阅读说明：本技术 一种胰岛素原前体蛋白的检测方法及其ppi单克隆抗体的制备方法 (Detection method of proinsulin precursor protein and preparation method of PPI monoclonal antibody thereof ) 是由 朱珠 张小倩 亓振国 方美姑 王长凤 赵雨 周伟 路江杰 熊爱军 于 2020-06-24 设计创作，主要内容包括：一种胰岛素原前体蛋白的检测方法及其PPI单克隆抗体的制备方法,本发明制备抗胰岛素原前体蛋白(PPI)抗体,在此基础上建立双抗体夹心ELISA,为重组人胰岛素中PPI的检测方法的确立奠定基础。本发明可用于重组人胰岛素生产的过程控制和产品放行控制。(The invention discloses a detection method of proinsulin precursor protein and a preparation method of a PPI monoclonal antibody thereof, wherein an anti-proinsulin Precursor Protein (PPI) antibody is prepared, and a double-antibody sandwich ELISA is established on the basis, so that a foundation is laid for establishing a detection method of PPI in recombinant human insulin. The invention can be used for the process control and product release control of recombinant human insulin production.)

1. A PPI monoclonal antibody is prepared through immunizing Balb/c mouse with proinsulin precursor protein as immunogen, mixing it with isovolume of Freund's complete adjuvant, emulsifying, immunizing once every 2 weeks for 3 weeks, detecting the titer of serum antibody, choosing high-titer mouse, injecting 100 ug of proinsulin precursor protein to the abdominal cavity 3 days before cell fusion, taking blood from eyeball, killing, separating immune spleen cells, fusing it with prepared mouse myeloma cells SP2/0, ELISA screening positive monoclonal hybridoma cells, 3 times of subcloning and ELISA detection, enlarging culture, creating strain, and culturing the hybridoma cells with stable secretion of target antibody by 1-5 × 10⁶Inoculating one or more of the prepared bacteria to Balb/c mice which are 6-8 weeks old and are subjected to intraperitoneal injection with Freund incomplete adjuvant, and collecting ascites by using a 10ml needle when the abdomen of the mice is obviously enlarged and the skin is stressed when touched by hands; and (4) centrifuging the ascites, collecting the supernatant, and purifying by using a Protein G gel column to obtain the purified PPI monoclonal antibody.

2. A method for detecting a proinsulin precursor protein, which is characterized in that: comprises diluting the purified PPI monoclonal antibody prepared in claim 1 to 1ug/ml with PBS buffer solution with pH 7.4, coating 100 ul/well in enzyme label plate, adding 150ul BSA solution with mass fraction of 2% into each well, blocking at 37 deg.C for 60min, and washing with PBST for 3 times; adding 100ul of proinsulin precursor protein water solution with the concentration of 1ug/ml into a detection hole, taking BSA solution with the mass concentration of 1% as a blank control, standing at 37 ℃ for 60min, and washing with PBST for 3 times; arranging according to a chessboard method, adding biotin-labeled PPI monoclonal antibody into detection holes and blank control respectively, standing at 37 ℃ for 60min at 100 ul/hole, and washing for 3 times by PBST; adding streptavidin into the detection hole and blank control, respectively, standing at 37 deg.C for 60min, washing with PBST for 3 times, adding TMB color development solution into the detection hole and blank control, keeping out of the sun for 5min, adding stop solution into the detection hole and blank control, reading the light absorption value at 450nm wavelength with enzyme labeling instrument, and taking the light absorption value of the detection hole greater than 2.1 times that of the blank control hole as a positive detection hole;

step 2: and (3) respectively using the PPI monoclonal antibody coated with the corresponding positive detection hole and the PPI monoclonal antibody marked by biotin as an optimal solid-phase antibody and a detection antibody, coating the determined optimal solid-phase antibody in an enzyme label plate, and adding proinsulin precursor protein with 100 muL series concentration: 82.5pg/mL, 41.25pg/mL, 20.65pg/mL, 10.31pg/mL, 5.16pg/mL, 2.58pg/mL, 1.29pg/mL, 0.645pg/mL, 0pg/mL, standing at 37 ℃ for 60min, PBST washing 3 times; then adding the optimal detection antibody of 1ug/ml and 100 ul/well to perform double-antibody sandwich ELISA operation, wherein each PPI concentration is 3 multiple wells; OD was averaged for each concentration₄₅₀In the ordinate, the PPI concentration is plotted in the abscissa, and a standard curve is fitted using SkanIt RE to determine the linear range of detection and the quantitative equation.

3. The method of detecting a proinsulin precursor protein according to claim 2, wherein: diluting the purified PPI monoclonal antibody prepared according to claim 1 with PBS buffer solution with pH 7.4 and concentration of 0.1M to concentration of 1mg/mL, ultrafiltering to remove interfering substances, adding water-soluble biotin, reacting at room temperature for 1 hr, and ultrafiltering to remove free biotin to obtain biotinylated PPI monoclonal antibody.

Technical Field

The invention belongs to the technical field of detection of proinsulin precursor protein in recombinant human insulin, relates to a detection method of proinsulin precursor protein and a preparation method of a PPI monoclonal antibody thereof, and particularly relates to a method for establishing double-antibody sandwich ELISA and a preparation method of the PPI monoclonal antibody thereof.

Background

Insulin participates in regulating glucose metabolism and controlling blood sugar balance, and is a main medicament clinically used for treating diabetes. Currently, most of the clinically used insulins are high-purity insulins obtained by implanting plasmids containing insulin DNA into escherichia coli or yeast by recombinant DNA technology to express a large amount of the plasmids to form inactive proinsulin precursor proteins (Pre-proinsulin, PPI), and then carrying out enzyme digestion and purification. The high-efficiency and stable expression of PPI is a precondition for obtaining high-yield insulin, but PPI which is not cut and removed by enzyme can seriously affect the quality safety of insulin. Therefore, the method has very important significance for measuring the PPI expression amount, and detecting and controlling the PPI residual amount in the processes of enzyme digestion and purification and insulin.

The european pharmacopoeia clearly indicates that PPI in insulin is to be detected and controlled, but no specific requirements are made on the detection method. At present, the commonly used methods mainly include radioimmunoassay, high performance liquid chromatography, and the like. The radioimmunoassay method is gradually eliminated due to the disadvantages of high price, easy environmental pollution, harm to human health and the like. The high performance liquid chromatography is convenient and accurate, is usually used for detecting the PPI content in the purification process, but has poor sensitivity, and cannot meet the requirement of PPI residue detection in the final product.

Disclosure of Invention

The invention aims to provide a detection method of proinsulin precursor protein and a preparation method of a PPI monoclonal antibody thereof.

In order to achieve the purpose and other related purposes, the preparation method of the PPI monoclonal antibody comprises the steps of immunizing a 6-8-week-old Balb/c mouse by using proinsulin precursor protein as immunogen, immunizing the mouse after mixing and emulsifying the mouse with 100 mu g of proinsulin precursor protein and isovolumetric Freund's complete adjuvant, immunizing the mouse once every 2 weeks, detecting serum antibody titer after immunizing two weeks for 3 times, selecting the mouse with high titer 3 days before cell fusion, injecting 100 mu g of proinsulin precursor protein per peritoneal cavity to enhance immunity, taking eyeball blood, killing, separating immune spleen cells, fusing the immune spleen cells and prepared mouse myeloma cells SP2/0, screening positive monoclonal hybridoma cells, carrying out amplification culture and strain establishment after 3 times of subcloning and ELISA detection of complete positivity, and selecting the hybridoma cells stably secreting target antibody by 1-5 × 10⁶Inoculating one or more of the prepared bacteria to Balb/c mice which are 6-8 weeks old and are subjected to intraperitoneal injection with Freund incomplete adjuvant, and collecting ascites by using a 10ml needle when the abdomen of the mice is obviously enlarged and the skin is stressed when touched by hands; and (4) centrifuging the ascites, collecting the supernatant, and purifying by using a Protein G gel column to obtain the purified PPI monoclonal antibody.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a method for establishing double-antibody sandwich ELISA comprises diluting the prepared PPI monoclonal antibody to 1ug/ml with PBS buffer solution with pH value of 7.4, coating 100 ul/well in an ELISA plate, adding 150ul BSA solution with mass fraction of 2% into each well, sealing at 37 deg.C for 60min, and washing with PBST for 3 times; adding 100ul of proinsulin precursor protein water solution with the concentration of 1ug/ml into a detection hole, taking BSA solution with the mass concentration of 1% as a blank control, standing at 37 ℃ for 60min, and washing with PBST for 3 times; arranging according to a chessboard method, adding biotin-labeled PPI monoclonal antibody into detection holes and blank control respectively, standing at 37 ℃ for 60min at 100 ul/hole, and washing for 3 times by PBST; adding streptavidin into the detection hole and blank control, respectively, standing at 37 deg.C for 60min, washing with PBST for 3 times, adding TMB color development solution into the detection hole and blank control, keeping out of the sun for 5min, adding stop solution into the detection hole and blank control, reading the light absorption value at 450nm wavelength with enzyme labeling instrument, and taking the light absorption value of the detection hole greater than 2.1 times that of the blank control hole as a positive detection hole;

step 2: and (3) respectively using the PPI monoclonal antibody coated with the corresponding positive detection hole and the PPI monoclonal antibody marked by biotin as an optimal solid-phase antibody and a detection antibody, coating the determined optimal solid-phase antibody in an enzyme label plate, and adding proinsulin precursor protein with 100 muL series concentration: 82.5pg/mL, 41.25pg/mL, 20.65pg/mL, 10.31pg/mL, 5.16pg/mL, 2.58pg/mL, 1.29pg/mL, 0.645pg/mL, 0pg/mL, standing at 37 ℃ for 60min, PBST washing 3 times; then adding the optimal detection antibody of 1ug/ml and 100 ul/well to perform double-antibody sandwich ELISA operation, wherein each PPI concentration is 3 multiple wells; OD was averaged for each concentration₄₅₀In the ordinate, the PPI concentration is plotted in the abscissa, and a standard curve is fitted using SkanIt RE to determine the linear range of detection and the quantitative equation.

The preferable technical scheme is as follows: diluting the purified PPI monoclonal antibody prepared according to claim 1 with PBS buffer solution with pH 7.4 and concentration of 0.1M to concentration of 1mg/mL, ultrafiltering to remove interfering substances, adding water-soluble biotin, reacting at room temperature for 1 hr, and ultrafiltering to remove free biotin to obtain biotinylated PPI monoclonal antibody.

Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:

1. the 6-strain PPI-resistant monoclonal antibody is prepared and obtained, a double-antibody sandwich ELISA detection method is initially established, and the method can be used for process control and product release control of recombinant human insulin production.

2. The linear range of the PPI quantitative curve detected by the double-antibody sandwich ELISA is 0.645-82.5pg/mL, the addition recovery rate is 89% -95%, and the detection limit is 3.06 pg/mL.

Drawings

FIG. 1 shows PPI monoclonal antibody titers.

FIG. 2 shows PPI monoclonal antibody purification. (A) Reduced SDS-PAGE displaying purification of monoclonal antibodies P1 and P2.1: 10. mu.L of P1; 2: 5. mu.L of P1; 3: 10. mu.L of P2; 4:5 μ Lof P2; (B) p3 and P4.1: 10. mu.L of P3; 2: 5. mu.L of P3; 3: 10. mu.L of P4; 4: 5. mu.L of P4; (C) p5.1: 10. mu.L of P5; 2: 5. mu.L of P5; (D) p6.1: 10. mu.L of P6; 2: 5. mu.L of P6; m molecular weight massstandards (kDa).

FIG. 3 shows the screening of paired antibodies.

FIG. 4 is paired antibody specificity. A: platinum antibodies are raised above the x-axis, detection antibodies isP 5. B: platinum antistide isP5, detection antistides areactual below the x-axis.

FIG. 5 is a PPI quantification curve.

Detailed Description

The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.

Please refer to fig. 1-5. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention. In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.