Method for screening paired antibodies by using coded magnetic beads and application

Abstract

The invention discloses a method for screening paired antibodies by using coded magnetic beads and application. The method comprises the following steps: respectively marking magnetic beads with different fluorescence intensities by different antibodies, co-incubating a magnetic bead mixed solution with antigen diluents with different concentrations, respectively mixing the incubated magnetic bead mixed solution with a plurality of fluorescently-marked antibodies, simultaneously detecting, and screening paired antibodies according to fluorescence values. According to the invention, high-throughput antibody pair screening can be carried out, optimal pairs in 50 pairs of monoclonal antibodies targeting the same antigen can be simultaneously screened in the system at most, and capture antibodies and detection antibodies between the paired antibodies can be determined at the same time.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for screening paired antibodies by using encoded magnetic beads and its application. Background Art

[0002] In immunoassay reagents, paired antibodies refer to a pair of antibodies that can bind to an antigen simultaneously, and are mainly used in the double-antibody sandwich method of the antibody sandwich method. Antibodies generated against different antigenic determinants may bind to different positions of the antigen, and the steric hindrance at the binding position will affect the detection sensitivity of the paired antibodies to the antigen. Therefore, it is crucial to screen highly sensitive monoclonal antibody pairs.

[0003] Currently, the most widely used immunoassay platforms for paired antibodies mainly include enzyme-linked immunosorbent assay, immunochromatography, chemiluminescence, flow cytometry fluorescence, etc. The most important stage in reagent research and development is the screening of the best antibody pairs. The general methods for screening antibody pairs currently are: (1) adsorbing and coating a sample containing a known antibody (capture antibody) on a solid-phase carrier and washing once; (2) adding the antigen to be detected, and if they are specific, binding will occur; (3) adding a labeled antibody that reacts specifically with the antigen to be detected (the labeling method is different for different application platforms); (4) finally adding a chromogenic solution or a fluorescent agent, and judging the content of the antigen to be detected by detecting the intensity of color development or fluorescence intensity.

[0004] A large number of antibody raw materials have emerged in the domestic and international markets. Screening the best antibody pair raw materials generally requires a large amount of manpower, material resources, and time.

[0005] Therefore, there is an urgent need to provide an efficient screening method to simultaneously screen the optimal pairing of antibodies from different sources, determine the capture antibody and the detection antibody between the paired antibodies, and shorten the reagent development time. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art and the actual needs, the present invention uses encoded magnetic beads to label antibodies respectively, and then performs paired screening with biotin- or fluorescence-labeled antibodies. The encoded magnetic beads have different fluorescence intensities and can be grouped during detection by a flow cytometer. Currently, the encoded magnetic beads can detect up to 50 multiplexes simultaneously on the flow platform. Therefore, the present invention can perform high-throughput antibody pair screening and can screen the best pairing among up to 50 pairs of monoclonal antibodies targeting the same antigen in the system at the same time.

[0007] To achieve the object of the present invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for screening paired antibodies, the method comprising: labeling different antibodies with magnetic beads having different fluorescence intensities, co-incubating the magnetic bead mixture with antigen dilutions of different concentrations, respectively mixing with a variety of fluorescence-labeled antibodies after incubation, and detecting simultaneously, and screening paired antibodies according to the fluorescence values.

[0009] Among them, the antibody used for magnetic bead labeling belongs to the capture antibody and is used to capture the antigen in the sample; the biotin-labeled antibody belongs to the detection antibody and is used to detect the captured antigen. The detection antibody can be labeled with biotin and then combined with SA-PE (SA (Streptavidin)-PE (P-phycoerythrin)), or directly labeled with a fluorescent agent.

[0010] The present invention can perform high-throughput antibody pair screening, and can screen the best pair among up to 50 pairs of monoclonal antibodies targeting the same antigen in the system at the same time, and can simultaneously determine the capture antibody and the detection antibody between the paired antibodies.

[0011] Preferably, the rotation speed of the co-incubation is 600 - 1000 rpm, the temperature is 25 - 39 °C, and the time is 30 - 180 min.

[0012] The specific point values within the range of 600 - 1000 rpm can be selected as 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, etc.

[0013] The specific point values within the range of 25 - 39 °C can be any temperature within the range of 25 °C - 39 °C.

[0014] The specific point values within the range of 30 - 180 min can be selected as 30 min, 40 min, 50 min, 80 min, 100 min, 120 min, 140 min, 160 min, 180 min, etc.

[0015] In the present invention, magnetic beads with different fluorescence intensities can be labeled with different antibodies. After the magnetic bead mixture is co-incubated with different concentrations of antigen diluents, it reacts with a variety of fluorescently labeled antibodies. It is also possible to incubate the co-incubation solution with different biotin-labeled antibodies and then add a fluorescent agent for reaction, and screen paired antibodies according to the fluorescence values.

[0016] Preferably, the magnetic beads labeled with different antibodies with different fluorescence intensities include: activation coupling and blocking.

[0017] Preferably, the activation coupling includes: separating and changing the liquid of the encoded magnetic beads by magnetic adsorption, changing them into a buffer solution, then mixing with coupling agent 1, and after mixing, mixing with antibody protein and incubating in the dark.

[0018] Preferably, the activation coupling further includes mixing with coupling agent 2 after mixing with coupling agent 1, and after mixing, mixing with antibody protein and incubating in the dark.

[0019] Preferably, the coupling agent 1 includes N-hydroxysuccinimide (NHS), and the coupling agent 2 includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC).

[0020] Preferably, the rotation speed for the dark incubation is 1000 - 2500 rpm, the temperature is 25 - 39 °C, and the time is 1 - 4 h.

[0021] Specific point values within the range of 1000 - 2500 rpm can be selected from 1000 rpm, 1200 rpm, 1400 rpm, 1600 rpm, 1800 rpm, 2000 rpm, 2200 rpm, 2500 rpm, etc.

[0022] Specific point values within the range of 25 - 39 °C can be any temperature within the range of 25 °C - 39 °C.

[0023] Specific point values within the range of 1 - 4 h can be selected from 1 h, 2 h, 3 h, 4 h, etc.

[0024] Preferably, the rotation speed for the blocking is 1000 - 2500 rpm, and the time for the blocking is 1 - 4 h.

[0025] Specific point values within the range of 1000 - 2500 rpm can be selected from 1000 rpm, 1200 rpm, 1400 rpm, 1600 rpm, 1800 rpm, 2000 rpm, 2200 rpm, 2500 rpm, etc. Specific point values within the range of 1 - 4 h can be selected from 1 h, 2 h, 3 h, 4 h, etc.

[0026] Preferably, the concentration of magnetic beads in the magnetic bead mixture is 1×10 6 -1×10 9 magnetic beads / mL.

[0027] Specific point values within the range of 1×10 6- 1×10 9 can be selected from 1×10 6 , 5×10 7 , 1×10 8 , 1×10 9 etc.

[0028] Preferably, the concentration of magnetic beads in the magnetic bead mixture is 1×10 7 -1×10 8 magnetic beads / mL.

[0029] Specific point values within the range of 1×10 7- 1×10 8 can be selected from 1×10 7 , 5×10 7 、1×108 etc.

[0030] Preferably, the rotation speed of the mixing is 600 - 1000 rpm, the temperature is 25 - 39 °C, and the time is 5 - 30 min.

[0031] Specific point values within the above 600 - 1000 rpm can be selected as 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, etc.

[0032] Specific point values within the above 25 - 39 °C can be any temperature within the range of 25 °C - 39 °C.

[0033] Specific point values within the above 5 - 30 min can be selected as 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, etc.

[0034] In a second aspect, the present invention provides an application of the method described in the first aspect in screening antibodies.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention can perform high - throughput antibody pair screening, and can screen for the best pairings among up to 50 monoclonal antibodies targeting the same antigen in the system simultaneously. Specific Embodiments

[0037] To further illustrate the technical means adopted by the present invention and its effects, the present invention will be further described below in conjunction with embodiments. It can be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0038] For those not specifying specific techniques or conditions in the examples, they shall be carried out according to the techniques or conditions described in the literature in this field, or according to the product specifications. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular commercial channels.

[0039] Example 1

[0040] Pairing and screening among 12 monoclonal antibodies targeting the same antigen.

[0041] Twelve monoclonal antibodies (1 - 12) targeting the same antigen need to screen for the best antibody pairings applicable to platforms such as enzyme immunoassay, luminescence, chromatography, flow - through fluorescence, etc.

[0042] Screening process:

[0043] 1. Magnetic bead labeling: Twelve different carboxyl-coded magnetic beads (coded A, B, C, D, E, F, G, H, I, J, K, L) were used to label twelve monoclonal antibodies, numbered A-1, B-2, C-3, D-4, E-5, F-6... L-12.

[0044] Labeling process: (1) Activation + coupling: Take 10 6 of each coded magnetic bead and place them in a 1.5 mL centrifuge tube. Add 100 μL of morpholineethanesulfonic acid buffer solution (MES) 5.0, mix well, and perform magnetic separation for 1 min to remove the supernatant (leave 10 μL), and repeat the washing 2 times. Add MES 5.0 to the magnetic beads to make up to 96 μL, mix well. EDC and NHS are respectively prepared into 50 mg / mL with MES 5.0, and 2 μL of each is taken and added to the magnetic beads (add NHS and mix well, then add EDC), mix well, and directly add the required antibody protein (9 μg), mix well, and centrifuge at 1500 rpm / room temperature / dark / 2 h (system magnetic bead concentration: 10 7 magnetic beads / mL). Perform magnetic separation for 1 min to remove the supernatant (leave 10 μL).

[0045] (2) Blocking: Add PBST containing Tween to make up to 100 μL, mix well, perform magnetic separation for 1 min to remove the supernatant (leave 10 μL), and repeat the washing 2 times. Add the blocking solution (phosphate buffer PBS, 1% bovine serum albumin BSA) to make up to 100 μL, mix well, centrifuge at 1500 rpm / room temperature / dark / 2 h (system magnetic bead concentration: 10 7 magnetic beads / mL), and perform magnetic separation for 1 min to remove the supernatant (leave 10 μL).

[0046] (3) Preservation: Add PBST to make up to 100 μL, mix well, perform magnetic separation for 1 min to remove the supernatant (leave 10 μL), and repeat the washing 2 times. Add the magnetic bead preservation solution (PBS, 0.1% BSA) to 10 μL, mix well, dilute to 10 7 / mL, and store at 8°C in the dark.

[0047] 2. Biotin labeling: Biotin was used to label twelve monoclonal antibodies, numbered Biotin-1, Biotin-2, Biotin-3, Biotin-4, Biotin-5, Biotin-6... Biotin-12.

[0048] Labeling process:

[0049] (1) Add 50 μg of the antibody, calculate the amount of PBS, add PBS and mix gently, add 2 μL of the required 10 mM biotin, centrifuge at 1500 rpm / room temperature / dark / 2 h (antibody concentration in the system is 1 mg / mL);

[0050] (2) Make up the labeled antibody to 500 μL with biotinylated antibody storage solution. After thorough mixing, store it at -20 °C (storage concentration: 0.1 mg / mL).

[0051] 3. Detection mode process: The antibodies on 12 kinds of capture magnetic beads are paired and detected with biotin-labeled antibodies respectively (as shown in Table 1). Specific steps: (1) Incubate 50 μL of the mixed solution of 12 kinds of capture magnetic beads (2500 beads / weight) with antigen dilutions of different concentrations at 800 rpm and 37 °C for 60 min. (2) Wash twice with PBST, add 50 μL of biotinylated antibody (2 μg / mL, only add one Biotin-1 for one detection, multiple Biotin-Abs can be detected at the same time), react at 37 °C for 30 min, and wash twice with PBST. (3) Add 50 μL of SA (Streptavidin)-PE (P-phycoerythrin) (2 μg / mL), 800 rpm, 37 °C, 5 min; (4) Wash twice with PBST, resuspend with 150 μL of 1×PBS, and detect the PE fluorescence intensity with BD CantoII.

[0052] Table 1

[0053]

[0054]

[0055] The incubation mode for each well of the 96-well detection plate is as follows. One plate can simultaneously perform the incubation of 12 antibodies paired with each other. Finally, detect with a flow cytometer.

[0056] Taking the detection results of the optimal pairing screening of 6 kinds of anti-IL6 monoclonal antibodies as an example, it is shown in Table 2.

[0057] Table 2

[0058]

[0059] Table 3

[0060]

[0061]

[0062] Table 4

[0063]

[0064]

[0065] Table 5

[0066]

[0067] Table 6

[0068]

[0069] Table 7

[0070]

[0071]

[0072] Screening and evaluation criteria: (1) Sensitivity: If the ratio of sample S1 / S0 (average value) > 2, it indicates that the antigen concentration detection of S1 can be significantly distinguished from the background value of S0, the binding sensitivity of the antibody pair to the antigen meets the requirements, and the detection limit can reach 1.6 pg / mL. If the ratio of sample S1 / S0 (average value) > 3, or even higher, it indicates higher detection sensitivity and the detection limit concentration is less than 1.6 pg / mL. (2) Linear range: The dilution factor of the antigen concentration from S7 to S6 is 2, and the dilution factor from S6 to S1 is 5. If the detection fluorescence value of S7 / S6 at the high dilution end can reach 2, it indicates that the binding linear high end of the antibody to the antigen concentration can reach the concentration of S7, which is 10,000 pg / mL. If it is significantly lower than 2, then continue to look at the ratio of S6 / S5. If the S6 / S5 ratio can reach 5 or > 4, it indicates that the binding linear high end of the antibody to the antigen concentration can reach the concentration of S6, which is 5,000 pg / mL. If it is significantly lower than 4, then the detection linear high end can only reach the concentration of S5, which is 1,000 pg / mL.

[0073] According to the above screening criteria, the statistical analysis of the above experimental results is as follows: From the results in Table 2, it can be seen that the sensitivity of the paired antibody B-2 / Biotin-1 meets the requirements, and the linear range is 1.6 - 5,000 pg / mL; from the results in Table 3, it can be seen that the paired antibodies D-4 / Biotin-2 and F-6 / Biotin-2 have higher sensitivity, and the linear range of both is 1.6 - 1,000 pg / mL; from the results in Table 4, it can be seen that the sensitivity of this group of paired antibodies is low and does not meet the requirements; from the results in Table 5, it can be seen that the sensitivity of the paired antibody C-3 / Biotin-4 in this group meets the requirements, and the linear range is 1.6 - 5,000 pg / mL; from the results in Table 6, it can be seen that the sensitivity of the paired antibody A-1 / Biotin-5 in this group meets the requirements, and the sensitivities of C-3 / Biotin-5 and F-6 / Biotin-5 are higher. The linear ranges of the three groups of paired antibodies are all 1.6 - 1,000 pg / mL; from the results in Table 7, it can be seen that the sensitivity of the paired antibody C-3 / Biotin-6 in this group meets the requirements, and the linear range is 1.6 - 5,000 pg / mL. [[ID=2,1]]

[0074] According to the above screening and evaluation criteria, the final conclusion of the best pairing screening of 6 kinds of IL6 monoclonal antibodies in this example is as follows:

[0075] (1) If the antibody pairs selected require a wide linear range for antigen detection in the sample and the sensitivity meets the requirements, then the antibody pairs we can choose are B-2 / Biotin-1, C-3 / Biotin-4, and C-3 / Biotin-6.

[0076] (2) If the antibody pairs selected require a lower linear range for antigen detection in the sample but a higher sensitivity, then the best antibody pairs we can choose are D-4 / Biotin-2, F-6 / Biotin-2, C-3 / Biotin-5, and F-6 / Biotin-5.

[0077] In summary, the present invention can perform high-throughput antibody pair screening, and can screen the best pairs among up to 50 monoclonal antibody pairs targeting the same antigen in the system simultaneously. And it can determine the capture antibody and the detection antibody between the paired antibodies at the same time.

[0078] The applicant declares that the present invention uses the above embodiments to illustrate the detailed method of the present invention, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for screening paired antibodies, characterized in that, The method includes: different antibodies are used to label magnetic beads with different fluorescence intensities, the magnetic bead mixture is co-incubated with antigen dilutions of different concentrations, after incubation, it is mixed with a variety of fluorescently labeled antibodies respectively, and detected simultaneously, and paired antibodies are screened according to the fluorescence values.

2. The method according to claim 1, wherein The rotation speed of the co-incubation is 600 - 1000 rpm, the temperature is 25 - 39 °C, and the time is 30 - 180 min.

3. The method according to claim 1, wherein The different antibodies respectively labeling magnetic beads with different fluorescence intensities includes: activation coupling and blocking.

4. The method according to claim 3, wherein The activation coupling includes: separating and changing the liquid of the encoded magnetic beads by magnetic adsorption, changing it into a buffer solution, then mixing with coupling agent 1, after mixing evenly, mixing with antibody protein and incubating in the dark.

5. The method according to claim 3 or 4, characterized in that The activation coupling also includes mixing with coupling agent 2 after mixing with coupling agent 1, after mixing evenly, mixing with antibody protein and incubating in the dark.

6. The method according to claim 4 or 5, characterized in that, The rotation speed of the incubation in the dark is 1000 - 2500 rpm, the temperature is 25 - 39 °C, and the time is 1 - 4 h.

7. The method according to any one of claims 3-6, characterized in that The rotation speed of the blocking is 1000 - 2500 rpm, and the time of the blocking is 1 - 4 h.

8. The method according to any one of claims 1 to 7, characterized in that, The concentration of magnetic beads in the magnetic bead mixture is 1×10 6 -1×10 9 magnetic beads / mL.

9. The method according to any one of claims 1-8, characterized in that, The rotation speed of the mixing is 600 - 1000 rpm, the temperature is 25 - 39 °C, and the time is 5 - 30 min.

10. Use of the method according to any one of claims 1 - 9 in screening antibodies.

Images