Antibody chip and surface plasmon resonance imaging combined high-throughput antibody affinity determination method
By combining antibody chips with surface plasmon resonance imaging (SPRi), the problems of low throughput, label dependence, and limited sensitivity in existing antibody affinity detection methods have been solved, achieving efficient and accurate high-throughput antibody affinity determination.
Patent Information
- Application Number
- CN202511155251.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-18
- Publication Date
- 2025-11-14
Smart Images
Figure CN120948419A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biochip technology, and mainly to a high-throughput method for determining antibody affinity based on the combined use of antibody chips and surface plasmon resonance imaging (SPRi). Background Technology
[0002] Antibody microarray technology, as an important tool in proteomics research, has shown great application potential in the biomedical field in recent years. Antibody microarrays immobilize specific antibodies in the form of microarrays on a solid support (such as a glass slide, nylon membrane, or polymer material), capturing target proteins in the sample and enabling qualitative and quantitative analysis of the target proteins by combining labeled signals (fluorescence, chemiluminescence, or enzyme-linked colorimetry). Signal readout techniques include fluorescence scanning, chemiluminescence, and surface plasmon resonance (SPR).
[0003] The core shortcomings of existing antibody affinity detection methods are as follows:
[0004] 1. ELISA (Enzyme-Linked Immunosorbent Assay)
[0005] It only provides endpoint data (kinetic parameters cannot be obtained), relies on labeled secondary antibodies to introduce errors, has low throughput, high sample consumption, limited sensitivity, and narrow dynamic range.
[0006] 2. Traditional SPR (such as Biacore)
[0007] With limited throughput, a single experiment typically tests 4-8 channels, and high-throughput modes (such as MX96) are extremely expensive (>1 million USD).
[0008] 3. BLI (Biolayer Interference Technology, such as Octet)
[0009] It is mainly semi-quantitative, and the accuracy of kinetic data is lower than that of SPR, especially its ability to resolve rapid binding / dissociation interactions is insufficient; there is a contradiction between throughput and sensitivity, and in high-throughput mode (such as 384 channels), the detection limit drops to the ng / mL level.
[0010] 4. ITC (Isothermal titration calorimetry)
[0011] It has extremely low throughput, with a single experiment taking several hours and only one pair of interactions can be analyzed; it requires large sample sizes and high concentrations of purified proteins (μM level), making it unsuitable for small clinical samples; it cannot distinguish between specific and non-specific bindings, and thermal changes may originate from multiple molecular interactions.
[0012] 5. Fluorescence polarization (FP)
[0013] Forced labeling and fluorescent labeling may alter antibody / antigen binding properties (e.g., steric hindrance); the dynamic range is limited, applicable only to medium affinity (K).D =10 -7 *10 -9 M) detection; low sensitivity, susceptible to autofluorescence interference, detection limit is usually in the nM range. Summary of the Invention
[0014] To address the problems existing in antibody affinity detection in the background art, this invention provides a high-throughput method for determining antibody affinity. This invention designs an antibody chip-surface plasmon resonance imaging (SPRi) coupled method for high-throughput antibody affinity detection, involving construction methods, applications, and detection methods, solving the technical problems of traditional methods (such as ELISA and BLI) due to low throughput, label dependence, or data limitations.
[0015] The technical solution adopted in this invention is:
[0016] The method involves placing the antibody chip in a surface plasmon resonance imaging instrument to measure and output the antibody affinity measurement results; the antibody chip is mainly composed of a photocrosslinked chip and an antibody.
[0017] The high throughput of this invention can achieve 3840 spots in the chip spotting area. If each sample is spotted with 2 replicate spots, then 1920 antibodies can be spotted, and 1920 antibody samples can be detected in a single run.
[0018] The antibody comprises several different antibody molecules, and the number can reach thousands.
[0019] In the antibody chip, the antibody is chemically bonded to the photocrosslinked chip.
[0020] In the antibody chip, several different antibody molecules are dotted onto the photocrosslinked chip via an Arrayjet device, so that several different antibody molecules exist simultaneously in the antibody chip.
[0021] In practice, the antibody concentration was standardized to 0.5 mg / mL, the solvent was PBS, and the antibody was transferred to a 384-well plate. The chip and the 384-well plate were placed in the designated position on the Arrayjet instrument. The photocrosslinking chip needed to be handled in the dark. After setting the spotting parameters, the instrument was started. The spotted chip was placed in a cold storage and allowed to stand overnight for fixation. Then, it was irradiated with a 365 nm UV lamp for 30 min to covalently crosslink the antibody to the chip surface. The chip was then washed sequentially with PBST (pH 7.4, 0.05% Tween 20), PBS, and deionized water for 15 min each. After blocking with 10 mg / mL BSA for 2 h, the chip was washed again with PBST (pH 7.4, 0.05% Tween 20), PBS, and deionized water for 15 min each. Finally, the chip was dried under nitrogen and stored for later use.
[0022] The chip is attached to the flow cell and then mounted onto the surface plasmon resonance imaging instrument. On the HTSystem, the program was set to sequentially flow gradient concentrations of HER2 extracellular fragment protein (1 nM-128 nM). When switching concentrations, the chip needed to be regenerated using 10 mM Gly-HCl (pH 2.0) and PBS containing 1% BSA. After the experiment, the surface plasmon resonance imaging (SCI) instrument output signal values. Based on the relationship between HER2 extracellular fragment protein concentration and signal value, the affinity K was fitted using the Langmuir adsorption equation. D value.
[0023] The antibody molecules are then chemically bonded onto the photocrosslinked chip in a solution with a concentration of 0.5-1 mg / mL using an Arrayjet device.
[0024] The aforementioned photocrosslinked chip refers to a chip in which antibodies dotted on the chip surface form covalent bonds with the chip surface through ultraviolet irradiation.
[0025] In practice, the optical crosslinking chip is from Suzhou Puxin Life Science Technology Co., Ltd.
[0026] In practice, the photocrosslinking chip is equipped with chemical bonds containing a blank control for experimental comparison. The blank control group is PBS, the solvent of the antibody.
[0027] The antibodies mentioned include fully human antibodies, mouse monoclonal antibodies, and / or rabbit monoclonal antibodies.
[0028] Specifically, HER2 antibodies in IgG form are used, including two fully human antibodies, two mouse monoclonal antibodies, and 39 rabbit monoclonal antibodies. The antibody species can be expanded to other species, and the types can include Fab, scFv, nanobody, and other antibody types.
[0029] For example, the antibodies are two mouse monoclonal antibodies (m66 and m75) and 39 rabbit monoclonal antibodies (r1-r40) obtained from mice and rabbits immunized with the extracellular domain (ECD) of the HER2 protein, as well as two monoclonal antibody drugs targeting HER2, trastuzumab and pertuzumab.
[0030] The affinity K is determined by fitting the Langmuir adsorption equation to the output signal value of the surface plasmon resonance imaging instrument based on the relationship between concentration and signal. D value.
[0031] The antibody chip provided by this invention is mainly composed of a photocrosslinked chip and an antibody chemically bonded together, with a buffer solution as a negative control.
[0032] This invention allows for the batch acquisition of affinity values for antibodies binding to antigens by circulating antigens at gradient concentrations. Furthermore, the surface plasmon resonance imaging instrument can simultaneously measure the affinity results of several different antibody molecules, thereby significantly improving efficiency and reducing measurement costs.
[0033] The antibody chip-SPRi coupling method provided by this invention for detecting antibody affinity has the advantages of simple operation, accurate detection results, low cost, and stable performance.
[0034] The beneficial effects of this invention are:
[0035] This invention uses the Arrayjet biochip spotting instrument to prepare antibody chips, and is based on the SPRi technology route of PlexArray. T The HT molecular interaction system uses gradient concentrations of antigen as the mobile phase to obtain batch antibody affinity parameters.
[0036] The innovative aspects and core technological breakthroughs of combining antibody chips with surface plasmon resonance imaging (SPRi) are as follows:
[0037] 1) Fusion of high-throughput parallel detection and dynamic data acquisition: Combining high-density antibody chips (hundreds to thousands of antibody / antigen microarrays) with SPRi wide-area imaging technology, real-time dynamic monitoring of hundreds of pairs of interactions can be achieved in a single experiment. The above processing solves the technical problems of traditional SPR (such as Biacore) being able to detect only 4-8 channels at a time and being unable to acquire dynamic parameters in ELISA / BLI high-throughput mode.
[0038] 2) Label-free and natural conformation preservation: The binding event is directly detected by utilizing the refractive index change of SPRi without the need for fluorescent / enzyme labeling, avoiding the interference of labeling on antibody-antigen conformation, and solving the problems of label-dependent ELISA and fluorescence polarization (FP) that may lead to false positives or masking of active sites.
[0039] 3) Ultra-high sensitivity and wide dynamic range: The detection limit is pushed to the fg / mL level by nano-plasma enhancement (such as gold nanoparticle modified chip), and combined with microfluidic gradient dilution to cover a dynamic range of 6 orders of magnitude, which solves the defects and problems of traditional SPR limited sensitivity (pg / mL) and sensitivity decrease (ng / mL) in BLI high-throughput mode. Attached Figure Description
[0040] Figure 1 The image shows the affinity results of four HER2 antibodies measured using the antibody chip-SPRi combination. The four antibodies are trastuzumab, pertuzumab, mouse monoclonal antibody (m66), and rabbit monoclonal antibody (r40).
[0041] Figure 2 ECG of 5 HER2 antibodies for ELISA 50 Result image.
[0042] Table 1 shows the affinity constants of 43 HER2 antibodies obtained by using the antibody chip-SPRi combination of the present invention. Detailed Implementation
[0043] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
[0044] The embodiments of the present invention are as follows:
[0045] Example 1:
[0046] A) The affinity of 43 antibodies for HER2 was determined using antibody chip-SPRi in high-throughput assay.
[0047] B) Using Arrayjet, 43 antibodies against HER2 (including trastuzumab, pertuzumab, two mouse monoclonal antibodies m66 and m75, and 39 rabbit monoclonal antibodies r1-r40) and blank controls (PBS buffer) were spotted onto an antibody chip, with 10 replicate spots.
[0048] C) Using Plexera PlexArray TM The HT molecular interaction system sequentially passed gradient concentrations of HER2ECD protein (1 nM, 2 nM, 4 nM, 8 nM, 16 nM, 32 nM, 64 nM, 128 nM), with a binding time of 285 s and a dissociation time of 930 s. The two regeneration buffers were 10 mM Gly-HCl pH 2.0 and PBS containing 1% BSA.
[0049] The test curves for the binding of four antibodies—trastuzumab, pertuzumab, m66, and r40—to HER2 are shown below. Figure 1 As shown. Figure 1 In the middle, the K of trastuzumab D The value was 0.283 nM, close to the reported ≈0.2 nM; the K value of pertuzumab was... D The value was 2.3 nM, close to the reported ≈3 nM, therefore it is believed that the K value measured by the antibody chip-SPRi coupling technology is... D The data is reliable.
[0050] Figure 1 As can be seen, the dissociation rate of m66 from HER2 protein is faster than that of the other three antibodies. Therefore, the affinity of m66 antibody for HER2 is 1-2 orders of magnitude lower than that of the other three antibodies.
[0051] Furthermore, 39 rabbit monoclonal antibodies (r1-r40), 2 mouse monoclonal antibodies (m66 and m75), and trastuzumab and pertuzumab were combined with HER2-bound K. D The results are shown in Table 1.
[0052] Table 1. Affinity constants of HER2 antibodies measured using antibody chip-SPRi co-processing.
[0053]
[0054] The above NaN indicates that no result can be detected.
[0055] Comparative Example 1:
[0056] EC50 of five HER2 antibodies was measured by ELISA. 50 .
[0057] The HER2 ECD protein was coated overnight at 0.2 μg / mL. After blocking, five antibodies were serially diluted 2-fold, with the highest concentration being 2 μg / mL. 3 μg / mL, serially diluted 2-fold to 2 μg / mL. -11 μg / mL, and ELISA was performed with the coated HER2 protein to calculate EC. 50 ( Figure 2 ).
[0058] Figure 2 As can be seen, the EC of the five antibodies 50 Value trend and K measured by SPRi D The trends were generally consistent: r40 showed the highest affinity for trastuzumab, followed by m75 and pertuzumab, with m66 showing the lowest affinity. The EC50 of an antibody was measured by ELISA. 50 48 wells are required, therefore a single 96-well plate can only measure EC of two antibodies. 50 .
[0059] But EC 50 Due to factors such as coating concentration, blocking conditions, and secondary antibody efficiency, it is impossible to obtain the true K. D Only obtaining endpoint data is possible, but it cannot detect dynamic changes in the binding process, and the throughput is low, the steps are cumbersome, and the operation time is long.
[0060] This invention combines SPRi technology with antibody microarrays, providing a highly sensitive, label-free, and real-time dynamic analysis platform for protein-protein interaction studies. The core advantages of the antibody microarray-SPRi co-processing technology are as follows:
[0061] 1. High-throughput and parallel detection capabilities:
[0062] The method of this invention can immobilize hundreds to thousands of antibodies on a single chip. Combined with SPRi's multi-point synchronous imaging technology, dozens to hundreds of interaction pairs can be analyzed simultaneously in a single experiment. SPRi does not require point-by-point scanning; it acquires binding / dissociation curves of all detection points in real time through wide-area imaging, significantly improving data acquisition efficiency (10-100 times faster than traditional SPR).
[0063] 2. Natural, unmarked, and highly sensitive with a wide range of sensitivity:
[0064] SPRi detection directly utilizes the refractive index change caused by molecular binding, avoiding interference from fluorescent / enzyme labeling on antibody-antigen conformation, preserving natural binding properties, and realizing label-free and natural state analysis.
[0065] With detection limits as low as pg / mL, it is suitable for the analysis of low-abundance antibodies or rare antigens, achieving high sensitivity and a wide dynamic range. Combined with microfluidic gradient dilution technology, a single experiment can cover a wide range from high affinity (K... D =10 - 12M) to low affinity (K) D =10 -6 The testing needs of M).
[0066] Therefore, this invention combines antibody microarrays with SPRi technology, achieving high-throughput, label-free, and fully kinetic analysis, thus overcoming the core bottlenecks of existing methods in terms of throughput, sensitivity, data depth, and cost. Its advantages are particularly prominent in antibody drug development, clinical diagnostics, and basic research, and it can become a benchmark method for next-generation antibody affinity detection.
[0067] The above specific embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.
[0068] The above description is only a preferred embodiment of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the claims of this patent application are included in the scope of this patent application.
Claims
1. A method for high-throughput determination of antibody affinity using antibody chip and surface plasmon resonance imaging, characterized in that: The method involves placing the antibody chip in a surface plasmon resonance imaging instrument to measure the affinity. The antibody chip is mainly composed of a photocrosslinked chip and an antibody.
2. The method for high-throughput determination of antibody affinity using antibody chip and surface plasmon resonance imaging according to claim 1, characterized in that: The antibody comprises several antibody molecules.
3. The method for high-throughput determination of antibody affinity using antibody chip and surface plasmon resonance imaging according to claim 1, characterized in that: In the antibody chip, the antibody is chemically bonded to the photocrosslinked chip.
4. The method for high-throughput determination of antibody affinity using antibody chip and surface plasmon resonance imaging according to claim 1, characterized in that: In the antibody chip, several antibody molecules are applied to the photocrosslinked chip via an Arrayjet device.
5. The method for high-throughput determination of antibody affinity using antibody chip and surface plasmon resonance imaging according to claim 1, characterized in that: The antibody molecules are then chemically bonded onto the photocrosslinked chip in a solution with a concentration of 0.5-1 mg / mL using an Arrayjet device.
6. The method for high-throughput determination of antibody affinity using antibody chip and surface plasmon resonance imaging according to claim 1, characterized in that: The aforementioned photocrosslinked chip refers to a chip in which antibodies dotted on the chip surface form covalent bonds with the chip surface through ultraviolet irradiation.
7. The method for high-throughput determination of antibody affinity using antibody chip and surface plasmon resonance imaging according to claim 1, characterized in that: The antibodies mentioned include fully human antibodies, mouse monoclonal antibodies, or rabbit monoclonal antibodies.