filter paper from the cathode to the anode, removing bubbles, clamping the plastic bracket, placing the plastic bracket into an electric rotating tank, switching on a power supply, and performing constant-current 400mA electric rotation for 1h in an ice bath;

3) sealing the electrotransformed sample membrane in TBST containing 5% skimmed milk powder at room temperature for 1 h;

4) placing the membrane in primary antibody with appropriate dilution times, and gently shaking at room temperature for 2-3h, or reacting at 4 deg.C overnight;

5) washing the membrane twice by TBST;

6) placing the membrane in a second antibody, and gently shaking for 1-2h at room temperature, or reacting overnight at 4 ℃;

7) washing the membrane twice by TBST;

4. development

Absorbing excessive moisture on the membrane, reacting the membrane in ECL reaction solution for 1min, absorbing moisture on the membrane, and developing the sample membrane by X-ray tabletting in a dark room.

That is, the results of the Western blot method for verifying the recognition specificity of the monoclonal antibody (21#) for the above-mentioned metabolin polypeptide are shown in FIG. 3.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

SEQUENCE LISTING

<110> Shenzhen advanced technology research institute

<120> artificial antigen of metabolite polypeptide, antibody and application

<130> 2010

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 16

<212> PRT

<213> Artificial sequence

<400> 1

Cys Tyr Leu Gly Ala Ser Val Pro Ser Pro Asp Pro Leu Glu Pro Thr

1 5 10 15

Claims

1. A metabolite polypeptide artificial antigen is characterized in that the metabolite polypeptide artificial antigen is obtained by coupling polypeptide and carrier protein activated by amine compounds; the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1 is shown.

2. The artificial antigen of a metabolite polypeptide according to claim 1, wherein the carrier protein is apocrin.

3. The artificial antigen of a metabolite polypeptide according to claim 2, wherein said amine-based compound is maleimide.