Detection methods for drug screening and characterization based on nanosurface plasmon resonance technology

Abstract

The application discloses a detection method for drug screening and characterization based on nano-surface plasmon resonance technology, and belongs to the technical field of antibody protein affinity detection and antibody drug screening and characterization. The detection method is as follows: a NanoSPR chip is coated with a fixed ligand, an analyte to be detected is added, and then sample injection detection is carried out; a specific fitting curve equation is used to obtain a final protein-protein, antigen-antibody, DNA-protein, etc. D kinetic simulation curve, and then a corresponding equilibrium affinity constant K D is calculated. The application firstly applies the nano-surface plasmon resonance technology to the affinity detection of protein-protein, antigen-antibody, DNA-protein, etc., and analyzes the kinetics of the binding process and the dissociation process at the same time, so that the final detection result is very close to the published equilibrium affinity constant K D , which indicates that the method has very high reliability and accuracy, and can be applied to antibody drug screening and characterization.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antibody protein affinity detection and antibody drug screening and characterization, in particular, a detection method for drug screening and characterization based on nano-surface plasmon resonance technology. BACKGROUND

[0002] Recognizing and detecting the interaction between compounds and proteins, i.e., the affinity between proteins (or antibodies) and antigens, is of great significance for discovering new uses of existing antibody drugs, discovering, developing and screening safe and effective antibody drugs; it can reveal unknown compound-protein interactions, help predict potential side effects, and analyze and detect antibody drug characterization. Existing protein affinity detection techniques usually use computer software simulation or statistical methods for evaluation and detection.

[0003] Surface plasmon resonance (SPR) is mainly based on the total reflection mode, which uses the attenuated total reflection prism coupling method to realize laser excitation of surface plasmon waves. By detecting the change of the total reflection angle, the extinction spectrum is moved, and then the information of the biochemical reaction is obtained. In the prior art, some people have used surface plasmon resonance chips to detect the affinity of specific binding between antibody proteins and antigens. For example, Chinese patent application CN112625091A provides a polypeptide sequence combined with porcine epidemic diarrhea virus Erns protein. The polypeptide sequence is obtained by diluting the porcine epidemic diarrhea virus Erns protein with PBS liquid and chip activation buffer, using the active ester method, and using an SPR detector equipped with a carboxyl chip to detect the interaction ability of the porcine epidemic diarrhea virus Erns protein and the target polypeptide to be tested.

[0004] Nano-surface plasmon resonance technology (NanoSPR) is a completely new qualitative and quantitative detection technology different from the plasmon resonance chip technology (SPR). It makes a row of micropores with a diameter of not more than 100 nm on the chip. Through the resonance coupling of incident light and metal nanostructure, the wavelength of surface plasmon resonance is sensitive to the dielectric environment around the nanostructure, and the detection of biochemical reactions is realized. When there is a difference in the refractive index between the adsorbed molecules and the surrounding environment, the reaction between the biological molecules adsorbed on the substrate surface and the target molecules will change the refractive index of the substrate surface, thereby causing a change in the resonance peak, and the detection of the target substance is realized. Therefore, the resonance analysis process of the nano-surface plasmon sensor does not require the complex optical system used in traditional SPR technology.

[0005] It is particularly important to construct a fast and reliable method for dynamically detecting protein binding and dissociation for drug screening and application, and early detection of viruses. There are many methods for detecting protein affinity by detecting protein binding and dissociation, but there are still limitations in application. So far, no one has applied NanoSPR technology to protein affinity detection. SUMMARY

[0006] In order to overcome the above problems of the prior art, the present application provides a method for detecting protein binding and dissociation, which is simple, label-free, high-throughput, portable and real-time, and can be applied to drug screening and characterization detection.

[0007] The method for detecting drug screening and characterization based on the NanoSPR technology comprises the following steps: coating a NanoSPR chip with a fixed ligand, combining an analyte to be detected to the NanoSPR chip, detecting a binding kinetic curve and a dissociation kinetic curve, and finally detecting drug screening and characterization.

[0008] Preferably, in the above method, the specific steps of coating the NanoSPR chip with the fixed ligand, combining the analyte to be detected to the NanoSPR chip, and measuring the binding kinetic curve and the dissociation kinetic curve comprise the following steps:

[0009] S1. washing a plurality of NanoSPR chips, adding a fixed ligand working solution to the NanoSPR chips, and standing after coating; discarding the waste fixed ligand working solution, sequentially adding a blocking solution and a protection solution for blocking and protection, and discarding the waste protection solution after drying.

[0010] S2. preparing different concentration gradients of the analyte working solution, and adding the analyte working solution with different concentrations to the NanoSPR chip obtained in step S1.

[0011] S3. performing kinetic detection at a specific wavelength, measuring the binding kinetic curve for 5-15 min, then measuring the dissociation kinetic curve for 15-30 min, and finally using a computer software to fit the equilibrium affinity constant K D .

[0012] The present application provides a NanoSPR biochip detection method for detecting the binding and dissociation of an analyte (such as ordinary proteins, antibodies, polypeptides, and small molecule compounds such as sulfonamides and aflatoxins in food) and a fixed ligand, which is simple, label-free, high-throughput, portable and real-time. The method applies the NanoSPR technology to the detection of the binding and dissociation of the analyte (mainly proteins), and detects the equilibrium affinity constant K DThe test results are basically consistent with the test results reported in the current literature by using common methods. Subsequently, by comparison with the Biacore system which has been commercialized, the results also show that the detection results obtained by the present application have very high reliability and accuracy. The binding and dissociation process of the to-be-tested analyte and the fixed ligand of the present method only needs 20-25 min, which can meet the requirement of rapid detection of protein kinetics, and provides an efficient and reliable method for real-time detection of protein binding and dissociation kinetics. This method can be widely used in the analysis of screening and characterization of antibody drugs, and provides a new means for the fields of antibody drug screening, early detection of viruses, etc.

[0013] The to-be-tested analyte can be an antigen, an antibody, a common protein, a common polypeptide, a small molecule substance commonly used for detection, etc. Specifically, the to-be-tested analyte can be a new coronavirus antibody protein, a tumor marker antibody protein, or a small molecule compound such as sulfonamide and aflatoxin in food inspection; and the fixed ligand is a substance that can bind with the to-be-tested analyte. The to-be-tested analyte does not necessarily have to be an antibody, and accordingly, the fixed ligand does not necessarily have to be an antigen. For example, if the to-be-tested analyte is an antigen, the fixed ligand is an antibody that can specifically bind with the antigen; if the to-be-tested analyte is an antibody, the fixed ligand is an antigen that can be specifically bound by the antibody. Since the binding and dissociation of the to-be-tested analyte (such as an antibody or an antigen) and the fixed ligand (such as an antigen or an antibody) is a dynamic process that occurs simultaneously and continuously, the innovation of the detection method of the present application is that not only the binding curve of the binding process of the to-be-tested analyte is obtained, but also the dissociation curve of the dissociation process of the to-be-tested analyte is simultaneously obtained, and the equilibrium affinity constant K D .

[0014] Before the formal detection method, first of all, the conventional method is used to prepare the NanoSPR chip according to the requirements, for example, the matrix arranged nano-pores are imprinted on the surface of the base plate, the top diameter of each nano-pore is generally 50-2000nm, the bottom diameter is 50-2000nm, the height is 100-800nm, and the spacing between adjacent nano-pores is 100-4000nm, then a 10nm titanium film layer, a 20nm silver film layer and a 10nm gold film layer are sequentially plated from bottom to top; for the analyte to be measured, the immobilized ligand capable of specifically binding therewith is determined, and different analytes have different immobilized ligands. In step S2, the one-step method is used, the analyte to be measured is combined with the immobilized ligand, and then directly placed into a spectrometer (such as XLement SPR100) for full-spectrum scanning. In step S3, the best wavelength (such as 575nm and 600nm) is selected by full-spectrum scanning, and then the kinetic detection is carried out, and finally the equilibrium affinity constant K D of the analyte to be measured and the immobilized ligand is obtained by using computer software to fit the equation.

[0015] Preferably, in step S1, the blocking solution is a complex solution prepared by dissolving at least one of bovine serum albumin, casein, milk and polyethylene glycol 20000 in a buffer solution, and the blocking method is to place at room temperature for 1-2h; the protective solution is a complex solution prepared by dissolving a sugar (such as dextran, glucose, sucrose or trehalose) in a buffer solution, and the protection method is to protect at 37℃ for 5-30min.

[0016] More preferably, in step S1, the buffer solution used is CBS buffer solution, PBS buffer solution, TBS buffer solution, Hanks buffer solution or HEPES buffer solution.

[0017] Preferably, in step S3, the dissociation solution used in the dissociation process is CBS buffer solution, PBS buffer solution, TBS buffer solution, Hanks buffer solution or HEPES buffer solution.

[0018] More preferably, in step S3, the dissociation solution contains a surfactant. For example, common Tween 20, Tween 80 and the like. The concentration of the surfactant is generally 0.5-1%. Preferably, in step S3, the fitting equation of the association rate constant K on and the dissociation rate constant K off is first obtained by using computer software fitting, which is

[0019] and

[0020] Then the equilibrium affinity constant K D of the protein to be measured is fitted according to the formula .The fitting equation is: Y = a + Bx, wherein [B] is the concentration of the protein to be detected, and a is the asymptote.

[0021] Preferably, the detection is performed using a microplate integrated with NanoSPR chips, wherein the NanoSPR chips are integrated on the bottom surface of each micro-well of the microplate. The microplate can be an 8-well, 16-well, 32-well, or 96-well microplate.

[0022] Based on the principle of the above detection method, as one of the specific step methods, the NanoSPR chip can be directly integrated into the micro-well of the microplate, and then the detection is performed according to the above steps S1-S3.

[0023] Preferably, the detection is performed using a NanoSPR chip assembly and a microplate, wherein the NanoSPR chip assembly comprises a plurality of NanoSPR chip columns arranged in at least one row, and each NanoSPR chip column is loaded with a NanoSPR chip on the top of the column head; the microplate comprises a plurality of micro-wells arranged in a matrix, and the bottom surface of each micro-well is made of transparent material; each row of micro-wells of the microplate is used to load a washing solution, a fixed ligand working solution, a blocking solution, a protective solution, a working solution of an analyte to be detected with different concentration gradients, and an elution solution.

[0024] As another specific step method, the detection can be performed using a NanoSPR chip assembly and a microplate, wherein the washing solution, the fixed ligand working solution, the working solution of the analyte to be detected with different concentrations, the elution solution, and the protective solution are respectively placed in each row of micro-wells of the microplate; if the chip column needs to be recovered, another row of micro-wells can be used to add a regeneration solution. When used, the downward NanoSPR chip column is inserted into the two rows of micro-wells loaded with the washing solution according to the above steps S1-S3, so that the NanoSPR chip is immersed in the washing solution to complete the washing, and then the micro-well loaded with the fixed ligand working solution is placed, so that the fixed ligand is coated and fixed on the surface of the NanoSPR chip. In the subsequent process, the corresponding micro-well is placed according to the predetermined steps, and the detection is performed on the machine, so as to complete the whole detection process.

[0025] Preferably, after step S3, the detection method further comprises a regeneration method of the NanoSPR chip, which is that the NanoSPR chip after the detection is eluted with an elution regeneration solution for 1-5 times, so as to realize the reuse of the NanoSPR chip.

[0026] Taking the detection of the binding site of the antibody and antigen as an example, the detection method provided by the application can be used for screening and characterization of the antibody by using the sandwich method, the tandem method, or the premix method. For example:

[0027] (1) using sandwich method, the fixed antibody is coated on the chip, the antigen is added and the first group of OD starting value is obtained by machine detection; the NanoSPR chip is taken out again, and other several kinds of antibodies to be screened (which can be combined with the antigen) are added for binding, and the full spectrum and OD endpoint value are detected again. By judging the difference between the two detection results, it can be judged whether the antibody to be screened is combined with the antigen, and further judging the specific binding effect, that is, whether the binding sites are the same; finally, the appropriate antibody is screened as a drug, or a pair of antibodies is used for sandwich method detection of antigen.

[0028] (2) using premix method, first, use the same way as the sandwich method above, coat the fixed antibody on the NanoSPR chip, then mix the antigen with several kinds of test antibodies to form several mixed test liquids, finally add these mixed test liquids to the NanoSPR chip, and detect the kinetic curve change respectively, which can judge whether the binding sites of the several different test antibodies, fixed ligands and antigens are completely the same, not completely the same or completely different.

[0029] (3) the principle of using tandem method is similar to sandwich method, except that the order of adding reagents is different. First, coat the antigen on the NanoSPR chip; then add the fixed antibody to obtain the corresponding kinetic curve; add different kinds of test antibodies to obtain several corresponding kinetic curves. By comparing the kinetic curves of the test antibodies and the fixed antibodies, it can be judged whether the binding sites of different test antibodies, fixed antibodies and antigens are the same.

[0030] Compared with the prior art, the advantages of the present application are that: the present application first applies the nano surface plasmon resonance technology to the affinity detection between the test analyte (antibody, ordinary protein, etc.) and the fixed ligand (antigen, protein, etc.), and through the simultaneous kinetic detection analysis of the binding process and the dissociation process, the equilibrium affinity constant K D is obtained by combining the binding curve equation and the dissociation curve equation, according to the above formula, the final equilibrium affinity constant K D is obtained, by detecting different kinds of antibody protein substances, and comparing the results with the published equilibrium affinity constant K D , it is found that the coincidence degree is very high, which shows that the antibody protein affinity detection method based on nano surface plasmon resonance technology has very high reliability and accuracy, and can be effectively applied to antibody drug screening and characterization, and provides a new means for antibody drug efficacy screening, early detection of viruses and other fields. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1Schematic diagram of the NanoSPR chip column and microplate of the NanoSPR chip assembly used in the specific embodiments of the present application;

[0032] Figure 2 Kinetic binding and dissociation processes and simulation curves of Herceptin and Her2 protein of Example 1.

[0033] Figure 3 Kinetic binding processes and simulation curves of anti-His tag antibody and his-tagged S-RBD protein of Example 2;

[0034] Figure 4 Kinetic binding and dissociation processes and simulation curves of his-tagged S-RBD protein and S-RBD antibody of Example 2;

[0035] Figure 5 Kinetic binding processes and simulation curves of Protein A and Fc-tagged ACE2 of Example 3;

[0036] Figure 6 Kinetic binding processes and simulation curves of Fc-tagged ACE2 and RBD protein of Example 3;

[0037] Figure 7 Kinetic binding and dissociation processes and simulation curves of SARS-CoV-2 N protein antibody and N protein of Example 4;

[0038] Figure 8 Kinetic binding and dissociation processes and simulation curves of wild-type SARS-CoV-2 RBD protein and Fc-tagged ACE2 of Example 5;

[0039] Figure 9 Kinetic binding processes and simulation curves of RBD protein of SARS-CoV-2 mutant and Fc-tagged ACE2 protein of Example 5;

[0040] Figure 10 OD value column chart of sandwich method screening antibody pairs of Example 6;

[0041] Figure 11 Binding kinetics chart of screening S protein antibody pairs of different epitopes using NanoSPR chip of Example 7;

[0042] Figure 12 Binding kinetics chart of screening S protein antibody pairs of different epitopes using NanoSPR chip combined with N protein antigen of Example 8. DETAILED DESCRIPTION

[0043] The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application.

[0044] The following embodiments are detected by using a microwell plate integrated with a NanoSPR chip (purchased from Junco (Shanghai) Medical Instrument Co., Ltd.) or by using a NanoSPR chip assembly and a microwell plate (such as shown in Figure 1 The used NanoSPR chip assembly is composed of 8 NanoSPR chip columns arranged in a row, and each NanoSPR chip column has a column head top fixedly pasted with a NanoSPR chip loaded thereon. The microwell plate is a 96-well microwell plate, and 96 microwells are arranged in a matrix on the microwell plate, with 8 microwells in each row and 12 rows in total. The bottom of each microwell is made of transparent material to facilitate the irradiation of the optical fiber probe of the full-spectrum spectrometer. The reagents added to each row of microwells are as follows:

[0045] The first and second rows of microwells: 150 μl of washing liquid, double distilled water;

[0046] The third row of microwells: 100 μl of fixed ligand working solution;

[0047] The fourth row of microwells: 150 μl of blocking solution;

[0048] The fifth row of microwells: 150 μl of protection solution;

[0049] The sixth row of microwells: fixed-concentration capture protein working solution (universal chip method);

[0050] The seventh row of microwells: analyte working solution to be detected;

[0051] The eighth row of microwells: 100 μl of dissociation solution; the ninth and tenth rows of microwells: 100 μl of regeneration solution each;

[0052] The eleventh and twelfth rows of microwells: PBST buffer, washing liquid.

[0053] Among the above reagents, the washing liquid is ultrapure water; the blocking solution is prepared by dissolving bovine serum albumin in a CBS buffer to form a 10 μg / ml complex solution, and then placing the complex solution at room temperature for 1 h; the protection solution is prepared by dissolving sucrose in a PBST buffer to form a 100 μg / ml complex solution, and the protection method is to protect at 37°C for 30 min;

[0054] The raw material composition of the CBS buffer solution is: potassium carbonate 8-12 g, sodium bicarbonate 15-20 g, preservative Proclin 300 5-10 ml, and purified water 500 ml.

[0055] The raw material composition of the PBS buffer solution is: disodium hydrogen phosphate dodecahydrate 29 g, sodium dihydrogen phosphate 2.72 g, potassium chloride 2 g, sodium chloride 80 g, preservative Proclin 300 5 ml, and double distilled water to 10 L.

[0056] The raw material composition of the PBST buffer solution is: disodium hydrogen phosphate dodecahydrate 29 g, sodium dihydrogen phosphate 2.72 g, potassium chloride 2 g, sodium chloride 80 g, Tween-20 5 ml, preservative Proclin 300 5 ml, and double distilled water to 10 L.

[0057] The raw material composition of the HBS-ET buffer solution is: 4-hydroxyethyl piperazine ethanesulfonic acid 0.12 g, sodium chloride 0.44 g, ethylenediaminetetraacetic acid 43.84 mg, and Tween 250 μl.

[0058] The dissociation solution is selected to be a CBS buffer solution containing Tween-20, and the concentration of Tween-80 is generally 0.5%. The regeneration solution is selected to be a PBS buffer solution containing Tween-20, and the concentration of Tween-80 is generally 0.5%.

[0059] The NanoSPR chip used is produced by Liangzhun (Shanghai) Medical Instrument Co., Ltd., and the surface of the base plate is imprinted with a matrix array of nanopores. The top diameter of each nanopore is about 200 nm, the bottom diameter is about 1000 nm, and the height is about 500 nm. The spacing between adjacent nanopores is about 1000 nm. Then, from bottom to top, a titanium film layer with a thickness of 10 nm, a silver film layer with a thickness of 20 nm, and a gold film layer with a thickness of 10 nm are sequentially plated.

[0060] Example 1: Measuring the equilibrium affinity constant K of tumor marker Her2 using the NanoSPR chip microplate D

[0061] 1. Experimental process

[0062] (1) Add 150 μl of washing solution to each microwell of the microwell plate integrated with the NanoSPR chip, and gently shake by hand for 6 times for cleaning, and then discard the waste liquid; repeat twice, and dry at room temperature for standby use;

[0063] (2) Dilute Herceptin (purchased from Beijing Baisaisi Biological Technology Co., Ltd.) to 15 μg / ml to prepare a working solution of the fixed ligand, and add 100 μl of the working solution of the fixed ligand to the microwell to cover the film, and then incubate overnight at 4°C in the refrigerator;

[0064] (3) Add 150 μl blocking solution to each microwell, and let stand at room temperature for 1 h, then discard the waste solution; add 150 μl protection solution, and protect at 37 °C for 30 min, then discard the waste solution, and let stand to dry;

[0065] (4) After washing twice with PBST buffer, dilute Her2 protein (human epidermal growth factor receptor 2, tumor marker, purchased from Beijing Bips Biotech Co., Ltd., analyte to be tested) to different concentrations (0.125, 0.25, 0.5, 1, 2, 4 μg / ml) with PBST buffer to prepare analyte working solution to be tested; take 100 μl of the analyte working solution to be tested of the six concentrations one by one, and add to each microwell of step (3); immediately use XLement SPR100 analyzer to start kinetic detection, and obtain raw data; use full-spectrum scanning to obtain the best wavelengths of 575 nm and 600 nm, combine at room temperature for 10 min, and dissociate for 15 min;

[0066] (6) Use origin 8.0 software to fit the raw data and obtain the fitting equation of affinity constant (K on ) and dissociation constant (K off ), respectively,

[0067]

[0068]

[0069] Then, according to the formula fitting, the fitting equation of the equilibrium affinity constant K D of the analyte Her2 protein is obtained.

[0070] Among them, K on is the association rate constant, K off is the dissociation rate constant; [B] is the concentration of Her2 protein; and a is the asymptote.

[0071] 2. Detection results

[0072] In this experiment, Herceptin was fixed on the surface of the NanoSPR chip to detect Her2 protein. The kinetic binding and dissociation process of Herceptin and Her2 protein and the simulation curve are shown in Figure 2 , and the K D of the two is obtained by simulation calculation.The value is 0.83 nM. The experimental results are the same as the experimental results provided by the raw material purchasing company (Beijing Bips Biotech Co., Ltd.) (0.83 nM, data source https: / / acrobiosystems.cn / P421-Human_Her2_%7C_ErbB2_Protein_His_Tag_MALS_verified.html). This shows that the NanoSPR chip microplate of the application is reliable and usable for kinetic detection.

[0073] Example 2: NanoSPR chip microplate detects the equilibrium affinity constant K of the S protein of the new coronavirus and the RBD antibody D

[0074] 1. Experimental process

[0075] (1) Add 150 μl of double distilled water to the 96-well NanoSPR chip, shake gently, discard the waste liquid, repeat once, and dry at room temperature for standby;

[0076] (2) Take the anti-His tag antibody (purchased from Beijing Yiqiao God Science and Technology Co., Ltd., fixed ligand) to 15 μg / ml to prepare a fixed ligand working solution, take 50 μl of the fixed ligand working solution to sample in the center of each microwell, and after covering the film, it is placed in the refrigerator at 4°C overnight;

[0077] (3) Discard the waste liquid in the microwell, add 150 μl of blocking solution to block at 37°C for 1 h, discard the waste liquid; add 150 μl of protective liquid to protect at 37°C for 30 min, then discard the waste liquid, and dry for standby.

[0078] (4) After washing twice with PBST buffer, add HBS-ET buffer to dilute the S-RBD protein with his tag (purchased from Beijing Yiqiao God Science and Technology Co., Ltd., antigen) to 20 μg / ml to prepare a working solution of the analyte capture protein to be tested; start the kinetic detection for 10 min with XLement SPR100 analyzer, shake at room temperature, then wash twice with PBST buffer for standby;

[0079] (5) PBST buffer was used to dilute the new coronavirus S-RBD antibody (analyte to be tested, antibody to receptor binding domain RBD, purchased from Beijing Yiqiao God Science and Technology Co., Ltd., which can bind to his-tagged new coronavirus S-RBD protein) to 7 different concentrations (1, 2, 4, 8, 16, 32, 64 μg / ml), 100 μl of 7 concentrations of D001 antibody protein (Beijing Yiqiao God Science and Technology Co., Ltd.) was added to the microwells, and then immediately placed in the XLement SPR100 analyzer, and the kinetic detection was started at 575 nm and 600 nm wavelengths. After 5 min of binding at room temperature, the dissociation solution was added and dissociated at room temperature for 15 min;

[0080] (6) The fitting equation of the association rate constant K on and the dissociation rate constant K off was obtained by using Origin 8.0 software, and the fitting method was the same as in Example 1.

[0081] 2, detection results

[0082] The kinetic binding and dissociation process of the anti-His tag antibody and the his-tagged new coronavirus S-RBD protein is shown in Figure 3 , and the kinetic binding and dissociation process of the his-tagged new coronavirus S-RBD protein and the new coronavirus S-RBD antibody is shown in Figure 4 . As can be seen from Figure 3 , 4 , the sandwich method of the present embodiment can accurately dynamically detect the dynamic binding and dissociation process of the anti-His tag antibody and the new coronavirus S-RBD protein, and the new coronavirus S-RBD protein and the new coronavirus S-RBD antibody, and can real-time monitor the detection process of the protein.

[0083] The K D of both obtained by Origin software simulation is 0.44 nM, which is similar to the reported results in the literature (Tan X, Lin C, Zhang J, et al. Rapid and quantitative detection of COVID-19 markers in micro-liter sized samples. 2020). The kinetic curve of the new coronavirus S-RBD protein connected to the anti-His tag antibody is shown in the following figure, indicating that the amount of new coronavirus S-RBD protein bound in each chip well is similar, and the consistency of different chip wells is good.

[0084] Example 3: NanoSPR chip microwell plate detects the equilibrium affinity constant K D

[0085] 1. Experimental process

[0086] (1) Add 150 μl double distilled water to the 96-well NanoSPR chip, shake gently, discard the waste liquid, repeat once, and dry at room temperature for standby;

[0087] (2) Dilute Protein A to 15 μg / ml with CBS buffer, mix well, take 50 μl sample and add to the center of each well, cover the film and incubate at 4°C overnight for standby;

[0088] (3) Discard the sample, add 150 μl blocking solution at 37°C for 1 h, then discard the waste liquid, add 150 μl protective liquid at 37°C for 30 min, discard the waste liquid, and dry for standby.

[0089] (4) After washing twice with PBST buffer, add 25 μl HBS-ET buffer to dilute the Fc-tagged ACE2 (Angiotensin Converting Enzyme 2, purchased from Jinser Biological Technology Co., Ltd.) to 20 μg / ml to prepare the protein capture working solution; start kinetic detection for 10 min with XLement SPR100 analyzer, shake at room temperature, then wash twice with PBST buffer for standby;

[0090] (5) Dilute RBD protein (novel coronavirus protein, purchased from Nanjing Bai Kang Biological Technology Co., Ltd.) to 7 different concentrations (1, 2, 4, 8, 16, 32, 64 μg / ml) with PBST buffer, add 100 μl of RBD protein at 7 concentrations to the microwells, immediately put into the XLement SPR100 spectrometer, start kinetic detection, and obtain the original data; use full-spectrum scanning to obtain the best wavelengths of 575 nm and 600 nm, bind for 5 min at room temperature, and dissociate for 15 min;

[0091] (6) Use Origin 8.0 software to obtain the fitting equation of affinity constant (K on ) and dissociation constant (K off ), which is the same as the fitting method of Example 1.

[0092] 2. Detection results

[0093] In this experiment, Protein A was directly immobilized on the chip surface, first by directional binding of Fc-tagged ACE2, then by directional binding of RBD protein at different concentrations, and the affinity was detected. The kinetic binding process and simulation curve of Protein A and Fc-tagged ACE2 are shown in Figure 5 , and the kinetic binding and dissociation process and simulation curve of Fc-tagged ACE2 and RBD protein are shown in Figure 6 .

[0094] Figure 5 In this embodiment, the Fc-tagged ACE2 is directly immobilized on the chip surface, and then different concentrations of RBD are detected, and the amount of Fc-tagged ACE2 bound in each chip well is similar. The RBD of different concentrations from manufacturer A is detected by using a common enzyme label instrument, and the kinetic curves of the two are obtained, and the Kd of Fc-tagged ACE2 and RBD is obtained by simulation. D The value is 0.98nM. The experimental results are consistent with those provided by the purchase company (Wuhan Yanjin Biotechnology Co., Ltd.). This shows that the experimental results of the kinetic detection of this embodiment using NanoSPR are reliable and usable.

[0095] Example 4: One-step detection of equilibrium affinity constant Kd of nucleocapsid protein (N protein) using NanoSPR chip assembly and microwell plate D

[0096] 1. Experimental process

[0097] (1) Insert each NanoSPR chip column (hereinafter referred to as chip column) of the NanoSPR chip assembly (hereinafter referred to as chip assembly) into the first row of microwells, and gently shake by hand for 6 times for cleaning. Take out the chip assembly and discard the waste liquid; insert it into the second row of microwells and clean it in the same way. Dry at room temperature for standby;

[0098] (2) Dilute SARS-CoV-2 N protein antibody (immobilized ligand, purchased from Beijing Yiqiao God Science and Technology Co., Ltd.) to 15μg / ml with CBS buffer to prepare immobilized ligand working solution. Take 100μl of immobilized ligand working solution and add it to each microwell in the third row. Insert the chip column to coat the film. After coating, incubate overnight at 4℃ in the refrigerator;

[0099] (3) Take out the chip column and discard the waste liquid. Insert it into the fourth row of microwells (blocking solution) and place it at room temperature for 1h. Discard the waste liquid. Insert it into the fifth row of microwells (protective solution) and protect it at 37℃ for 30min. Discard the waste liquid and dry it for standby;

[0100] (4) Dilute N protein (nucleocapsid protein, purchased from Beijing Yiqiao God Science and Technology Co., Ltd.) with PBST buffer to prepare six concentrations (1, 2, 4, 8, 16, 32μg / ml) of antibody protein working solution. Take 100μl of each of the six concentrations of antibody protein working solution and add it to the microwells in the sixth row. Insert the chip column quickly and immediately place it in the XLementSPR100 spectrometer to start the kinetic detection and obtain the original data. Use full-spectrum scanning to obtain the best wavelengths of 575nm and 600nm. Combine at room temperature for 5min. Place the chip column in the dissociation solution (eighth row of microwells) and dissociate for 15min;

[0101] (5) Obtain the association rate constant Kd using Origin 8.0 softwareon and dissociation rate constant K off The fitting equation is the same as that in Embodiment 1.

[0102] (6) When the chip column and the chip need to be recycled, the chips of the chip column are eluted in the micro-holes of the 9th and 10th rows in turn, so as to be reused;

[0103] (7) The used chip column can be additionally soaked in a sucrose (PBS buffer) solution with a certain concentration for 2-15 min, dried and sealed for storage.

[0104] 2. Test results

[0105] The kinetic binding and dissociation process of the SARS-CoV-2 N protein antibody and the N protein and the simulation curve are shown in Figure 7 From Figure 7 , it can be seen that the NanoSPR one-step method of the embodiment can well detect N proteins of different concentrations, and the equilibrium affinity constant K D of the two obtained by Origin software simulation is 1.40 nM, which is very close to the K D value provided by the purchasing company.

[0106] Embodiment 5: Using the NanoSPR chip assembly and the micro-hole plate to detect the equilibrium affinity constant K D

[0107] 1. Experimental process

[0108] (1) Each chip column of the chip assembly was cleaned and dried at room temperature for standby, and the cleaning method was the same as that in step (1) of Embodiment 4;

[0109] (2) Protein A (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., fixed ligand) was diluted with CBS buffer to 15 μg / ml to prepare a fixed ligand working solution, 100 μl of the fixed ligand working solution was taken and added to each micro-hole of the 3rd row, and the chip column was inserted to coat the film, and after coating, it was placed in a refrigerator at 4℃ overnight;

[0110] (3) The chip column was taken out and the waste liquid was shaken off, and then inserted into the 4th row of micro-holes (150 μl of blocking solution) and placed at 37℃ for 2 h, and the waste liquid was shaken off; then inserted into the 5th row of micro-holes (150 μl of protection solution) and protected at 37℃ for 30 min, and the waste liquid was shaken off, dried and standby;

[0111] (4) After washing twice with PBST buffer (the 11th row of microwells), dilute the Fc-tagged ACE2 (angiotensin-converting enzyme 2, purchased from Jinserui Biotechnology Co., Ltd.) into HBS-ET buffer to 20 μg / ml to prepare the test protein capture working solution; take 25 μl of the test protein capture working solution and add it to the 6th row of microwells, shake at room temperature for 10 min, and then wash twice with PBST buffer (the 11th and 12th rows of microwells) for standby;

[0112] (5) Dilute the RBD protein (novel coronavirus protein, purchased from Wuhan Yanjin Biotechnology Co., Ltd., which is the RBD protein of the mutant strain of the novel coronavirus) into PBST buffer to 7 different concentrations (1, 2, 4, 8, 16, 32, and 64 μg / ml), and add 100 μl of the RBD protein at the seven concentrations to the 7th row of microwells, insert the chip column, and then immediately put it into the XLement SPR100 analyzer, start the kinetic detection using 575 nm and 600 nm wavelengths, bind at room temperature for 5 min, and then put it into the dissociation solution (the 8th row of microwells) for dissociation for 15 min;

[0113] In addition, the same detection steps were used to simultaneously detect different concentrations of wild-type RBD protein of the novel coronavirus (purchased from Wuhan Yanjin Biotechnology Co., Ltd.) (1, 2, 4, 8, and 16 μg / ml);

[0114] (6) The binding rate constant K on and the dissociation rate constant K off were obtained using Origin 8.0 software, and the fitting equation was the same as the fitting method in Example 1.

[0115] 2. Detection results

[0116] In this experiment, the kinetic binding process and simulation curve of the Fc-tagged ACE2 and the wild-type RBD protein of the novel coronavirus are shown in Figure 8 , the kinetic binding and dissociation process and simulation curve of the RBD protein of the mutant strain of the novel coronavirus and the Fc-tagged ACE2 are shown in Figure 9 , and the kinetic binding process and simulation curve of Pro A and the Fc-tagged ACE2 have been measured in Example 3, so they are omitted. Figure 8 、 9 In the above-mentioned two K D values are 2.05 nM and 3.07 nM, respectively; it is shown that the affinity of the wild-type RBD protein to ACE2 is greater than that of the RBD protein of the mutant strain of the novel coronavirus to ACE2, and the affinities of the two are similar to the results detected by the microwell plate method.

[0117] Example 6: Screening of antibodies using a microwell sandwich method

[0118] 1. Screening method:

[0119] (1) Add 150 μl of washing solution to each microwell of the microwell plate integrated with the NanoSPR chip, gently shake by hand for 6 times for cleaning, and discard the waste liquid; repeat twice, and dry at room temperature for standby;

[0120] (2) Dilute the first antibody (new coronavirus N protein antibody, purchased from Beijing Yiqioshen State Technology Co., Ltd.) to 20 μg / ml with CBS buffer to prepare the working solution of the fixed ligand, take 2 μl and add it to the microwell to coat the film, and incubate at 4℃ overnight;

[0121] (3) Add 150 μl of blocking solution to each microwell, and incubate at 37℃ for 1 h, then discard the waste liquid; add 150 μl of protective solution (PBST, 5% sucrose), and protect at 37℃ for 1 h, then discard the waste liquid and dry for standby;

[0122] Blocking solution (PBS, 1% BSA, 3% ZT, 0.1% Tween-20, pH 8.0);

[0123] (4) Wash the microwell plate with 150 μl of PBST buffer solution for 1 time, add 50 μl of PBST buffer solution, and read the wavelength starting point at 500-700 nm;

[0124] (5) Dilute the sandwich antigen to be tested (new coronavirus N protein antigen, purchased from Beijing Yiqioshen State Technology Co., Ltd.) to 15 μg / ml with diluent (PBS, 1% PEG6000), and add 50 μl to the microwell, respectively, and shake the plate for 10 min;

[0125] (6) Discard the combined sample antigen sample in the hole, wash with PBST buffer solution for 3 times to completely wash away the uncombined antigen; add 50ul of PBST buffer solution to each hole, respectively, and read the OD value starting point at 575 nm and 595 nm wavelength;

[0126] (7) Dilute the antibody mAb (purchased from Jinersi Biological Technology Co., Ltd.) with different binding sites to 10 μg / ml with diluent, respectively, and add 50 μl to the microwell, respectively, and shake the plate for 10 min;

[0127] (8) Discard the liquid in the hole and wash it with 150 μl of PBST for 1 time, add 50 μl of PBST solution to each hole, respectively, and read the OD value starting point at 575 nm and 595 nm wavelength;

[0128] 2. Screening results

[0129] As Figure 10As shown, the size of the OD difference (OD end value minus OD start value) can be used to determine whether different antibody mAbs have different epitopes with the first antibody and whether they can be used as an antibody pair. The larger the resonance peak reaction, the better the pairing effect of the antibody mAb and the first antibody, i.e., the antibody pair can be used to detect the antigen by sandwich method. The smaller the resonance peak reaction, the worse the pairing effect of the antibody mAb and the first antibody. No resonance peak reaction indicates that the antibody mAb and the first antibody have the same binding site with the sandwich antigen to be detected, and cannot be used as an antibody pair to detect the antigen by sandwich method.

[0130] Example 7: Screening of new coronavirus S protein antibodies with different epitopes by using the outside-premixing method of microwell plate

[0131] 1. Screening method

[0132] (1) Add 150 μl of washing solution to each microwell of the microwell plate integrated with NanoSPR chip, gently shake by hand for 6 times for cleaning, and discard the waste liquid; repeat twice, and dry at room temperature for standby;

[0133] (2) Dilute the first antibody (purchased from Beijing Yiqiao God Science and Technology Co., Ltd.) to 20 μg / ml with CBS buffer to prepare a working solution of fixed ligand, take 2 ul and add it to the microwell to coat the film, and incubate at 4°C overnight;

[0134] (3) Add 150 μl of blocking solution to each microwell, incubate at 37°C for 1 h, and discard the waste liquid; then add 150 μl of protective solution (PBST, 5% ZT), protect at 37°C for 1 h, discard the waste liquid, and dry for standby;

[0135] Blocking solution (PBS, 1% BSA, 3% ZT, 0.1% Tween-20, pH 8.0);

[0136] (4) Wash the microwell plate with 150 μl of PBST buffer solution for 1 time, add 50 μl of PBST buffer solution, and read the wavelength start at 500-700 nm;

[0137] (5) Dilute RBD protein (V08H, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.) with diluent (PBS, 1% PEG6000) to 7.5 μg / ml, and add 40 μl to each microwell of the EP tube or microwell plate; dilute SARS-CoV-2 S protein antibodies S1-23mAb and S1-29mAb (purchased from Hangzhou Xianzhi Biological Technology Co., Ltd.), S1-D001mAb and S1-D003mAb (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.), and S1-2B1mAb (purchased from Chongqing Taosheng Technology Co., Ltd.) with different binding sites to 20 μg / ml, and add 50 μl to each microwell of the corresponding EP tube or microwell plate, respectively, and react at 37°C and 1000 rpm for 10 min;

[0138] (6) Add 50 μl of the reaction solution of step (5) to different chip microwell plates, and immediately place them in an XLement SRP100 analyzer for kinetic shaking detection at room temperature for 10 min;

[0139] 2. Screening results

[0140] As shown in Figure 11 , compared with the curve of S1-23mAb, the larger the kinetic curve reaction, the more different the binding sites of the corresponding SARS-CoV-2 S protein antibody and RBD protein from those of S1-23mAb and RBD; the smaller the curve, the more similar the binding sites; and the smooth kinetic curve without binding indicates that the binding sites are the same.

[0141] Example 8: Screening of SARS-CoV-2 S protein antibodies with different epitopes by using a microwell plate in series

[0142] 1. Screening method

[0143] (1) Add 150 μl of washing solution to each microwell of the microwell plate integrated with a NanoSPR chip, and gently shake by hand for 6 times to clean and remove the waste liquid; repeat twice, and dry at room temperature for standby;

[0144] (2) Dilute RBD protein (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.) with CBS buffer to 20 μg / ml to prepare a working solution of immobilized ligand, and add 2 μl to each microwell to coat the film, and incubate at 4°C overnight;

[0145] (3) Add 150 μl of blocking solution to each microwell, and incubate at 37°C for 1 h, and then discard the waste liquid; then add 150 μl of protective solution (PBST, 5% ZT), and protect at 37°C for 1 h, and then discard the waste liquid and dry for standby;

[0146] Blocking solution (PBS, 1% BSA, 3% ZT, 0.1% Tween-20, pH 8.0)

[0147] (4) Wash once with 150 μl PBST buffer for microplate;

[0148] (5) Dilute the new crown S protein antibody S1-D001 mAb (purchased from Beijing Yiqiao God Science and Technology Co., Ltd.) to 30 μg / ml with diluent (PBS, 1% PEG6000), add 50 μl per well, and place it in the XLement SRP100 analyzer. Kinetic shaking detection for 10 min at room temperature;

[0149] (6) Take out the microplate, wash once with PBST buffer, dilute different manufacturers' S1 mAb (Hangzhou Xianzhi's S1-23 mAb, S1-29 mAb, Beijing Yiqiao's S1-D001 mAb, S1-D003 mAb, Chongqing Taisheng's S1-2B1 mAb) to 30 μg / ml, add 50 μl per well, and place it in the XLement SRP100 analyzer. Kinetic shaking detection for 10 min at room temperature;

[0150] 2. Screening results

[0151] As shown in Figure 12 , compared with the curve of S1-D001 mAb, the kinetic curve reaction is large, which indicates that the corresponding new crown S protein antibody and RBD protein binding site are different from S1-D001 mAb and RBD; The curve with small reaction indicates that the binding site is partially the same; The kinetic curve is smooth without binding, which indicates that the binding site is the same.

[0152] From the screening detection results of the above embodiments 1-8, it can be found that the detection method provided by the present application can effectively detect the affinity degree of various antibody proteins and receptors / antigens, and has very high accuracy and reliability. By using this method, the best antibody protein drug can be effectively screened, and the screening, characterization and other detection evaluation of antibody drugs can be completed.

Claims

1. A method for detection of drug screening and characterization based on nanosurface plasmon resonance technology, characterized in that, First, the NanoSPR chip is coated with a fixed ligand, and then the analyte to be detected is combined with the NanoSPR chip to obtain the binding kinetics curve and the dissociation kinetics curve, and finally the drug screening and characterization detection are performed. The specific steps include: S1, washing a plurality of NanoSPR chips, adding a fixed ligand working solution to the NanoSPR chip, and standing after coating; discarding the waste fixed ligand working solution, and sequentially adding a blocking solution and a protection solution for blocking and protection, and then discarding the waste protection solution and drying for use; S2, preparing different concentration gradients of the analyte working solution, and adding the analyte working solution with different concentrations to the NanoSPR chip obtained in step S1; S3, using 575nm and 600nm wavelengths for kinetics detection, and measuring the binding kinetics curve for 5-15min; then measuring the dissociation kinetics curve for 15-30min; finally, using computer software, the equilibrium affinity constant is obtained by fitting the binding kinetics curve equation and the dissociation kinetics curve equation; In step S3, the fitting equation of the binding rate constant and the dissociation rate constant is first obtained by computer software fitting, which is ； Then according to the formula The fitting equation of the equilibrium affinity constant K of the analyte to be tested is obtained D ; wherein [B] is the concentration of the protein to be tested, and a1 and a2 are asymptote constants.

2. The method of claim 1, wherein the method is a method of drug screening and characterization based on nanosurface plasmon resonance technology. In step S1, the blocking solution is a composite solution prepared by dissolving at least one of bovine serum albumin, casein, milk, and polyethylene glycol 20000 in a buffer solution, and the blocking method is to stand at room temperature for 1-2h; the protection solution is a composite solution prepared by dissolving a sugar in a buffer solution, and the protection method is to protect at 37℃ for 5-30min.

3. The method of claim 2, wherein the method is a method of drug screening and characterization based on nanosurface plasmon resonance technology. In step S1, the buffer solution used is CBS buffer solution, PBS buffer solution, TBS buffer solution, Hanks buffer solution, or HEPES.

4. The method of claim 1, wherein the method is a method of drug screening and characterization based on nanosurface plasmon resonance technology. In step S3, the dissociation solution used in the dissociation process is CBS buffer solution, PBS buffer solution, TBS buffer solution, Hanks buffer solution, or HEPES buffer solution.

5. The method of claim 4, wherein the method is a method of drug screening and characterization by nanosurface plasmon resonance technology. In step S3, the buffer solution used for dissociation contains a surfactant.

6. The method of detection of drug screening and characterization based on nano surface plasmon resonance technology according to any one of claims 1-5, characterized in that, The detection is performed using a microwell plate integrated with a NanoSPR chip, and the NanoSPR chip is integrated on the bottom surface of each microwell of the microwell plate; Alternatively, a NanoSPR chip assembly and a microwell plate are used for detection, the NanoSPR chip assembly contains at least one row of a plurality of NanoSPR chip columns, and the top of each NanoSPR chip column is loaded with a NanoSPR chip; the microwell plate contains a plurality of microwells arranged in a matrix, and the bottom surface of each microwell is made of transparent material; each row of microwells of the microwell plate is used to load a washing solution, a fixed ligand working solution, a blocking solution, a protection solution, different concentration gradients of an analyte working solution, and a dissociation solution.

7. The method of claim 1, wherein the method is a method of drug screening and characterization based on nanosurface plasmon resonance technology. After step S3, the regeneration method of the NanoSPR chip is also included, which is to elute the NanoSPR chip after detection with an elution regeneration solution for 1-5 times, so that the NanoSPR chip can be repeatedly used.

8. The method of claim 1, wherein the method is a method of drug screening and characterization based on nanosurface plasmon resonance technology. The drug screening and characterization detection are performed using a sandwich method, a series connection method, or a premix method.

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