阅读说明：本技术 一种制备肌红蛋白配对单抗的方法及由该方法制备得到的配对单抗 (Method for preparing myoglobin pairing monoclonal antibody and pairing monoclonal antibody prepared by method ) 是由 熊宁 张晓杰 李光 于 2019-09-18 设计创作，主要内容包括：本发明公开了一种制备肌红蛋白配对单抗的方法及由该方法制备得到的配对单抗。所述方法包括分泌抗肌红蛋白单克隆抗体的杂交瘤细胞的制备以及筛选配对单抗的步骤。本发明采用羊抗MYO纯化IgG预包被酶标板,再加入MYO抗原,对MYO抗原进行间接包被。与直接包被MYO抗原相比,间接包被的MYO抗原暴露出的位点发生了变化,突破了之前的表位偏好性,有利于筛选到针对新的MYO抗原表位的单抗,从而提高了MYO配对单抗的筛选几率。(The invention discloses a method for preparing a myoglobin pairing monoclonal antibody and the pairing monoclonal antibody prepared by the method. The method comprises the steps of preparing hybridoma cells secreting the anti-myoglobin monoclonal antibody and screening paired monoclonal antibodies. The invention adopts sheep anti-MYO purified IgG to pre-coat the ELISA plate, and then MYO antigen is added to indirectly coat the MYO antigen. Compared with direct coating of MYO antigen, the site exposed by indirect coating of MYO antigen is changed, the previous epitope preference is broken through, monoclonal antibody aiming at new MYO antigen epitope is screened, and thus the screening probability of MYO paired monoclonal antibody is improved.)

1. A method for preparing a myoglobin pairing monoclonal antibody is characterized by comprising the following steps: comprises the steps of preparing hybridoma cells secreting anti-myoglobin monoclonal antibodies and screening paired monoclonal antibodies; when the hybridoma secreting the anti-myoglobin monoclonal antibody is screened by using an indirect ELISA method, a goat anti-MYO purified IgG coats an enzyme label plate, then a MYO antigen is added, then hybridoma cell culture supernatant and goat anti-mouse IgG-HRP are sequentially added, a color development reading plate is performed to obtain the hybridoma secreting the anti-myoglobin monoclonal antibody, and then monoclonal antibodies capable of being paired are screened from the hybridoma.

2. The method of claim 1, wherein: the preparation of hybridoma cells secreting anti-myoglobin monoclonal antibodies was carried out according to the following steps:

1) cell fusion: separating spleen of mouse with immune myoglobin antigen, preparing B cell suspension, mixing with SP2/0 cell, centrifuging, and performing cell fusion culture;

2) enzyme label plate coating: na for goat anti-MYO purified IgG₂CO₃-NaHCO₃Diluting the buffer solution, adding the diluted buffer solution into an enzyme label plate, coating the enzyme label plate at 2-8 ℃ overnight, and washing the plate for 1 time by PBST;

3) adding MYO antigen: adding MYO antigen into the ELISA plate after PBST, incubating at 37 ℃, and washing the plate for 1 time by PBST;

4) adding hybridoma cell culture supernatant: aseptically sampling the fusion cell culture supernatant, adding the fusion cell culture supernatant into an enzyme label plate, incubating at 37 ℃, and washing the plate for 1 time by PBST;

5) addition of goat anti-mouse IgG-HRP: diluting goat anti-mouse IgG-HRP to working concentration with PBST, incubating at 37 ℃, and washing the plate for 2 times with PBST;

6) developing and reading the plate: addition of H₂O₂Developing the substrate with-TMB at 37 ℃ in the absence of light, adding 2M H₂SO₄And (3) stopping color development, placing the mixture in an enzyme labeling instrument, and reading a plate at 450nm/630nm double-wavelength to obtain the hybridoma secreting the anti-myoglobin monoclonal antibody.

3. The method of claim 2, wherein: spleen of immunized myoglobin antigen mouse is separated, B cell suspension is prepared, mixed with SP2/0 cell and centrifuged, and PEG is used₁₄₅₀Cell fusion was performed, and culture was performed in DMEM medium containing 2% v/v HAT and 10% v/v fetal bovine serum, and on day 4 after fusion, the medium was changed, and the medium was gently aspirated, and changed to DMEM medium containing 1% v/v HT and 10% v/v fetal bovine serum.

4. The method of claim 2, wherein: step 2) goat anti-MYO purified IgG 50mM Na₂CO₃-NaHCO₃Buffer (pH9.6) diluted to 5ug/ml, 100 ul/well, 2-8 ℃ overnight coating, PBST washing plate 1Secondly; in step 3), the MYO antigen is diluted to 1ug/ml and 100 ul/well by PBST, incubated at 37 ℃ for 30min, and washed by PBST for 1 time.

5. The method of claim 2, wherein: in step 5) the goat anti-mouse IgG-HRP was diluted with PBST to working concentration, 100 ul/well, incubated at 37 ℃ for 30min, and washed 2 times with PBST.

6. The method of claim 1 or 2, wherein: the screening of the paired monoclonal antibodies is carried out according to the following steps:

1) coating with purified monoclonal antibody: purifying an ascites monoclonal antibody of a hybridoma cell secreting the anti-myoglobin monoclonal antibody, diluting the purified monoclonal antibody, adding the diluted purified monoclonal antibody into a new enzyme label plate, coating overnight, and washing the plate for 1 time by PBST;

2) loading MYO antigen: adding MYO antigen into the ELISA plate after PBST, incubating at 37 ℃, and washing the plate for 1 time by PBST;

3) loading HRP enzyme-labeled monoclonal antibody: diluting the HRP enzyme-labeled monoclonal antibody matched with the coated monoclonal antibody by PBST, adding the diluted HRP enzyme-labeled monoclonal antibody into an enzyme-labeled plate, incubating at 37 ℃, and washing the plate for 2 times by PBST;

4) developing and reading the plate: addition of H₂O₂Developing the substrate with-TMB at 37 ℃ in the absence of light, adding 2M H₂SO₄And (3) stopping color development, placing the sample in an enzyme-labeling instrument, reading a plate with 450nm/630nm double-wavelength, and determining that the sample can be matched if the wavelength of 450nm/630nm is more than or equal to 1.0.

7. The method of claim 6, wherein: 50mM Na is used for purifying the monoclonal antibody in the step 1)₂CO₃-NaHCO₃Buffer (pH9.6) diluted to 5ug/ml, 100 ul/hole, 2-8 ℃ overnight coating, PBST washing plate 1 times.

8. The method of claim 6, wherein: diluting MYO antigen with PBST in step 2) to 1ug/ml and 100 ul/well, incubating at 37 deg.C for 30min, and washing plate with PBST for 1 time; in the step 3), the HRP enzyme-labeled monoclonal antibody matched with the coated monoclonal antibody is diluted to the working concentration by PBST, 100 ul/hole is incubated for 30min at 37 ℃, and the PBST is used for washing the plate for 2 times.

9. Myoglobin paired monoclonal antibody screened according to the method of any one of claims 1-8, wherein preferably, the paired monoclonal antibody is secreted by two hybridoma cells, i.e., MYO 3F1 and MYO 5A 8;

wherein, the hybridoma cell strain MYO 3F1 is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.18184 and the preservation time of 2019, 7 months and 5 days;

wherein, the hybridoma cell strain MYO 5A8 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.18185 and the preservation time of 2019, 7 months and 5 days.

10. Use of the myoglobin pairing mab of claim 9 in the preparation of a reagent for detecting myoglobin antigen, wherein preferably said reagent is suitable for ELISA double antibody sandwich assay.

Technical Field

The invention relates to a preparation method of a monoclonal antibody, in particular to a method for preparing a myoglobin pairing monoclonal antibody, and also relates to the pairing monoclonal antibody prepared by the method. The invention belongs to the field of biotechnology.

Background

Myoglobin (Myoglobin, MYO) is a heme protein consisting of a polypeptide chain of 153 amino acids and an iron-containing heme prosthetic group, is present in tissues such as skeletal muscle and cardiac muscle of mammals, and is mainly used for storing and distributing oxygen. Because Mb has a small molecular weight, can be quickly released from damaged cells, the blood concentration of Mb rapidly rises within 1-3 hours after AMI (acute myocardial infarction) disease occurs, the peak value is reached within 6-7 hours, and nearly all MYO of patients suffering from Acute Myocardial Infarction (AMI) rises within l2 hours, and the rise range is larger than that of each myocardial enzyme, so that the Mb can be used as an early diagnosis marker of AMI, and the myocardial infarction area and thrombolytic curative effect can be estimated according to the marker.

At present, the domestic MYO detection methods include Radioimmunoassay (RIA), ELISA, fluorescence immunoassay and the like. The ELISA has the advantages of high sensitivity, strong specificity, accurate quantification, simple operation, suitability for simultaneous detection of multiple specimens, no need of special equipment and instruments and the like, and has wide application prospect. High-quality MYO monoclonal antibody (mAb) has been prepared abroad, and a MYO double-antibody sandwich ELISA kit is successfully developed, but the kit is expensive and has higher detection cost. The principle of ELISA double antibody sandwich method (enzyme linked immunosorbent assay-sandwich technique) is that specific antibody is combined on solid phase carrier to form solid phase antibody, then combined with corresponding antigen in serum to be detected to form immune complex, after washing, enzyme labeled antibody is added to combine with antigen in immune complex to form enzyme labeled antibody-antigen-solid phase antibody complex, substrate is added for color development to judge antigen content, and in the process of double antibody sandwich matching, only 2 monoclonal antibodies which aim at different epitopes and have far space positions are possible to form matching. Therefore, the key point of preparing the MYO double-antibody sandwich ELISA kit lies in obtaining high-quality paired monoclonal antibodies, but the preparation difficulty of the myoglobin paired monoclonal antibodies is high, and the main reason is that when the myoglobin is coated by a conventional method, the dominant sites exposed on an enzyme label plate by the antigen are excessively concentrated, so that the identification sites of the screened monoclonal antibodies are also concentrated on a few MYO sites, and the screened monoclonal antibodies have strong preference, and therefore, paired monoclonal antibody cell strains are difficult to screen.

In order to screen more MYO paired monoclonal antibodies, the invention adopts sheep anti-MYO purified IgG to pre-coat an enzyme label plate, and then MYO antigen is added to indirectly coat the MYO antigen. Compared with direct coating of MYO antigen, the site exposed by indirect coating of MYO antigen is changed, the previous epitope preference is broken through, monoclonal antibody aiming at new MYO antigen epitope is screened, and thus the screening probability of MYO paired monoclonal antibody is improved.

Disclosure of Invention

The invention aims to provide a novel method for preparing a myoglobin pairing monoclonal antibody, which can overcome the defects of concentrated recognition sites, strong preference and the like of the monoclonal antibody screened by the existing method.

In order to achieve the purpose, the invention adopts the following technical means:

the invention relates to a method for preparing a myoglobin pairing monoclonal antibody, which comprises the steps of preparing hybridoma cells secreting an anti-myoglobin monoclonal antibody and screening the pairing monoclonal antibody; when the hybridoma secreting the anti-myoglobin monoclonal antibody is screened by using an indirect ELISA method, a goat anti-MYO purified IgG coats an enzyme label plate, then a MYO antigen is added, then hybridoma cell culture supernatant and goat anti-mouse IgG-HRP are sequentially added, a color development reading plate is performed to obtain the hybridoma secreting the anti-myoglobin monoclonal antibody, and then monoclonal antibodies capable of being paired are screened from the hybridoma.

In the method of the present invention, preferably, the hybridoma secreting an anti-myoglobin monoclonal antibody is prepared by the following steps:

1) cell fusion: separating spleen of mouse with immune myoglobin antigen, preparing B cell suspension, mixing with SP2/0 cell, centrifuging, and performing cell fusion culture;

2) enzyme label plate coating: na for goat anti-MYO purified IgG₂CO₃-NaHCO₃Diluting the buffer solution, adding the diluted buffer solution into an enzyme label plate, coating the enzyme label plate at 2-8 ℃ overnight, and washing the plate for 1 time by PBST;

3) adding MYO antigen: adding MYO antigen into the ELISA plate after PBST, incubating at 37 ℃, and washing the plate for 1 time by PBST;

4) adding hybridoma cell culture supernatant: aseptically sampling the fusion cell culture supernatant, adding the fusion cell culture supernatant into an enzyme label plate, incubating at 37 ℃, and washing the plate for 1 time by PBST;

5) addition of goat anti-mouse IgG-HRP: diluting goat anti-mouse IgG-HRP to working concentration with PBST, incubating at 37 ℃, and washing the plate for 2 times with PBST;

6) developing and reading the plate: addition of H₂O₂Developing the substrate with-TMB at 37 ℃ in the absence of light, adding 2M H₂SO₄And (3) stopping color development, placing the mixture in an enzyme labeling instrument, and reading a plate at 450nm/630nm double-wavelength to obtain the hybridoma secreting the anti-myoglobin monoclonal antibody.

Among them, preferably, spleen of a mouse immunized with myoglobin antigen is isolated, B cell suspension is prepared, mixed with SP2/0 cells and centrifuged, cell fusion is performed with PEG1450, culture is performed with DMEM medium containing 2% v/v HAT and 10% v/v fetal bovine serum, the medium is changed on day 4 after fusion, the original medium is gently aspirated, and culture is performed with DMEM medium containing 1% v/v HT and 10% v/v fetal bovine serum.

Wherein, preferably, the goat anti-MYO purified IgG in step 2) is treated with 50mM Na₂CO₃-NaHCO₃Diluting buffer solution (pH9.6) to 5ug/ml, 100 ul/well, coating overnight at 2-8 deg.C, and washing plate with PBST for 1 time; in step 3), the MYO antigen is diluted to 1ug/ml and 100 ul/well by PBST, incubated at 37 ℃ for 30min, and washed by PBST for 1 time.

Among them, preferably, in step 5), the goat anti-mouse IgG-HRP was diluted with PBST to a working concentration of 100 ul/well, incubated at 37 ℃ for 30min, and washed 2 times with PBST.

In the method of the present invention, preferably, the screening of the matsumab is performed according to the following steps:

1) coating with purified monoclonal antibody: purifying an ascites monoclonal antibody of a hybridoma cell secreting the anti-myoglobin monoclonal antibody, diluting the purified monoclonal antibody, adding the diluted purified monoclonal antibody into a new enzyme label plate, coating overnight, and washing the plate for 1 time by PBST;

2) loading MYO antigen: adding MYO antigen into the ELISA plate after PBST, incubating at 37 ℃, and washing the plate for 1 time by PBST;

3) loading HRP enzyme-labeled monoclonal antibody: diluting the HRP enzyme-labeled monoclonal antibody matched with the coated monoclonal antibody by PBST, adding the diluted HRP enzyme-labeled monoclonal antibody into an enzyme-labeled plate, incubating at 37 ℃, and washing the plate for 2 times by PBST;

4) developing and reading the plate: addition of H₂O₂Developing the substrate with-TMB at 37 ℃ in the absence of light, adding 2M H₂SO₄And (3) stopping color development, placing the sample in an enzyme-labeling instrument, reading a plate with 450nm/630nm double-wavelength, and determining that the sample can be matched if the wavelength of 450nm/630nm is more than or equal to 1.0.

Wherein, preferably, 50mM Na is used for purifying the monoclonal antibody in the step 1)₂CO₃-NaHCO₃Buffer (pH9.6) diluted to 5ug/ml, 100 ul/hole, 2-8 ℃ overnight coating, PBST washing plate 1 times.

Wherein, preferably, the MYO antigen in the step 2) is diluted to 1ug/ml and 100 ul/well by PBST, incubated for 30min at 37 ℃, and washed by PBST for 1 time; in the step 3), the HRP enzyme-labeled monoclonal antibody matched with the coated monoclonal antibody is diluted to the working concentration by PBST, 100 ul/hole is incubated for 30min at 37 ℃, and the PBST is used for washing the plate for 2 times.

Furthermore, the invention also provides myoglobin pairing monoclonal antibody obtained by screening according to any one of the methods, wherein preferably, the pairing monoclonal antibody is secreted and produced by two hybridoma cells, namely MYO 3F1 and MYO 5A 8;

wherein, the hybridoma cell strain MYO 3F1 is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.18184 and the preservation time of 2019, 7 months and 5 days;

wherein, the hybridoma cell strain MYO 5A8 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.18185 and the preservation time of 2019, 7 months and 5 days.

Furthermore, the invention also provides the application of the myoglobin pairing monoclonal antibody in preparing a reagent for detecting the myoglobin antigen, wherein preferably, the reagent is suitable for an ELISA double-antibody sandwich method.

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

1. because the coating antibody and the enzyme-labeled secondary antibody are essentially IgG from the same genus, the goat anti-MYO purified IgG pre-coating plate is adopted, so that the MYO antigen can be captured, indirect coating is completed, and the normal use of goat anti-mouse IgG-HRP (enzyme-labeled secondary antibody) is not influenced;

2. the indirect coating method of antibody pre-coating changes the preference of exposing the antigen epitope by the conventional coating method, obtains monoclonal antibody cell strains aiming at different MYO epitopes, improves the pairing screening probability, and finally successfully obtains paired cell strains.

Drawings

FIG. 1A is a schematic representation of MYO antigen;

FIG. 1B is a conventional screening method: direct coating of MYO antigen, and the subsequent steps are performed according to indirect ELISA: 1) Adding the supernatant; 2) enzyme-labeled secondary antibody; 3) developing color;

FIG. 1C shows a screening method of the present invention: coating goat anti-MYO purified IgG, adding MYO antigen, and performing indirect ELISA operation according to the following steps: 1) adding the supernatant; 2) enzyme-labeled secondary antibody; 3) and (4) developing color.

FIG. 1D is a schematic diagram of MYO mab pairing;

FIG. 2 shows the result of the pairing of mAb MYO-3F1 with MYO-5A 8.

Strain preservation information:

the myoglobin monoclonal antibody hybridoma cell strain is named as MYO 3F1 and classified and named as myoglobin monoclonal antibody hybridoma cell strain, is preserved in China General Microbiological Culture Collection Center (CGMCC), is addressed to the institute of microbiology, China academy of sciences, No. 3, China academy of sciences, North China, south China, Yangtze, China, West Asian province, No.1, the China, the preservation number is CGMCC No.18184, and the preservation time is 7 months and 5 days in 2019.

The myoglobin monoclonal antibody hybridoma cell strain is named as MYO 5A8 and classified and named as myoglobin monoclonal antibody hybridoma cell strain, and is preserved in China General Microbiological Culture Collection Center (CGMCC), the address of the microorganism research institute of China academy of sciences No. 3 of Ministry of China, West Lu 1 of the south China, the City, the preservation number is CGMCC No.18185, and the preservation time is 7 months and 5 days in 2019.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.