High-throughput screening method for screening adaptive TR-FRET application development antibody

Abstract

The invention provides a high-throughput screening method for screening an antibody adapted to TR-FRET application development. Conventional antibody screening methods include ELISA, SPR and the like, the methods are long in time consumption, high in sample demand and tedious in operation, and the screened antibodies are not necessarily suitable for TR-FRET application and development. The method is an antibody screening method developed and optimized based on the TR-FRET technology, and has the characteristics of simple steps, low sample demand, easy realization of high-throughput automatic detection and the like; and the technical methods are completely consistent, so that the screened antibody is completely suitable for TR-FRET application and development, and the affinity and matching degree are better. In addition, the antibody screened by the method is also suitable for ELISA and FACS detection application and development. The method can be used for screening research and development tool antibodies, diagnosis antibodies, antibody drugs, nano antibodies and the like, so that the sample consumption and the manpower demand are greatly reduced while the screening efficiency is improved and the project progress is accelerated.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and particularly to a method for detecting and screening antibodies based on the TR-FRET technology. The screened antibodies are fully compatible with the application development of TR-FRET. The present invention is also applicable to the screening of antibodies for ELISA and FACS applications. Background Art

[0002] Currently, the main application directions of commercial antibodies include: WB, IP, IHC, IF, ChIP, ELISA, FACS, and they have not been verified for TR-FRET applications. If one wants to develop a TR-FRET detection and analysis method using commercial antibodies, it is necessary to purchase and try multiple antibodies, and the results are often not satisfactory. Eventually, it is still necessary to re-develop and screen antibodies.

[0003] During the antibody development process, multiple rounds of hybridoma cloning and screening experiments are required. The commonly used method at present is ELISA, that is, enzyme-linked immunosorbent assay. Due to methodological differences, the high-affinity monoclonal antibodies screened by ELISA often have lower affinity than expected when used for TR-FRET application development, and even positive signals may not be detected.

[0004] The steps for antibody screening using the ELISA method are as follows:

[0005] (1) Coat a multi-well plate suitable for ELISA with an antigen, and the coating incubation time ranges from 1 hour to overnight;

[0006] (2) Wash the coated multi-well plate 3 - 4 times with 1X PBS;

[0007] (3) Add the supernatant of the hybridoma clone to be tested into the coated multi-well plate, and incubate for 1 - 3 hours. Generally, 100 - 200 µL of supernatant is required;

[0008] (4) Wash the multi-well plate 3 - 4 times with 1X PBS;

[0009] (5) Add an HRP-conjugated secondary antibody and incubate for 1 - 2 hours;

[0010] (6) Wash the multi-well plate 3 - 4 times with 1X PBS;

[0011] (7) Add the substrate of HRP and react for a certain period of time (usually 30 minutes), then add a reaction termination solution;

[0012] (8) Read the values on an enzyme-linked immunosorbent assay analyzer. The signal will gradually decay after more than half an hour;

[0013] (9) Select positive hybridoma clones according to the OD values.

[0014] As a traditional method, ELISA has some drawbacks that are difficult to overcome. For example: 1. The experimental steps are numerous and time-consuming, requiring multiple washings of the plate, sample addition, color development, etc., which usually takes 1 day; 2. It is difficult to achieve high throughput; 3. There is a risk that some binding sites are shielded in the way of solid-phase coating of antigens, resulting in missed positive signals, that is, false negative results; 4. Incomplete washing will result in non-specific binding, causing false positive results; 5. Due to the large number of steps, it is easy to cause experimental errors, resulting in poor repeatability and instability of the results; 6. The required sample volume is relatively high. If multiple methods need to be tested, there may be a shortage of samples; 7. The antibodies screened have a bias towards different methods in subsequent applications. Especially when the antibodies are applied to the development of TR-FRET detection methods, the results are not ideal.

[0015] To overcome the deficiencies of the prior art, the present invention provides an antibody detection and screening method based on TR-FRET technology, which can be used to screen antibodies suitable for TR-FRET application development. Through the present invention, high-throughput screening of samples to be tested can be achieved in a short time, providing more economical and reliable screening results. In addition, this method can also be used for the relative affinity analysis of commercial antibodies or other finished antibodies, especially for screening antibodies developed using the TR-FRET method. Summary of the Invention

[0016] TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) is the abbreviation of homogeneous time-resolved fluorescence technology, KeyTec ® The TR-FRET technology is a proprietary technology independently innovated and upgraded by Pharmaron (Shanghai) Biotechnology Co., Ltd. on the basis of the existing TR-FRET technology.

[0017] TR-FRET is based on two major technologies: time-resolved fluorescence (TRF) and fluorescence resonance energy transfer (FRET). TRF utilizes the characteristic of rare earth elements with long half-lives (millisecond level, with a 6-order-of-magnitude difference from ordinary fluorescence in the nanosecond level). Therefore, the background can be excluded by delaying 50 - 100 microseconds. FRET utilizes the energy transfer between two fluorescent groups, which are respectively called the energy donor (Donor) and the energy acceptor (Acceptor). When the Donor is excited by an external light source, if it is close to the Acceptor, it can transfer the energy resonantly to the Acceptor, causing it to be excited and emit emission light with a specific wavelength of 665 nm. The rare earth elements (such as europium Eu, terbium Tb) of the traditional TR-FRET energy donor are encapsulated in chelates, KeyTec ® There are two types of TR-FRET fluorescence energy donors, KeyTec ®Solar Eu and KeyTec ® Solar Tb is respectively and permanently stably encapsulated in a cryptand compound, which is designed and modified with a special structure to enhance the stability and luminescence intensity of the energy donor. After being excited by laser, the emission spectra of europium and terbium have a peak at 620 nm. There are two types of acceptors. One is the modified allophycocyanin with the commercial name KeyTec ® HX, with a molecular weight of about 60 kD; the other is a small molecule compound with the commercial name KeyTec ® LA, with a molecular weight of about 1 kD. KeyTec ® HX and KeyTec ® The excitation light of LA overlaps with the emission light of the Donor to a certain extent. After being excited, it will form a specific peak at 665 nm, and the emitted fluorescence is red. KeyTec ® The reaction system of TR-FRET is a liquid homogeneous phase. Using an optimized reaction buffer can keep the biological samples (antibodies, proteins, polypeptides, etc.) in the detection system as close as possible to their natural conformations and biological activities, more truly expose the binding sites or active sites, avoid the probability of false positives / false negatives, and screen out truly effective positive antibodies. TR-FRET data processing and calculation are generally recommended to be expressed using the ratio (Ratio), that is, 665 nm / 620 nm; to make the data convenient for reading and comparison, multiply by the amplification factor 10,000 uniformly, that is, TR-FRET Ratio = signal 665 nm / signal 620 nm * 10,000. Through ratio calculation, dividing the binding signal (665 nm) by the fluorescence energy donor signal (620 nm) in the corresponding well can reduce the data fluctuations and deviations caused by experimental operations, making the data more stable and the repeatability better.

[0018] The basic principle of the present invention is as Figure 1 shown. The antibody detection and screening system contains a verified tagged antigen, an anti-tag antibody conjugated with KeyTec ® Solar Eu, and an anti-species secondary antibody conjugated with KeyTec ® LA. After adding the sample to be screened (hybridoma cell supernatant or purified antibody, commercial antibody, etc.; monoclonal or polyclonal antibodies are all acceptable) to the detection system, the antibody that can specifically recognize the antigen will bind to the antigen, thereby forming an interacting quaternary complex, enabling KeyTec ® Solar Eu (Donor) and KeyTec ®When the LA (Acceptor) comes closer, a TR-FRET signal is generated. The higher the signal, the higher the affinity between the antibody and the antigen (at the same concentration and in the absence of the hook effect). The test samples include, but are not limited to, hybridoma cell supernatants or purified antibodies, monoclonal or polyclonal antibodies, and antibodies of various species. Among them, the tags carried by the antigen include common tags such as His, GST, FLAG, etc., and the anti-species secondary antibodies include common secondary antibodies such as Anti-mouse IgG antibody, Anti-rat IgG antibody, Anti-rabbit IgG antibody, etc.

[0019] The general reaction system of the present invention is shown in Table 1. The total reaction volume of 20 µL is for the 384-well assay plate and can be adjusted according to the detection mode. For example, the corresponding volume for a 1536-well assay plate is 8 - 10 µL; the proportions of the various components in the reaction system can also be adjusted according to the detection requirements; the other three components except the test sample can be premixed, making the operation steps more streamlined (more easily automated) and with less error.

[0020] Table 1 KeyTec ® Conventional reaction system for TR-FRET antibody screening experiment

[0021] Reagent (Component) Volume (Total reaction system: 20 µL) Sample to be measured 5 µL Antigen with tag (Antigen-tag) 5 µL <![CDATA[Conjugated with KeyTec ® Anti-tag antibody of Solar Eu (mAb anti Tag - Solar Eu)]]> 5 µL <![CDATA[Anti-species IgG secondary antibody (pAb anti-species IgG - LA) conjugated with KeyTec ® LA]]> 5 µL

[0022] The general operation steps of the present invention are as follows:

[0023] (1) Add the prepared tagged antigen (the use concentration has been optimized in advance) to the corresponding wells of the assay plate;

[0024] (2) Add the test sample (diluted or undiluted hybridoma supernatant, purified antibody at a specific concentration);

[0025] (3) Add the prepared anti-tag antibody conjugated with KeyTec ® Solar Eu;

[0026] (4) Add the prepared anti-species secondary antibody conjugated with KeyTec ® LA;

[0027] (5) Seal the assay plate with a sealing film to prevent liquid evaporation and incubate at room temperature for a specific time (30 minutes or more):

[0028] (6) Read the values on a microplate reader compatible with TR-FRET to obtain data and perform data analysis.

[0029] To meet the requirements of high-throughput automated detection or reduce manual labor, the steps of the present invention can also be streamlined as follows:

[0030] (1) Premix the prepared labeled antigen, the anti-tag antibody conjugated with KeyTec ® Solar Eu, and the anti-species secondary antibody conjugated with KeyTec ® LA in a certain proportion and mix evenly, then add them to the corresponding wells of the analysis plate;

[0031] (2) Add the sample to be tested;

[0032] (3) Seal the analysis plate with a sealing film to prevent liquid evaporation, and incubate at room temperature for a specific time (30 minutes or more);

[0033] (4) Read the values on a microplate reader adapted for TR-FRET and obtain data for data analysis.

[0034] KeyTec ® The technical characteristics of KeyTec

[0035] (1) Simple operation: Only need to add samples and detection reagents, and detect after incubation without washing, which can be completed within 1 hour;

[0036] (2) Easy to achieve high-throughput automated operation: Compatible with 96, 384 or 1536-well plate operations;

[0037] (3) Low sample requirement, the minimum reaction system can reach 8 µL, and the sample volume is 2 µL;

[0038] (4) Stable signal: Can be continuously detected multiple times with good repeatability;

[0039] (5) Low false positive and false negative rates: Homogeneous detection, which does not affect the properties of the sample to be tested itself;

[0040] (6) The antibodies obtained by screening can be fully adapted to the development and application of the TR-FRET method.

[0041] The advantages of the present invention are as follows:

[0042] (1) Provide a new antibody screening method and meet the detection requirements of high efficiency, rapidity, simplicity, flexibility, and low sample demand;

[0043] (2) Applicable to various sample types, antibodies with different matrices (cell supernatant, cell lysate, conventional buffer), different species (mouse anti, rabbit anti, goat anti, etc.), monoclonal or polyclonal antibodies can be detected;

[0044] (3) Greatly saves the experimental workload and time: The experimental operation has changed from the original processes of coating, blocking, and multiple washings of plates to simply adding samples and detection reagents now. The experimental time has been shortened from the original one day to 1 - 2 hours.

[0045] (4) Greatly improves the experimental throughput: The detection of samples has been increased from the original 96 - well ELISA or single (or multiple) samples in SPR to 384 - well or even 1536 - well, thus enabling large - scale sample screening in a short time.

[0046] (5) The results are more reliable: In homogeneous detection, the antigen protein (or small - molecule antigen) presents its natural conformation and fully exposes the binding sites in the liquid phase system, effectively avoiding the generation of false positives and false negatives.

[0047] (6) The signal is more stable: The fluorescence lasts for a long time and can be detected after overnight without affecting the detection results; it can be continuously read multiple times and the signal remains stable, enabling the study of reaction kinetics; while the detection results of ELISA are restricted by time, too long or too short time will affect the detection results; the reading generally needs to be completed within half an hour, and when detecting a large number of samples, it is difficult to control the signal consistency and stability between different detection plates.

[0048] (7) Due to being based on the same methodology, the antibodies screened using the present invention can be fully adapted when developing and applying subsequent TR - FRET detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is for KeyTec ® Schematic diagram of the principle of TR - FRET antibody screening;

[0050] Figure 2 is the result graph of the affinity comparison of anti - GST - tag antibodies (KeyTec ® TR - FRET vs ELISA);

[0051] Figure 3 is the detailed data of the affinity comparison of anti - GST - tag antibodies (KeyTec® TR - FRET vs ELISA). DETAILED DESCRIPTION OF THE INVENTION

[0052] The technical solution of the present invention will be clearly and completely described below through examples. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The raw materials used in the embodiments can all be purchased commercially or prepared by conventional methods.

[0053] Example 1: Development of cytokine antibodies - Multiple rounds of positive hybridoma screening

[0054] In the development of antibodies against human interleukin 8 (human IL8), KeyTec ® TR-FRET technology was used for the primary screening of monoclonal samples. The steps are as follows:

[0055] (1) Prepare a small number of monoclonal antibodies by the traditional hybridoma method. In the primary screening stage of monoclonal antibodies after hybridoma cell fusion, the cells were seeded in a 384-well cell culture plate according to the conventional method;

[0056] (2) Culture the cells under conventional conditions until they reach the detection stage;

[0057] (3) Pre-mix his-IL8 (tagged antigen), rabbit mAb anti-His-SolarEu, and goat pAb anti-Mouse IgG Fc-LA at the working concentration using the detection buffer, and add 15 µL of the pre-mixed working solution to each well in a 384-well assay plate;

[0058] (4) Add 5 µL of the cell culture supernatant (test sample) from step (2) to the corresponding wells in the assay plate;

[0059] (5) Set up a negative control (NC): 5 µL of blank medium, and a positive control (PC): 5 µL of a positive antibody with average affinity;

[0060] (6) Seal the assay plate well with a high-permeability pressure-sensitive sealing film and incubate at room temperature for 30 minutes or more;

[0061] (7) Read the data on a microplate reader compatible with TR-FRET and perform calculation and analysis.

[0062] Generally, a TR-FRET Ratio value of the sample to be tested that is 3 times or higher than that of the negative control is considered positive. However, in order to screen for antibodies with better affinity, the higher the TR-FRET Ratio, the better, and it is preferably significantly higher than that of the positive control. Table 2 shows some of the results of this example, and the ELISA method was used for comparison at the same time. Among them, for the clone numbered 16G2-4N14, the ELISA result showed positive, while the TR-FRET result showed negative. Later, it was verified to be a negative clone through experiments, that is, the ELISA result was a false positive.

[0063] Table 2 Comparison of TR-FRET and ELISA Results in Hybridoma Screening for the Development of Anti-Human Interleukin 8 Antibodies

[0064] Clone number TR-FRET Ratio ELISA Value Clone number TR-FRET Ratio ELISA Value PC (Positive control) 31,669 1.944 NC (Negative control) 2,521 0.058 3A11-1A23 45,913 2.059 1B12-1C12 12,970 1.727 3A10-1A22 44,469 2.068 16G2-4N14 2,678 2.023 2E9-1J9 43,459 1.981 5H5-2O5 3,578 1.407 3B5-1C17 29,760 1.568 13G4-4M4 3,511 1.253 7F3-2K15 26,789 2.034 15B12-4C24 2,696 1.263 13H2-4O2 24,365 1.822 13F6-4K6 2,619 1.000 4C10-1F22 17,663 1.768 3A4-1A16 2,601 1.253 8G11-2N23 17,177 2.026 6G6-2N6 2,231 0.535 4D7-1H19 16,060 1.654 8F9-2L21 2,025 0.994

[0065] Example 2: Affinity Comparison and Screening of Anti-GST Tag Antibodies (Primary Antibodies, Finished Antibodies)

[0066] When screening commercially available antibodies on the market, the present invention can be directly used for antibody affinity comparison and screening. For example, anti-GST tag antibodies are commonly used tool antibodies, and there are many manufacturers selling this product. In this example, 19 anti-GST tag mouse monoclonal antibodies commercially available and self-screened were tested. The specific steps are as follows:

[0067] (1) Dilute all anti-GST tag antibodies with PBS buffer to a specific concentration (generally between 1 and 50 nM), and add 5 μL of the antibody to be tested to each well in a 384-well assay plate;

[0068] (2) Dilute the double-tagged protein with N-GST and C-His with PBS buffer to a specific concentration (optimized in advance), and add 5 μL to each well in a 384-well assay plate;

[0069] (3) Premix rabbit mAb anti-His-Solar Eu and goat pAb anti-MouseIgG Fc-LA at the working concentration with the detection buffer, and add 10 μL of the premixed working solution to the corresponding wells in a 384-well assay plate;

[0070] (4) Set up a negative control (NC): diluted with 5 μL of buffer, and a positive control (PC): 5 μL of a positive antibody with general affinity;

[0071] (5) Seal the assay plate well with a high-permeability pressure-sensitive sealing film and incubate at room temperature for 30 minutes or more;

[0072] (6) Read the data on a microplate reader suitable for TR-FRET and perform calculation and analysis.

[0073] From Figure 2 and Figure 3 As can be seen from the results shown, the results of affinity tests using the two methods of TR-FRET and ELISA are not completely consistent, and there are significant differences in the affinity rankings obtained by the two methods. Among them, the ELISA test results of Clone #2, #3, #5, #11, #15, #18, and #19 are negative, but the TR-FRET test results are positive. Clone #2 and #3, which have the highest TR-FRET affinity rankings, are also among them. Thus, it can be seen that if only the ELISA method is used in the monoclonal antibody screening stage, many TR-FRET positive clones with good affinity will be lost, resulting in less than ideal results when the selected antibodies are used for TR-FRET application development. If the TR-FRET method is used in the monoclonal antibody screening stage, the efficiency of screening antibodies suitable for TR-FRET application development can be improved.

[0074] As described above, using the high-throughput screening method of the present invention to screen antibodies suitable for TR-FRET applications can achieve streamlined steps, low sample requirements, low false negatives, and high-throughput automated detection. The present invention can be used to screen research tool antibodies, diagnostic antibodies, antibody drugs, nanobodies, etc. While improving the screening efficiency and accelerating the project progress, it greatly reduces sample consumption and manpower requirements.

Claims

1. A method for high-throughput screening of antibodies adapted for TR-FRET applications, which method itself is based on TR-FRET technology. It is characterized in that, The TR-FRET technology is an optimization and upgrade of fluorescent dyes by Pharma Yuan (Shanghai) Biotechnology Co., Ltd. based on the original TR-FRET technology, and its registered trademark is KeyTec ® TR-FRET. The data processing of the present invention adopts the method of TR-FRET Ratio = signal 665 nm / signal 620 nm*10000. Through ratio calculation, by dividing the combined signal (665 nm) by the fluorescence energy donor signal (620 nm) in the corresponding well, the data fluctuations and deviations caused by experimental operations can be reduced, the data is more stable, and the repeatability is better. The antibodies screened using the present invention are fully compatible with the application development of TR-FRET and can also be used for the application development of technologies such as ELISA and FASC. The present invention is applicable to the detection and analysis of various sample types, and antibodies with different solution matrices (cell supernatant, cell lysate, conventional buffer), different species (mouse anti-, rabbit anti-, goat anti-, etc.), monoclonal or polyclonal antibodies can all be detected.

2. According to claim 1, wherein: This invention is a technology that has been optimized and upgraded in terms of fluorescent dyes based on the original TR-FRET technology, and its registered trademark is KeyTec ® TR-FRET.

3. According to claim 1, wherein: The present invention is a method for screening antibodies that are fully suitable for the development of TR-FRET applications.

4. According to claim 1, wherein: The present invention can replace the traditional ELISA screening method, and the antibodies screened can also be used in technical applications such as ELISA and FACS.

5. According to claim 1, characterized in that: Data processing in the present invention adopts the method of TR-FRET Ratio = signal 665 nm / signal 620 nm * 10000. The corresponding fluorescence wavelength signal values of this processing method include but are not limited to 665 nm, 620 nm, 615 nm, 520 nm, 488 nm, and the multiplication coefficients include but are not limited to 10000, 1000, 100.

6. According to claim 1, characterized in that: The present invention is applicable to the screening of antibodies in various solution matrix types, including but not limited to antibodies in cell supernatants, cell lysates, and conventional buffers.

7. According to claim 1, wherein: The present invention is applicable to the screening of antibodies of different species types, including but not limited to mouse, rat, rabbit, goat, sheep, chicken, donkey.

8. According to claim 1, characterized in that: The present invention is applicable to the screening of antibodies of different clone types, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, nanobodies, and engineered antibodies.

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