Immunoaffinity Active Protein Purification Device and Method Based on Surface Plasmon Resonance

Abstract

The surface plasmon resonance immunosorbent affinity active protein purification device based on the present invention comprises: a first six-way selector valve, a second six-way selector valve, a first four-way proportional valve, a first pump, a separation column, a damper, an ultraviolet detector, a second four-way proportional valve, a surface plasmon resonance spectroscopy detector, a second pump, a third pump, a two-way selector valve, a collector, a waste liquid bottle, a six-way valve, a controller and a column thermostat. The liquid outlet holes of the first six-way selector valve and the second six-way selector valve are respectively connected to one of the liquid inlet holes in the first four-way proportional valve. The liquid outlet hole of the first four-way proportional valve is connected to the first hole of the six-way valve through the first pump. The first hole of the six-way valve is sequentially connected to the second hole and the liquid inlet of the separation column. The first path of the separation column is sequentially connected to the ultraviolet detector and the two-way selector valve. The two liquid outlet holes of the two-way selector valve are respectively connected to the collector and the waste liquid bottle. The second path of the separation column is sequentially connected to the damper, one of the liquid inlet holes in the second four-way proportional valve and the sample injection end of the surface plasmon resonance spectroscopy detector.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical analysis, and particularly to an immunoaffinity active protein purification device and method based on surface plasmon resonance. Background Art

[0002] As an important class of biological macromolecules, proteins are the main bearers of life activities, and the functions of proteins are indispensable in various life activities in vivo, such as nutritional metabolism, enzymatic catalysis, hormones, immunity, genetics, variation, etc. Given the important role of proteins in living organisms, they have become detection targets in many fields. For example, in in vitro diagnosis, more than half of the common in vitro diagnosis items are proteins; in food safety, more than 160 foods contain food allergens that can cause allergic reactions, and more than 90% of these allergens are proteins. According to the domestic food label identification management regulations, the corresponding protein allergens should be detected and identified; in the biomedical field, more than 150 protein drugs have been approved for marketing, more than 400 protein drugs are in the clinical research stage, and more than 3,000 are in the pre-clinical research stage. Whether it is in vitro diagnosis, food safety, or drug safety, these are all related to public health and national economy and people's livelihood, and the accurate comparability of protein test results in related fields is the cornerstone of ensuring public health and safety.

[0003] In terms of structure, proteins are a class of biological macromolecules composed of amino acids connected end to end. The polypeptide chain undergoes further folding and coiling, as well as binding to other protein subunits, to form more complex secondary, tertiary, and quaternary structures. The function of proteins depends more on their structure and activity, and exerts the expected function through binding to corresponding receptors, etc. Therefore, when the higher-order structure of a protein changes and it cannot bind to the receptor, that is, when the protein is inactivated, it will no longer have activity. So in protein research, more often, active proteins are desired.

[0004] To obtain the target protein, it is usually necessary to separate and purify the raw materials to separate the target protein from other proteins or components. The equipment used for protein purification is the protein purification system. Usually, the protein purification system consists of an injection system, a pump system, a detection system, a collection system, and control software. The detection system usually includes pH, conductivity, and ultraviolet detectors. After the protein sample to be separated and purified is injected, the target protein is separated from other proteins or components by means of molecular sieve, ion exchange, affinity chromatography, reverse phase, etc. According to the changes in the ultraviolet or pH and conductivity signals of the sample, the target protein is collected. Obviously, the detection and separation processes focus on the physical and chemical purity of the protein. In the traditional protein purification process, the activity of the protein is not detected, and it cannot be ensured that the collected protein must have activity. In order to determine whether the collected protein has activity, additional protein activity detection is required later, which increases the cost of separation and purification. Therefore, in order to improve the efficiency of protein separation and purification and accurately obtain the target protein with activity, it is urgent to develop and build a device that can detect the activity of proteins in real time during the protein separation and purification process and collect according to the activity of the protein, rather than relying solely on physical and chemical purity, so as to collect the target protein with activity. Summary of the Invention

[0005] The invention objective of the present application is to provide an immunoadsorbent active protein purification device and technology based on surface plasmon resonance technology, aiming to overcome the drawback that the current protein purification system cannot monitor and collect active proteins and ensure that the collected target protein has the expected immunoadsorbent activity.

[0006] To achieve the invention objective of the present application, the following technical solutions are adopted in the present application:

[0007] A purification device for immunologically-affine active proteins based on surface plasmon resonance according to the present invention comprises: a first six-way selector valve, a second six-way selector valve, a first four-way proportional valve, a first pump, a separation column, a damper, an ultraviolet detector, a second four-way proportional valve, a surface plasmon resonance spectroscopy detector, a second pump, a third pump, a two-way selector valve, a collector, a waste liquid bottle, a six-way valve, a controller, and a column oven. The liquid outlets of the first six-way selector valve and the second six-way selector valve are respectively connected to one of the liquid inlets in the first four-way proportional valve. The liquid outlet of the first four-way proportional valve is connected to the first hole of the six-way valve through the first pump. The first hole of the six-way valve is sequentially connected to the second hole and the liquid inlet of the separation column. The sample enters the fourth hole of the six-way valve through a sampling needle. The fourth hole is sequentially connected to the third hole and the sixth hole equipped with a quantitative loop. The sixth hole is connected to the fifth hole, and the fifth hole is connected to the waste liquid bottle. The excess sample enters the waste liquid bottle through the fifth hole. The first hole, the second hole, the third hole, the fourth hole, the fifth hole, and the sixth hole of the six-way valve are sequentially arranged around the six-way valve. The liquid outlet of the separation column is divided into a first path and a second path. The first path is sequentially connected to the ultraviolet detector and the two-way selector valve. The two liquid outlets of the two-way selector valve are respectively connected to the collector and the waste liquid bottle. The second path is sequentially connected to the damper and one of the liquid inlets in the second four-way proportional valve. The liquid outlet in the second four-way proportional valve is connected to the sample injection end of the surface plasmon resonance spectroscopy detector. The other two liquid inlets in the second four-way proportional valve are respectively connected to the second pump and the third pump. The column oven is installed outside the separation column to maintain the temperature of the separation column. The controller is respectively connected to the first six-way selector valve, the second six-way selector valve, the first four-way proportional valve, the first pump, the ultraviolet detector, the second four-way proportional valve, the surface plasmon resonance spectroscopy detector, the second pump, the third pump, the two-way selector valve, and the six-way valve. The controller controls the separation or purification process of the protein sample solution to be separated and purified.

[0008] The purification device for immunologically-affine active proteins based on surface plasmon resonance according to the present invention, wherein: the two-way selector valve comprises: one liquid inlet and two liquid outlets, wherein the liquid inlet is connected to the ultraviolet detector, and the two liquid outlets are respectively selectively connected to one of the collector and the waste liquid bottle.

[0009] The purification device for immunologically-affine active proteins based on surface plasmon resonance according to the present invention, wherein: the first six-way selector valve and the second six-way selector valve comprise: one liquid outlet and six liquid inlets, and the liquid outlet is respectively selectively communicated with one of the six liquid inlets; the first four-way proportional valve and the second four-way proportional valve comprise: one liquid outlet and four liquid inlets, and the liquid outlet is respectively selectively communicated with one of the four liquid inlets.

[0010] The purification device for immunologically affinity active proteins based on surface plasmon resonance of the present invention, wherein: the six-way valve includes: six holes, the first hole is connected to the first pump, the second hole is connected to the separation column, the third hole is connected to the quantitative loop, the fourth hole is connected to the sampling needle, the fifth hole is connected to the waste liquid bottle, the sixth hole is communicated with the quantitative loop. When mobile phases A, B, C, and D are fed to the separation column, the first hole is connected to the second hole, the second hole is connected to the third hole, the fourth hole is connected to the fifth hole, and mobile phases A, B, C, and D are sent into the separation column, and the protein sample solution to be separated and purified enters the quantitative loop; after the six-way valve rotates 60°, the first hole is connected to the sixth hole, the second hole is connected to the third hole, the fourth hole is connected to the fifth hole, and the protein sample solution to be separated and purified is sent into the separation column; the first pump is a high-pressure pump or a peristaltic pump; the second pump and the third pump are peristaltic pumps.

[0011] The purification device for immunologically affinity active proteins based on surface plasmon resonance of the present invention, wherein: the separation column is a purification column or a chromatographic column; the collector is a disk-type rotary collector, and the sample tubes are placed on the turntable, and the target active protein is collected into the sample tubes.

[0012] The method for collecting proteins using the purification device for immunologically affinity active proteins based on surface plasmon resonance of the present invention, wherein: it includes:

[0013] (1) Preparation of the sample solution

[0014] The total protein concentration of the protein sample solution to be separated and purified is roughly measured by ultraviolet spectrophotometry, and it is diluted or centrifugally concentrated according to the total protein concentration. The protein sample solution to be separated and purified is dissolved in a phosphate, acetate buffer solution or water so that its total protein concentration is (1 - 5) mg / mL, and the content of organic solvent is not higher than 5%.

[0015] (2) Antibody conjugation

[0016] First, the monoclonal antibody of the target protein is conjugated to the detection chip of the surface plasmon resonance spectrometer. The affinity between the monoclonal antibody of the above target protein and the target protein is not higher than 10 -5 M -1 , and the monoclonal antibody of the target protein is first diluted to (10 - 50) μg / mL with a 10 mM sodium acetate buffer solution with a pH of (4.0 - 5.5) and then reserved;

[0017] The detection chip is a commercially available chip. The gold foil surface of the detection chip has been fixed with carboxymethyl dextran. After the above carboxymethyl dextran is activated by NHS / EDC and catalyzed to undergo an esterification reaction, it covalently binds to the amino group on the monoclonal antibody of the above target protein, thereby ensuring that the monoclonal antibody of the target protein is conjugated to the gold foil surface;

[0018] (III) Screening of the regeneration solution

[0019] In order to reuse the detection chip multiple times, after detecting the target protein with a surface plasmon resonance spectroscopy detector, the target protein adheres to the surface of the detection chip. The detection chip needs to be cleaned with a regeneration solution. Select at least three concentrations of the regeneration solution, which is a NaOH solution with a concentration of (10 - 50) mM. After detecting the target protein sample with the detection chip for 120 s, then inject the above regeneration solution for 30 s. Each concentration is repeated for 5 cycles, and the optimal regeneration conditions are selected according to the degree to which the signal returns to the baseline, that is: the concentration of the optimal regeneration solution, the optimal regeneration temperature is (20 - 30) °C, the sample cell temperature is (4 - 25) °C, and the regeneration solution is injected into the detection chip at a flow rate of (5 - 100) μL / min;

[0020] (IV) Purification, concentration and collection of the protein sample solution to be separated and purified

[0021] (a) Sampling of the protein sample solution to be separated and purified

[0022] The protein sample solution to be separated and purified enters the six-port valve through the sampling needle of the fourth hole. After the protein sample solution to be separated and purified passes through the quantitative loop of the sixth hole, the protein sample solution to be separated and purified overflowing from the quantitative loop is sent to the waste liquid bottle through the fifth hole, and then by the counterclockwise rotation of the six-port valve, the protein sample solution in the quantitative loop of the sixth hole enters the liquid inlet of the separation column. The injection volume of the quantitative loop is 10 μL - 10000 μL;

[0023] (b) Mobile phases A, B, C and D

[0024] Mobile phases A1 - A6 and D1 - D6 respectively enter one liquid inlet hole of the first six-way selector valve and the second six-way selector valve, then enter their liquid outlet holes and one liquid inlet hole of the first four-way selector valve in sequence. Mobile phases B and D respectively enter one liquid inlet hole of the first four-way proportioning valve. One or several of mobile phases A, B, C and D are mixed in proportion in the first four-way proportioning valve, and then enter the first hole and the second hole of the six-port valve in sequence and then enter the liquid inlet of the separation column for the balance, separation, elution or regeneration of the separation column. The flow rate of the first pump is (0.1 - 100) ml / min;

[0025] (c) Separation of the protein sample solution to be separated and purified

[0026] The protein sample solution to be separated and purified and one or several of mobile phases A, B, C and D respectively enter the separation column. The target protein in the protein sample solution to be separated and purified is separated from other compounds mixed with it, and the separated target protein solution is detected by an ultraviolet detector and a surface plasmon resonance spectroscopy detector respectively;

[0027] (d), Detection of the target protein solution

[0028] The first-way target protein solution coming out of the separation column enters the ultraviolet detector. The ultraviolet detector detects the absorption signal of the target protein at a wavelength of 280 nm. When the protein in the solution flows through the above ultraviolet detector, a peak-shaped signal will appear; the second-way target protein solution coming out of the separation column enters one of the liquid inlet holes of the second quaternary proportion valve after passing through the damper. The other two liquid inlet holes of the second quaternary proportion valve are respectively connected to the second pump and the third pump for transporting the buffer solution and the regeneration solution. By switching the second quaternary proportion valve, the target protein solution separated from the separation column is mixed with the buffer solution and then enters the surface plasmon resonance spectroscopy detector. The signals of the surface plasmon resonance spectroscopy detector and the ultraviolet detector are sent to the controller;

[0029] (e), Collection of the target protein solution

[0030] When the signal of the surface plasmon resonance spectroscopy detector exceeds the baseline signal by (10 - 50)% and the signal of the ultraviolet detector exceeds (10 - 100) mAU, the target protein solution obtained from the separation column is the target active protein solution. The two-way selector valve is switched to communicate with the collector to collect the target active protein solution obtained from the separation column; otherwise, the two-way selector valve is switched to communicate with the waste liquid bottle, and the target protein obtained from the separation column is sent into the waste liquid bottle;

[0031] (5), Regeneration of the detection chip

[0032] After the collection of the target protein is completed, the third pump pumps the NaOH regeneration solution with a concentration of (10 - 50) mM into the surface plasmon resonance spectroscopy detector at a flow rate of (5 - 100) μL / min, and the detection chip is regenerated according to the above-selected regeneration conditions.

[0033] The method for collecting proteins using the purification device for immunologically affinitive active proteins based on surface plasmon resonance according to the present invention, wherein: the separation column is a chromatographic column or a purification column, which are commercially available pre-packed columns or chromatographic columns or purification columns filled in the laboratory by oneself, and the temperature of the column oven is between room temperature and 90 °C.

[0034] The method for collecting proteins using the purification device for immunologically affinitive active proteins based on surface plasmon resonance according to the present invention, wherein: the buffer solution is HBS-EP+; the flow rates of the buffer solution HBS-EP+ and the target protein solution are (10 - 50) μL / min.

[0035] The method for collecting proteins using the purification device for immunologically-affine active proteins based on surface plasmon resonance of the present invention, wherein: the controller respectively collects the flow rates and pressures of the first pump, the second pump and the third pump, the intensity of the absorption signal of the ultraviolet detector, the signal intensity of the surface plasmon resonance spectroscopy detector, and the status information of the first six-way selector valve, the second six-way selector valve, the first quaternary proportional valve, the second quaternary proportional valve and the two-way selector valve, and according to the above information collected and the input control instructions, respectively adjusts the start / stop, flow rate of the first pump, the second pump and the third pump, and switches the selected states or the opening ratios of the first six-way selector valve, the second six-way selector valve, the first quaternary proportional valve, the second quaternary proportional valve and the two-way selector valve, and displays this information on the computer.

[0036] The method for collecting proteins using the purification device for immunologically-affine active proteins based on surface plasmon resonance of the present invention, wherein: the pH of the solution dissolving the monoclonal antibody is usually between the pKa (3.5) of the surface of the detection chip and the isoelectric point of the monoclonal antibody. Before the monoclonal antibody is conjugated to the detection chip, the target protein monoclonal antibody needs to be diluted with sodium acetate buffer solutions of different pH values respectively and injected onto the surface of the carboxymethyl dextran detection chip fixed respectively, and the binding signal intensity is observed. The sodium acetate buffer solution with a higher response value of pH is selected for the actual conjugation operation. After the antibody conjugation is completed, the detection chip conjugated with the antibody is rinsed with HBS-EP+ buffer solution to complete the antibody conjugation operation.

[0037] Aiming at the disadvantages that the current protein purification system cannot monitor the activity of proteins in real time, cannot collect based on protein activity, and cannot ensure that the collected proteins have the expected immunological affinity activity, the present invention provides a protein purification device and technology based on surface plasmon resonance spectroscopy technology, so as to monitor and collect the active proteins in the sample in real time, ensure that the collected target proteins have the expected immunological affinity activity, improve the protein purification efficiency, reduce the purification cost, and solve social resources. Description of the Drawings

[0038] Figure 1 is the purification device for immunologically-affine active proteins based on surface plasmon resonance of the present invention;

[0039] Figure 2 is a schematic diagram of the connection state of the flow-through valve when the mobile phases A, B, C and D are fed into the separation column;

[0040] Figure 3 is a schematic diagram of the connection state of the flow-through valve when the protein sample solution to be separated and purified is fed into the separation column;

[0041] Figure 4 is a diagram of the screening conditions for the pH conditions of insulin antibody conjugation.

[0042] In Figures 1 to 3 it, reference numeral 1 is the first six-way selector valve; reference numeral 2 is the second six-way selector valve; reference numeral 3 is the first quaternary proportional valve; reference numeral 4 is the first pump; reference numeral 5 is the separation column; reference numeral 6 is the damper; reference numeral 7 is the ultraviolet detector; reference numeral 8 is the second quaternary proportional valve; reference numeral 9 is the surface plasmon resonance spectroscopy detector; reference numeral 10 is the second pump; reference numeral 11 is the third pump; reference numeral 12 is the two-way selector valve; reference numeral 13 is the collector; reference numeral 14 is the waste liquid bottle; reference numeral 15 is the six-port valve; reference numeral 16 is the controller; reference numeral 17 is the column oven. Detailed implementation manners

[0043] To further illustrate the present invention, specific descriptions are given in combination with the following embodiments.

[0044] As Figure 1As shown in the figure, the purification device for immunologically affinity active proteins based on surface plasmon resonance of the present invention comprises: a first six-way selector valve 1, a second six-way selector valve 2, a first quaternary proportioning valve 3, a first pump 4, a separation column 5, a damper 6, an ultraviolet detector 7, a second quaternary proportioning valve 8, a surface plasmon resonance spectroscopy detector 9, a second pump 10, a third pump 11, a two-way selector valve 12, a collector 13, a waste liquid bottle 14, a six-way valve 15, a controller 16 and a column thermostat 17. The liquid outlet holes of the first six-way selector valve 1 and the second six-way selector valve 2 are respectively connected to one of the liquid inlet holes in the first quaternary proportioning valve 3. The liquid outlet hole of the first quaternary proportioning valve 3 is connected to the first hole of the six-way valve 15 through the first pump 4. The first hole of the six-way valve 15 is successively connected to the second hole and the liquid inlet of the separation column 5. The sample enters the fourth hole of the six-way valve 15 through a sampling needle. The fourth hole is successively connected to the third hole and the sixth hole equipped with a quantitative loop. The sixth hole is connected to the fifth hole, and the fifth hole is connected to the waste liquid bottle 14. The excess sample enters the waste liquid bottle 14 through the fifth hole. The first hole, the second hole, the third hole, the fourth hole, the fifth hole and the sixth hole of the six-way valve 15 are successively arranged around the six-way valve 15. The liquid outlet of the separation column 5 is divided into a first path and a second path. The first path is successively connected to the ultraviolet detector 7 and the two-way selector valve 12. The two liquid outlet holes of the two-way selector valve 12 are respectively connected to the collector 13 and the waste liquid bottle 14. The second path is successively connected to the damper 6 and one of the liquid inlet holes in the second quaternary proportioning valve 8. The liquid outlet hole in the second quaternary proportioning valve 8 is connected to the sample injection end of the surface plasmon resonance spectroscopy detector 9. The other two liquid inlet holes in the second quaternary proportioning valve 8 are respectively connected to the second pump 10 and the third pump 11. The column thermostat 17 is installed outside the separation column 5 for maintaining the temperature of the separation column 5. The controller 16 is respectively connected to the first six-way selector valve 1, the second six-way selector valve 2, the first quaternary proportioning valve 3, the first pump 4, the ultraviolet detector 7, the second quaternary proportioning valve 8, the surface plasmon resonance spectroscopy detector 9, the second pump 10, the third pump 11, the two-way selector valve 12 and the six-way valve 15. The controller 16 controls the separation or purification process of the protein sample solution to be separated and purified.

[0045] The two-way selector valve 12 comprises: one liquid inlet hole and two liquid outlet holes, wherein the liquid inlet hole is connected to the ultraviolet detector 7, and the two liquid outlet holes are respectively selectively connected to one of the collector 13 and the waste liquid bottle 14.

[0046] The first six-way selector valve 1 and the second six-way selector valve 2 comprise: one liquid outlet hole and six liquid inlet holes, and the liquid outlet hole is respectively selectively communicated with one of the six liquid inlet holes; the first quaternary proportioning valve 3 and the second quaternary proportioning valve 8 comprise: one liquid outlet hole and four liquid inlet holes, and the liquid outlet hole is respectively selectively communicated with one of the four liquid inlet holes.

[0047] As Figure 2 and Figure 3As shown in the figure, the six-way valve 15 includes: six holes. The first hole is connected to the first pump 4, the second hole is connected to the separation column 5, the third hole is connected to the quantitative loop, the fourth hole is connected to the sampling needle, the fifth hole is connected to the waste liquid bottle 14, and the sixth hole communicates with the quantitative loop. As Figure 2 shown, when the mobile phases A, B, C, and D are fed to the separation column 5, the first hole is connected to the second hole, the second hole is connected to the third hole, and the fourth hole is connected to the fifth hole. The mobile phases A, B, C, and D are sent into the separation column 5, and the human insulin sample to be separated and purified enters the quantitative loop. As Figure 3 shown, after the six-way valve 15 rotates counterclockwise by 60°, the first hole is connected to the sixth hole, the second hole is connected to the third hole, and the fourth hole is connected to the fifth hole. The human insulin sample to be separated and purified is sent into the separation column 5; the first pump 4 is a high-pressure pump or a peristaltic pump; the second pump 10 and the third pump 11 are peristaltic pumps.

[0048] The separation column 5 is a purification column or a chromatographic column; the collector 13 is a disk-type rotary collector. The sample tubes are placed on the turntable, and the target active protein is collected into the sample tubes.

[0049] The first six-way selector valve 1, the second six-way selector valve 2, the first quaternary proportioning valve 3, the first pump 4, the separation column 5, the damper 6, the ultraviolet detector 7, the second quaternary proportioning valve 8, the surface plasmon resonance spectroscopy detector 9, the second pump 10, the third pump 11, the two-way selector valve 12, the collector 13, the waste liquid bottle 14, the six-way valve 15, the controller 16, and the column incubator 17. All of the above parts are off-the-shelf products.

[0050] The method for collecting the separated target active protein of human insulin with immunoaffinity activity by using the purifying device for immunoaffinity active protein based on surface plasmon resonance of the present invention includes:

[0051] (1) Preparation of the sample solution

[0052] For the human insulin sample solution to be separated and purified, the concentration of the sample is roughly measured by ultraviolet spectrophotometry. The total protein concentration is about 3.5 mg / mL, which contains human insulin, deaminated insulin, insulin with disulfide bond mismatch, insulin dimers and polymers, insulin with amino acid residue insertions or deletions, as well as other protein impurities and organic impurities. The content of organic solvents is not higher than 5%, and its HPLC purity is about 90%. Before purification, the immunoaffinity activity of this sample is measured by using the Biacore T200 surface plasmon resonance spectrometer and the CFCA method of GE Company. Its immunoaffinity activity concentration is 1.72 mg / mL, and the specific activity is 0.49;

[0053] (2) Antibody conjugation

[0054] In order to detect insulin with immune affinity activity, it is first necessary to conjugate the monoclonal antibody against insulin to the detection chip of the surface plasmon resonance spectroscopy detector 9. The insulin antibody to be conjugated is a murine monoclonal antibody, which is purchased from Nanjing Genscript Corporation. The affinity of the monoclonal antibody of the above-mentioned target protein for the target protein is not higher than 10 -5 M -1 . For conjugation, first, dilute the insulin monoclonal antibody to 40 μg / mL with 10 mM sodium acetate buffer solution at pH = 4.0 for standby;

[0055] The detection chip is a commercially available detection chip purchased, and carboxymethyl dextran has been immobilized on its gold foil surface. After the carboxymethyl dextran on the detection chip is activated by NHS / EDC and catalyzes the esterification reaction, it can covalently bind to the amino group on the antibody to be conjugated, thereby conjugating the insulin antibody to the gold foil surface. To ensure the conjugation effect, the pH of the ligand solution is usually between the pKa (3.5) of the detection chip surface and the isoelectric point of the ligand. Because the carboxymethylated surface of the detection chip has a net negative charge when the pH is greater than 3.5, and at this time the ligand with a net positive charge can gradually approach the detection chip surface through electrostatic interaction, making it easier for the ligand to bind to the activated detection chip. The isoelectric point of the insulin monoclonal antibody used in the experimental conjugation is approximately 8.0. Therefore, the selection of an acidic sodium acetate buffer solution can achieve the conjugation of the insulin monoclonal antibody. To further investigate the conjugation conditions of the insulin monoclonal antibody, first, the pH of the sodium acetate buffer solution used for conjugation was screened. In this experiment, sodium acetate solutions with pH values of 4.0, 4.5, 5.0, and 5.5 were selected for screening. Dilute the insulin monoclonal antibody to 40 μg / mL with sodium acetate buffer solutions of different pH values, and inject them onto the surface of the detection chip immobilized with carboxymethyl dextran, and observe the binding signal intensity. The binding conditions of the antibody under different pH conditions are as Figure 4 shown. During the experiment, it was found that when the pH was 4.0 or 4.5, the response values were both relatively high. Therefore, a sodium acetate solution with pH = 4.0 was used for conjugation during conjugation.

[0056] After activating the detection chip with 100 μL of NHS / EDC mixture, set the conjugation temperature to 25 °C and the flow rate to 5 μL / min. Inject the diluted and prepared human insulin antibody solution into the detection chip, and let it react for 720 s. Then inject 100 μL of 1 M ethanolamine hydrochloride-NaOH solution, and let it continue to react with the detection chip for 720 s to block the remaining unbound active carboxymethyl on the detection chip surface. After the antibody conjugation is completed, rinse the detection chip conjugated with the antibody with 1000 μL of HBS-EP+ buffer solution to complete the antibody conjugation operation;

[0057] (III) Screening of regeneration solution

[0058] To enable the detection chip to be reused multiple times, it is necessary to remove the bound analyte for surface regeneration within each cycle and maintain the activity of the ligand. Effective regeneration is crucial for obtaining accurate analytical data. Insufficient regeneration of the detection chip will cause abnormal response values in the next cycle, and after multiple repeated injections, the detection chip will produce a memory effect, resulting in an increase in the baseline and a decrease in the binding response, thus affecting the accuracy of the experiment. When the regeneration effect is too strong, it will affect the activity of the ligand and further affect the repeatability performance of the detection chip.

[0059] In this experiment, NaOH solution was used as the regeneration solution, and the optimal concentration of the regeneration solution was screened within five concentration ranges of 16, 17, 18, 19, and 20 mmol / L. The experimental procedure was as follows: Set the instrument temperature to 25 °C and the sample cell temperature to 10 °C. After injecting a 2 μg / mL human insulin sample at a flow rate of 30 μL / min for 120 s, inject the regeneration solution for 30 s. Repeat each concentration 5 times to determine the optimal regeneration conditions.

[0060] The experiment found that when using 18 mM NaOH, the binding signal and antibody activity were relatively stable, with good repeatability, and both the baseline and binding level could be maintained stably. Therefore, 18 mM NaOH solution is the appropriate regeneration solution.

[0061] (IV) Purification, concentration, and collection of the human insulin sample to be separated and purified

[0062] (a) Injection of the human insulin sample to be separated and purified

[0063] As Figure 2 shown, the human insulin sample to be separated and purified enters the six-port valve 15 through the injection needle of the fourth hole. After passing through the quantitative loop of the sixth hole, the human insulin sample to be separated and purified overflowing from the quantitative loop is sent to the waste liquid bottle 14 through the fifth hole. After the six-port valve 15 rotates to the Figure 3 position shown, the human insulin sample to be separated and purified enters the inlet of the separation column 5. The human insulin sample to be separated and purified was purified 3 times using this device in total. The volume of the quantitative loop was 1000 μL during the first ion exchange chromatography purification, 100 μL during the second reversed-phase chromatography purification, and 100 μL during the third gel filtration purification;

[0064] (b) Mobile phases A, B, C, and D

[0065] Mobile phases A1 - A6 and D1 - D6 enter one inlet hole of the first six-way selector valve 1 and the second six-way selector valve 2 respectively, then enter their outlet holes and one inlet hole of the first four-way selector valve 3 in sequence. Mobile phases B and D enter one inlet hole of the first four-way proportioning valve 3 respectively. As Figure 1and 2 As shown, one or several of mobile phases A, B, C, and D are proportionally mixed in the first quaternary proportioning valve 3, and then successively enter the first hole and the second hole of the six-port valve 15 and then enter the liquid inlet of the separation column 5 for the equilibration, separation, elution, or regeneration of the separation column 5. The flow rate of the first pump 4 is 0.1 - 100 ml / min;

[0066] When purifying insulin, a total of three purifications were carried out. The first purification was by ion exchange chromatography with a pump flow rate of 1 mL / min. A1 to A6 were stepwise eluted with elution buffers containing 0.1, 0.2, 0.3, 0.4, 0.5, and 1.0 mol / L NaCl and 20 mmol / L Tris-HCl respectively. The second purification was by reverse phase with mobile phases B: 0.2 mol / L sodium sulfate buffer: acetonitrile (82:18) and C: acetonitrile: water (50:50), and mobile phases B and C were eluted according to the gradient in Table 1. The third purification was by molecular sieve exclusion with mobile phase DI: Ix PBS buffer solution.

[0067] Table 1 Liquid chromatography mobile phase gradient

[0068] Time / min B％ C％ 0 78 22 36 78 22 61 33 67 67 33 67

[0069] (c), Separation of the human insulin sample to be separated and purified

[0070] The human insulin sample to be separated and purified and one or several of mobile phases A, B, C, and D enter the separation column 5 respectively. The target protein in the human insulin sample to be separated and purified is separated from other compounds mixed with it, and the separated target protein solution is detected by an ultraviolet detector 7 and a surface plasmon resonance spectroscopy detector 9 respectively;

[0071] When purifying the human insulin sample, the temperature of the column oven was set at 40°C. When performing ion exchange chromatography purification for the first time, the purification column used was a Q-Trap ion exchange chromatography column; when performing reverse phase purification for the second time, the purification column used was a Vydac C8 chromatographic column; when performing molecular sieve purification for the third time, the purification column used was a TSKgel G2000SWxl gel exclusion chromatographic column;

[0072] (d), Detection of the target protein solution

[0073] The first path of the target protein solution emerging from the separation column 5 enters the ultraviolet detector 7. The ultraviolet detector 7 detects the absorption signal of the target protein at a wavelength of 280 nm. When the protein in the solution flows through the above-mentioned ultraviolet detector 7, a peak-shaped signal will be presented; the second path of the target protein solution emerging from the separation column 5 enters one of the liquid inlet holes of the second quaternary proportioning valve 8 after passing through the damper 6. The other two liquid inlet holes of the second quaternary proportioning valve 8 are respectively connected to the second pump 10 and the third pump 11 for delivering the buffer solution and the regeneration solution. By switching the second quaternary proportioning valve 8, the target protein solution separated from the separation column 5 is mixed with the buffer solution and then enters the surface plasmon resonance spectroscopy detector 9. The signals of the surface plasmon resonance spectroscopy detector 9 and the ultraviolet detector are sent to the controller 16. A detection chip conjugated with human insulin monoclonal antibody is placed on the surface plasmon resonance spectroscopy detector. HBS-EP+ and 18 mM NaOH are respectively used as the running buffer solution and the detection chip regeneration solution, and they are injected into the flow path system by a syringe pump. The flow rates of both solutions are set to 30 μL / min;

[0074] (e), Collection of the target protein solution

[0075] When the signal of the surface plasmon resonance spectroscopy detector 9 exceeds the baseline signal by 10 - 50% and the signal of the ultraviolet detector exceeds 10 - 100 mAU, the target protein solution obtained from the separation column 5 is the target active protein solution. The two-way selection valve 12 is switched to communicate with the collector 13, and the target active protein solution obtained from the separation column 5 is collected. The target active human insulin is collected into the sample tube; otherwise, the two-way selection valve 12 is switched to communicate with the waste liquid bottle 14, and the target protein obtained from the separation column 5 is sent into the waste liquid bottle 14;

[0076] (V), Regeneration of the detection chip

[0077] After the collection of the target protein is completed, the third pump 11 pumps the NaOH regeneration solution with a concentration of 10 - 50 mM into the surface plasmon resonance spectroscopy detector 9 at a flow rate of (5 - 100) μL / min, and the detection chip is regenerated according to the above-selected regeneration conditions.

[0078] After the purification of the human insulin sample to be separated and purified is completed, the immunological affinity activity of human insulin needs to be verified. The process is as follows:

[0079] Repeat the above process of collecting and purifying the active protein until all the human insulin samples are purified and collected. Then, all the collected active human insulin solutions are combined and freeze-dried, re-dissolved in water, and the immunological affinity activity of this sample is measured again using the Biacore T200 surface plasmon resonance spectrometer of GE Company and the CFCA method. Its immunological affinity activity concentration is 2.81 mg / mL, and the specific activity is 0.80.

[0080] For comparison, the above collection process was repeated with the same sample, except that the target protein was collected only based on the response value of the ultraviolet detector exceeding 50 mAU as the basis for starting the collection valve. After the collected liquid was combined, freeze-dried and redissolved, the immunoaffinity activity of the sample was also measured again using the Biacore T200 surface plasmon resonance spectrometer and the CFCA method of GE Company. The immunoaffinity activity concentration was 2.41 mg / mL and the specific activity was 0.69.

[0081] The controller 16 respectively collects the flow rates and pressures of the first pump 4, the second pump 10 and the third pump 11, the intensity of the absorption signal of the ultraviolet detector 7, the signal intensity of the surface plasmon resonance spectrometer detector 9, and the status information of the first six-way selector valve 1, the second six-way selector valve 2, the first four-way proportional valve 3, the second four-way proportional valve 8 and the two-way selector valve 12. According to the above information collected and the input control instructions, it respectively adjusts the start / stop, flow rate of the first pump 4, the second pump 10 and the third pump 11, and switches the selected states or the opening ratios of the valves of the first six-way selector valve 1, the second six-way selector valve 2, the first four-way proportional valve 3, the second four-way proportional valve 8 and the two-way selector valve 12, and displays this information on the computer.

[0082] Working principle of the protein purification device

[0083] This device can achieve the purification and collection of the target active protein. The protein mixture to be separated and purified is injected into the six-port valve 15 through the injection needle of the injection unit. The volume exceeding the quantitative loop will overflow and flow into the waste liquid bottle 14. After the sample injection is completed, the six-port valve 15 rotates to inject the sample in the quantitative loop into the flow path. Driven by a peristaltic pump or a high-pressure pump, the main flow path, in coordination with the actions of the multi-channel solvent inlet holes and the inlet hole, injects a mobile phase with an appropriate ratio into the flow path to push the sample into the separation column 5. According to the properties of the target protein, a chromatographic column with different principles such as molecular sieve, ion exchange, and affinity chromatography can be selected to separate the protein mixture, and then it enters the detection unit in sequence. After the flow path enters the detection unit, it is divided into two paths. One path enters the ultraviolet detector 7, and when the protein passes through, an ultraviolet absorption peak will be generated; the other path simultaneously enters the surface plasmon resonance spectroscopy detector 9. When there is an active target protein in the flow path, the target protein will bind to the antibody against the target active protein immobilized on the gold foil surface of the detection chip, thereby causing a change in the surface plasmon resonance signal. At this time, both the ultraviolet detector 7 and the surface plasmon resonance detector 9 can detect signals. If it is a non-target protein or an inactivated target protein, since it cannot bind to the antibody against the target active protein, it will not cause a change in the surface plasmon resonance signal. At this time, only a signal can be observed on the ultraviolet detector 7, and no signal can be observed on the surface plasmon resonance detector 9. According to whether an immunoaffinity signal is detected on the surface plasmon resonance detector 9, the two-way selection valve 12 is switched. When there is an active target protein, rotate the two-way selection valve 12 to the collection position, and collect the target active protein through the disk-type rotary collector.

[0084] Thus, by using the protein purification device based on the surface plasmon resonance technology of the present invention, the active protein components can be effectively collected, and compared with the traditional purification method relying only on the ultraviolet detector, the purification efficiency of the protein and the yield of the active protein are greatly improved.

[0085] The above-described embodiments are only descriptions of the preferred embodiments of the present invention patent, and do not limit the scope of the present invention patent. Without departing from the design spirit of the present invention patent, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention patent shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A purification device for immunologically affinity active proteins based on surface plasmon resonance, which comprises: a first six-way selector valve (1), a second six-way selector valve (2), a first quaternary proportioning valve (3), a first pump (4), a separation column (5), a damper (6), an ultraviolet detector (7), a second quaternary proportioning valve (8), a surface plasmon resonance spectroscopy detector (9), a second pump (10), a third pump (11), a two-way selector valve (12), a collector (13), a waste liquid bottle (14), a six-way valve (15), a controller (16) and a column thermostat (17). The liquid outlet holes of the first six-way selector valve (1) and the second six-way selector valve (2) are respectively connected to one of the liquid inlet holes in the first quaternary proportioning valve (3). The liquid outlet hole of the first quaternary proportioning valve (3) is connected to the first hole of the six-way valve (15) through the first pump (4). The first hole of the six-way valve (15) is successively connected to the second hole and the liquid inlet of the separation column (5). The sample enters the fourth hole of the six-way valve (15) through a sampling needle. The fourth hole is successively connected to the third hole and the sixth hole equipped with a quantitative loop. The sixth hole is connected to the fifth hole. The fifth hole is connected to the waste liquid bottle (14). The excess sample enters the waste liquid bottle (14) through the fifth hole. The first hole, the second hole, the third hole, the fourth hole, the fifth hole and the sixth hole of the six-way valve (15) are successively arranged around the six-way valve (15). The liquid outlet of the separation column (5) is divided into a first path and a second path. The first path is successively connected to the ultraviolet detector (7) and the two-way selector valve (12). The two liquid outlet holes of the two-way selector valve (12) are respectively connected to the collector (13) and the waste liquid bottle (14). The second path is successively connected to the damper (6) and one of the liquid inlet holes in the second quaternary proportioning valve (8). The liquid outlet hole in the second quaternary proportioning valve (8) is connected to the injection end of the surface plasmon resonance spectroscopy detector (9). The other two liquid inlet holes in the second quaternary proportioning valve (8) are respectively connected to the second pump (10) and the third pump (11). The column thermostat (17) is installed outside the separation column (5) to maintain the temperature of the separation column (5). The controller (16) is respectively connected to the first six-way selector valve (1), the second six-way selector valve (2), the first quaternary proportioning valve (3), the first pump (4), the ultraviolet detector (7), the second quaternary proportioning valve (8), the surface plasmon resonance spectroscopy detector (9), the second pump (10), the third pump (11), the two-way selector valve (12) and the six-way valve (15). The controller (16) controls the separation or purification process of the protein sample solution to be separated and purified.

2. The purification device for immunologically affinity active proteins based on surface plasmon resonance according to claim 1, characterized in that: the two-way selector valve (12) comprises: one liquid inlet hole and two liquid outlet holes, wherein the liquid inlet hole is connected to the ultraviolet detector (7), and the two liquid outlet holes are respectively selectively connected to one of the collector (13) and the waste liquid bottle (14).

3. The purification device for immunologically affinity active proteins based on surface plasmon resonance according to claim 2, characterized in that: The first six-way selector valve (1) and the second six-way selector valve (2) include: one liquid outlet hole and six liquid inlet holes, and the liquid outlet hole is selectively communicated with one of the six liquid inlet holes respectively; the first four-way proportional valve (3) and the second four-way proportional valve (8) include: one liquid outlet hole and four liquid inlet holes, and the liquid outlet hole is selectively communicated with one of the four liquid inlet holes respectively.

4. The purification device for immunologically-affinity active protein based on surface plasmon resonance according to claim 3, characterized in that: The six-way valve (15) includes: six holes. The first hole is connected to the first pump (4), the second hole is connected to the separation column (5), the third hole is connected to the quantitative loop, the fourth hole is connected to the sampling needle, the fifth hole is connected to the waste liquid bottle (14), and the sixth hole is communicated with the quantitative loop. When the mobile phases A, B, C, and D are fed to the separation column (5), the first hole is connected to the second hole, the second hole is connected to the third hole, and the fourth hole is connected to the fifth hole, and the mobile phases A, B, C, and D are sent into the separation column (5), and the protein sample solution to be separated and purified enters the quantitative loop; after the six-way valve (15) rotates 60°, the first hole is connected to the sixth hole, the second hole is connected to the third hole, and the fourth hole is connected to the fifth hole, and the protein sample solution to be separated and purified is sent into the separation column (5); the first pump (4) is a high-pressure pump or a peristaltic pump; the second pump (10) and the third pump (11) are peristaltic pumps.

5. The purification device for immunologically-affinity active protein based on surface plasmon resonance according to claim 4, characterized in that: The separation column (5) is a purification column or a chromatographic column; the collector (13) is a disk-type rotary collector, and the sample tubes are placed on the turntable, and the target active protein is collected into the sample tubes.

6. A method for collecting proteins using the purification device for immunologically-affinity active protein based on surface plasmon resonance according to claim 5, characterized in that: including: (1) Preparation of the sample solution The total protein concentration of the protein sample solution to be separated and purified is roughly measured by ultraviolet spectrophotometry, diluted or centrifugally concentrated according to the total protein concentration, and the protein sample solution to be separated and purified is dissolved in a phosphate, acetate buffer solution or water so that its total protein concentration is 1-5 mg / mL, and the content of the organic solvent is not higher than 5%; (2) Antibody coupling First, couple the monoclonal antibody of the target protein to the detection chip of the surface plasmon resonance spectroscopy detector (9). The affinity of the monoclonal antibody of the target protein for the target protein is not higher than 10 -5 M -1 . First, dilute the monoclonal antibody of the target protein to 10 - 50 μg / mL with a 10 mM sodium acetate buffer solution with a pH of 4.0 - 5.5 and set it aside for later use; The detection chip is a commercially available chip. Carboxymethyl dextran has been immobilized on the gold foil surface of the detection chip. After the carboxymethyl dextran is activated by NHS / EDC and catalyzed to undergo an esterification reaction, it covalently binds to the amino group on the monoclonal antibody of the target protein, thereby ensuring that the monoclonal antibody of the target protein is coupled to the gold foil surface; (3) Screening of the regeneration solution In order to enable the detection chip to be reused multiple times, after detecting the target protein with the surface plasmon resonance spectroscopy detector (9), the target protein adheres to the surface of the detection chip. The detection chip needs to be cleaned with a regeneration solution. Select at least three concentrations of the regeneration solution, and the regeneration solution is a NaOH solution with a concentration of 10 - 50 mM. After detecting the target protein sample with the detection chip for 120 s, inject the above regeneration solution for 30 s. Each concentration is repeated for 5 cycles, and the optimal regeneration conditions are selected according to the degree to which the signal returns to the baseline, that is: the concentration of the optimal regeneration solution, the optimal regeneration temperature is 20 - 30 °C, the sample cell temperature is 4 - 25 °C, and the regeneration solution is injected into the detection chip at a flow rate of 5 - 100 μL / min; (IV). Purification, concentration and collection of the protein sample solution to be separated and purified (a). Sampling of the protein sample solution to be separated and purified The protein sample solution to be separated and purified enters the six-port valve (15) through the sampling needle of the fourth hole. After the protein sample solution to be separated and purified passes through the quantitative loop of the sixth hole, the protein sample solution to be separated and purified overflowing from the quantitative loop passes through the fifth hole and is sent to the waste liquid bottle (14). Then, by rotating the six-port valve (15) counterclockwise, the protein sample solution in the quantitative loop of the sixth hole enters the liquid inlet of the separation column (5). The sampling volume of the quantitative loop is 10 μL - 10000 μL; (b). Mobile phases A, B, C and D Mobile phases A1 - A6 and D1 - D6 respectively enter one liquid inlet hole of the first six-way selector valve (1) and the second six-way selector valve (2), then enter their liquid outlet holes and one liquid inlet hole of the first quaternary proportion valve (3) in sequence. Mobile phases B and D respectively enter one liquid inlet hole of the first quaternary proportion valve (3). One or several of mobile phases A, B, C and D are mixed in proportion in the first quaternary proportion valve (3), and then enter the first hole and the second hole of the six-port valve (15) in sequence and then enter the liquid inlet of the separation column (5) for the balance, separation, elution or regeneration of the separation column (5). The flow rate of the first pump (4) is 0.1 - 100 ml / min; (c). Separation of the protein sample solution to be separated and purified The protein sample solution to be separated and purified and one or several of mobile phases A, B, C and D respectively enter the separation column (5). The target protein in the protein sample solution to be separated and purified is separated from other compounds mixed with it, and the separated target protein solution is detected by the ultraviolet detector (7) and the surface plasmon resonance spectroscopy detector (9) respectively; (d). Detection of the target protein solution The first path of the target protein solution emerging from the separation column (5) enters the ultraviolet detector (7). The ultraviolet detector (7) detects the absorption signal of the target protein at a wavelength of 280 nm. When the protein in the solution flows through the above-mentioned ultraviolet detector (7), a peak-shaped signal will be presented. The second path of the target protein solution emerging from the separation column (5) enters one of the liquid inlet holes of the second quaternary proportion valve (8) after passing through the damper (6). The other two liquid inlet holes of the second quaternary proportion valve (8) are respectively connected to the second pump (10) and the third pump (11) that deliver the buffer solution and the regeneration solution. By switching the second quaternary proportion valve (8), the target protein solution separated from the separation column (5) is mixed with the buffer solution and then enters the surface plasmon resonance spectroscopy detector (9). The signals of the surface plasmon resonance spectroscopy detector (9) and the ultraviolet detector are sent to the controller (16). (e), Collection of the target protein solution When the signal of the surface plasmon resonance spectroscopy detector (9) exceeds the baseline signal by 10 - 50% and the ultraviolet detector signal exceeds 10 - 100 mAU, the target protein solution obtained from the separation column (5) is the target active protein solution. The two-way selection valve (12) is switched to communicate with the collector (13) to collect the target active protein solution obtained from the separation column (5); otherwise, the two-way selection valve (12) is switched to communicate with the waste liquid bottle (14) to send the target protein obtained from the separation column (5) into the waste liquid bottle (14). (V), Regeneration of the detection chip After the collection of the target protein is completed, the third pump (11) pumps the NaOH regeneration solution with a concentration of 10 - 50 mM into the surface plasmon resonance spectroscopy detector (9) at a flow rate of 5 - 100 μL / min to perform regeneration treatment on the detection chip according to the above-selected regeneration conditions.

7. The method for collecting proteins as claimed in claim 6, wherein: The separation column is a chromatographic column or a purification column, which are commercially available pre-packed columns or chromatographic columns or purification columns self-packed in the laboratory, and the temperature of the column oven is between room temperature and 90 °C.

8. The method for collecting proteins as claimed in claim 7, wherein: The buffer solution is HBS-EP+; the flow rates of the buffer solution HBS-EP+ and the target protein solution are 10 - 50 μL / min.

9. The method for collecting proteins as claimed in claim 8, wherein: The controller (16) collects the flow rates and pressures of the first pump (4), the second pump (10) and the third pump (11) respectively, collects the intensity of the absorption signal of the ultraviolet detector (7), the signal intensity of the surface plasmon resonance spectroscopy detector (9), and the status information of the first six-way selector valve (1), the second six-way selector valve (2), the first quaternary proportional valve (3), the second quaternary proportional valve (8) and the two-way selector valve (12). According to the above information collected and the input control instructions, it adjusts the start / stop and flow rate of the first pump (4), the second pump (10) and the third pump (11) respectively, and switches the selected states or the opening ratios of the valves of the first six-way selector valve (1), the second six-way selector valve (2), the first quaternary proportional valve (3), the second quaternary proportional valve (8) and the two-way selector valve (12), and displays this information on the computer.

10. The method for collecting proteins according to claim 9, characterized in that: The pH of the solution for dissolving the monoclonal antibody is usually between the pKa of 3.5 on the surface of the detection chip and the isoelectric point of the monoclonal antibody. Before the monoclonal antibody is conjugated to the detection chip, the target protein monoclonal antibody needs to be diluted with sodium acetate buffer solutions of different pH values respectively and injected onto the surface of the carboxymethyl dextran detection chip fixed respectively. Observe the binding signal intensity, and select the sodium acetate buffer solution with a higher response value of pH for the actual conjugation operation. After the antibody conjugation is completed, the detection chip conjugated with the antibody is rinsed with HBS-EP+ buffer solution to complete the antibody conjugation operation.

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