Screening method for detecting monoclonal antibody pairing based on high-throughput flow cytometry

Abstract

The invention provides a screening method for detecting monoclonal antibody pairing based on a high-throughput flow cytometry. The screening method comprises the following steps: (1) incubating and combining Protein G-labeled magnetic fluorescent encoding microspheres with captured antibodies; (2) adding confining liquid to seal blank sites of the microspheres after incubation; (3) after closing, adding an antigen for incubation; (4) after antigen incubation, adding a detection antibody for incubation; and (5) incubating the detection antibody, adding a fluorescent secondary antibody SA-PE, incubating, and detecting by adopting a flow cytometer after the incubation is finished. According to the screening method, the experiment process can be shortened, experiment consumables and labor cost are reduced, and experiment verification results show that the matching rate of primary screening is 85% or above compared with Elisa, and the matching rate of secondary screening is 100%. Therefore, according to the method, antibody pair screening work of different projects can be skillfully carried out, the streaming screening result can be kept consistent with Elisa double antibody sandwich, and the accuracy rate is high.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody screening, and in particular to a screening method based on high-throughput flow cytometer detection of monoclonal antibody pairing. Background Art

[0002] The Elisa double antibody sandwich method is a relatively mature, low-difficulty and generally applicable antibody pair screening method and is used by most laboratories. However, in actual operations, a large number of monoclonal antibodies are screened in many projects. When the Elisa double antibody sandwich method is used for monoclonal antibody cross-pairing screening, there is an exponential relationship between the experimental scheme and the number of antibodies, resulting in a very large workload and cumbersome operating steps.

[0003] Flow cytometry is also widely used in the detection of cytokines. The principle is also double antibody sandwich, but the source of antibody raw materials is usually obtained by Elisa method. At present, flow cytometry is not used for a large number of antibody pair screening. The reason is that the double antibody sandwich experiment of flow cytometry needs to replace the ELISA plate and the fluorescent magnetic beads similar to cells as the matrix material, and the type and performance requirements of the matrix material are relatively high. Therefore, in order to solve the problems of cumbersome operation steps and large workload when screening monoclonal antibody pairs by Elisa double antibody sandwich method, it is imperative to develop a monoclonal antibody pair screening method based on flow cytometry. Summary of the invention

[0004] The present invention proposes a screening method for detecting monoclonal antibody pairing based on high-throughput flow cytometry, which not only solves the problems of complicated operation steps and large workload when screening monoclonal antibody pairs by Elisa double antibody sandwich method, but also can greatly improve the detection efficiency.

[0005] The technical solution of the present invention is achieved in this way:

[0006] The present invention provides a screening method for monoclonal antibody pairing based on high-throughput flow cytometry detection, comprising the following steps:

[0007] (1) The magnetic fluorescent coded microspheres labeled with Protein G are incubated and combined with the capture antibody;

[0008] (2) After incubation, a blocking solution is added to block the blank sites of the microspheres;

[0009] (3) After blocking, add antigen and incubate;

[0010] (4) adding detection antibody after antigen incubation for incubation;

[0011] (5) After incubation with the detection antibody, the fluorescent secondary antibody SA-PE was added for incubation, and then detected by flow cytometry.

[0012] Based on the above technical solution, preferably, the preparation steps of the magnetic fluorescent coded microspheres labeled with Protein G in step (1) are:

[0013] (1) Activation of magnetic fluorescent coded microspheres: Add magnetic fluorescent coded microspheres to MES solution and mix well. After removing the supernatant, add to EDC / NHS mixed solution and incubate.

[0014] (2) Magnetic fluorescent coded microsphere labeling: After the incubated microspheres are washed, Protein G is added for incubation to obtain magnetic fluorescent coded microspheres labeled with Protein G.

[0015] On the basis of the above technical solution, preferably, the EDC and NHS are uniformly mixed in a volume ratio of 1:1.

[0016] On the basis of the above technical solution, preferably, 10 6 The amount of Protein G used in each of the magnetic fluorescent encoded microspheres is 200 μg.

[0017] On the basis of the above technical solution, preferably, the incubation time in the process of preparing the magnetic fluorescent encoded microspheres labeled with Protein G is 20 min-2 h.

[0018] On the basis of the above technical solution, preferably, the mass volume ratio of the capture antibody to the magnetic fluorescent encoded microspheres labeled with Protein G is 1:1-2 (in units of μg and μL, respectively).

[0019] More preferably, the mass volume ratio of the magnetic fluorescent encoded microspheres labeled with Protein G to the capture antibody is 1:1.

[0020] Based on the above technical solution, preferably, the blocked microspheres in step (3) are divided into two groups, one of which is added with antigen, and the other is added with the same volume of diluent, and incubated with shaking at room temperature.

[0021] Based on the above technical solution, preferably, the blocking solution in step (2) is FBS with a volume fraction of 2%-3%; and the detection antibody in step (4) is diluted with a mixture of FBS with a volume fraction of 0.5%-1% and gelatin with a volume fraction of 0.5%-1%.

[0022] More preferably, the blocking solution in step (2) is 2% FBS by volume; and the detection antibody in step (4) is diluted with a mixture of 1% FBS by volume and 1% gelatin by volume.

[0023] Based on the above technical solution, preferably, in step (5), the fluorescent secondary antibody SA-PE is diluted with PBS at a volume ratio of 1:5000-6000.

[0024] More preferably, in step (5), the fluorescent secondary antibody SA-PE is diluted with PBS at a volume ratio of 1:5000.

[0025] Based on the above technical solution, preferably, the incubation time in steps (1) to (5) is 30 min-1 h.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention first divides the carboxyl magnetic bead microspheres into different microspheres according to the intensity of the autofluorescence signal, and each microsphere can correspond to a capture antibody. When the one-to-one corresponding microspheres are connected to the capture antibody, they can be combined into 1 group. This step is the key to reducing the workload when the flow screening antibody pair is performed, and antigen binding, detection antibody binding, fluorescent secondary antibody binding are performed in sequence, and finally the final PE fluorescence signal intensity of each microsphere is detected by flow detection to determine the screening result. The screening method of the present invention can not only shorten the experimental process, but also reduce experimental consumables and labor costs, and the experimental verification results show that the matching rate of the initial screening compared with Elisa reaches more than 85%, and the matching rate of the rescreening is 100%. Therefore, the method can skillfully carry out the antibody screening work of different projects, and the flow screening results can be consistent with the Elisa double antibody sandwich, with a high accuracy rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 Flow cytometry was used to detect 9 types of microsphere clusters. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] 1. Magnetic fluorescent coded microspheres labeling protein G

[0033] 1. Activation of magnetic fluorescent coded microspheres

[0034] Take 100 μL of different types of magnetic fluorescent coded microspheres (purchased from Weidu Biotechnology, catalog number: MFB100501A~MFB100519A, see the table below for specific information) in a 2.0 mL EP tube, add 1 mL of 100 mM MES, mix well and remove the supernatant by magnetic suction; mix 50 mg / mL EDC and 50 mg / mL NHS at a ratio of V:V=1:1, take 100 μL and add them to the EP tube respectively, and incubate at room temperature for 20 min;

[0035] Fluorescently coded microspheres AFC fluorescence intensity MFB100501A ～800W MFB100505A ～400W MFB100507A ～200W MFB100508A ～100W MFB100513A ～50W MFB100516A ～25W MFB100517A ～10W MFB100518A ～5W MFB100519A ～2W

[0036] 2. Magnetic fluorescent coded microsphere labeling

[0037] After incubation, the microspheres were magnetically aspirated to remove the supernatant, the microspheres were washed with 1 mL of 50 mM MES, the supernatant was magnetically aspirated to remove, 2 mg of Protein G was added, and incubated at room temperature for 2 h;

[0038] 3. Magnetic fluorescent coded microspheres sealing

[0039] After incubation, the microspheres were magnetically aspirated to remove the supernatant, the microspheres were washed with 1 mL of PBS, the supernatant was magnetically aspirated to remove the supernatant, 1 mL of 0.1 M Tris-HCl solution was added, and incubated at room temperature for 30 min;

[0040] 4. Magnetic fluorescence coded microsphere counting

[0041] After incubation, the microspheres were magnetically aspirated to remove the supernatant, and the microspheres were washed with 1 mL of PBS. The supernatant was magnetically aspirated to remove the supernatant, and 1 mL of PBS was added to mix. 2 μL of each type of microspheres were mixed, diluted to 200 μL with PBS, and counted by flow cytometry to obtain the content of different types of microspheres (pieces / mL). Finally, the microspheres were diluted to 1×10 4 / μL, store at 4℃ away from light.

[0042] 2. Flow Cytometry Screening of Monoclonal Antibodies

[0043] 1. The labeled magnetic fluorescent coded microspheres bind to the capture antibody

[0044] For example, assuming there are 27 monoclonal antibodies, add 30 μL of microspheres and 30 μg of capture antibody in a 1:1 ratio into an EP tube containing 500 μL of PBS and incubate with shaking for 1 hour.

[0045] 2. After the microspheres bind to the capture antibody, the blank sites of the microspheres are blocked

[0046] After the microspheres were bound to the capture antibody, the supernatant was removed by magnetic aspiration, and 1.0 mL of a homemade blocking solution (2% FBS by volume) was added to each tube and incubated for 0.5 h.

[0047] 3. Add a certain concentration of antigen for incubation

[0048] The supernatant was removed by magnetic suction, and the 27 microspheres bound to the capture antibody were divided into three groups. The first group was capture antibodies No. 1-9, the second group was capture antibodies No. 10-18, and the third group was capture antibodies No. 19-27. The 9 microspheres in each group were mixed separately and then divided into 2 groups. The antigen was diluted to 0.5 μg / mL with a homemade diluent (1% FBS + 1% gelatin), 1 mL of the antigen with a concentration of 0.5 μg / mL was added to one group, and the same volume of diluent (1% FBS + 1% gelatin) was added to the other group, and incubated at room temperature with shaking.

[0049] From the above scheme, it can be seen that in the experimental operation, flow cytometry combines 27 groups of capture antibodies into 3 groups, greatly reducing the experimental content (compared with Elisa). In the final detection, the magnetic fluorescent encoding microspheres are used to separate them into 9 groups for analysis by specific fluorescence differences (see Figure 1 ). The actual detection differences between Elisa and flow cytometry were compared through the data of two projects.

[0050] 4. Add detection antibody for incubation

[0051] After diluting the 27 groups of detection antibodies with homemade diluent (1% FBS + 1% gelatin), take 100 μL / well into a 96-well plate, remove the supernatant from the microspheres incubated in step 3 by magnetic aspiration, add 270 μL of diluent and shake to mix, then take 10 μL / well and add it to the 96-well plate, and incubate at room temperature for 30 minutes.

[0052] 5. Add fluorescent secondary antibody SA-PE for incubation

[0053] Remove the supernatant from the microspheres that have completed incubation in step 4 by magnetic aspiration, dilute the fluorescent secondary antibody SA-PE with PBS at a volume ratio of 1:5000, take 100 μL / well into a 96-well plate, and incubate at room temperature with shaking for 30 minutes. After the incubation is completed, remove the supernatant by magnetic aspiration, and add 100 μL / well PBS to the 96-well plate to mix.

[0054] 6. Flow cytometry test and result analysis.

[0055] Example 2

[0056] 1. Magnetic fluorescent coded microspheres labeling protein G

[0057] 1. Activation of magnetic fluorescent coded microspheres

[0058] Take 100 μL of each magnetic fluorescent encoded microsphere in a 2.0 mL EP tube, add 1 mL of 100 mM MES, mix well and remove the supernatant by magnetic suction; mix EDC and NHS at a ratio of V:V=1:1, take 100 μL and add it to the EP tube respectively, and incubate at room temperature for 20 min;

[0059] 2. Magnetic fluorescent coded microsphere labeling

[0060] After incubation, the microspheres were magnetically aspirated to remove the supernatant, the microspheres were washed with 1 mL of 50 mM MES, the supernatant was magnetically aspirated to remove, 2 mg of Protein G was added respectively, and incubated at room temperature for 2 h.

[0061] 3. Magnetic fluorescent coded microspheres sealing

[0062] The supernatant of the incubated microspheres was removed by magnetic suction, the microspheres were washed with 1 mL of PBS, the supernatant was removed by magnetic suction, 1 mL of 0.1 M Tris-HCl solution was added, and incubated at room temperature for 30 min.

[0063] 4. Magnetic fluorescence coded microsphere counting

[0064] After incubation, the microspheres were magnetically removed from the supernatant, washed with 1 mL of PBS, and the supernatant was magnetically removed. 1 mL of PBS was added to mix well. 2 μL of each type of microspheres were mixed, diluted to 200 μL with PBS, counted with a flow cytometer, and finally diluted to 1×10 4 Pieces / μL.

[0065] 2. Flow Cytometry Screening of Monoclonal Antibodies

[0066] 1. The labeled magnetic fluorescent coded microspheres bind to the capture antibody

[0067] The protein G-labeled microspheres and capture antibodies were added to an EP tube containing 500 μL PBS at a ratio of 1:2 (μL:μg) and incubated with shaking for 1 h.

[0068] 2. After the microspheres bind to the capture antibody, the blank sites of the microspheres are blocked

[0069] After the microspheres were bound to the capture antibody, the supernatant was removed by magnetic aspiration, and 1.0 mL of a homemade blocking solution (2% FBS by volume) was added to each tube and incubated for 0.5 h.

[0070] 3. Add a certain concentration of antigen for incubation

[0071] The antigen was diluted to 0.5 μg / mL using a homemade diluent (1% FBS + 0.5% gelatin). After incubation of the microspheres, the supernatant was removed by magnetic aspiration, and the 9 microspheres in each group were mixed and divided into 2 groups. One group was added with 1 mL of 0.5 μg / mL antigen, and the other group was added with the same volume of diluent, and incubated at room temperature with shaking.

[0072] 4. Add detection antibody for incubation

[0073] After diluting the detection antibody with a homemade diluent (1% FBS + 0.5% gelatin), take 100 μL / well into a 96-well plate, magnetically remove the supernatant from the microspheres that have been incubated in step 3, add 270 μL of the diluent and shake to mix, then take 10 μL / well and add it to the 96-well plate, and incubate at room temperature for 30 minutes.

[0074] 5. Add fluorescent secondary antibody SA-PE for incubation

[0075] Remove the supernatant from the microspheres that have completed incubation in step 4 by magnetic aspiration, dilute the fluorescent secondary antibody SA-PE with PBS at a volume ratio of 1:5k, take 100 μL / well into a 96-well plate, and incubate at room temperature with shaking for 30 minutes. After the incubation is completed, remove the supernatant by magnetic aspiration, and add 100 μL / well PBS into the 96-well plate to mix.

[0076] 6. Flow cytometry test and result analysis.

[0077] Example 3

[0078] 1. Magnetic fluorescent coded microspheres labeling protein G

[0079] 1. Activation of magnetic fluorescent coded microspheres

[0080] Take 100 μL of magnetic fluorescent coded microspheres in a 2.0 mL EP tube, add 1 mL of 100 mM MES, mix well and remove the supernatant by magnetic suction; mix EDC and NHS at a ratio of V:V=1:1, take 100 μL and add them to the EP tube respectively, and incubate at room temperature for 20 min;

[0081] 2. Magnetic fluorescent coded microsphere labeling

[0082] After incubation, the microspheres were magnetically aspirated to remove the supernatant, the microspheres were washed with 1 mL of 50 mM MES, the supernatant was magnetically aspirated to remove, 2 mg of Protein G was added respectively, and incubated at room temperature for 2 h.

[0083] 3. Magnetic fluorescent coded microspheres sealing

[0084] The supernatant of the incubated microspheres was removed by magnetic suction, the microspheres were washed with 1 mL of PBS, the supernatant was removed by magnetic suction, 1 mL of 0.1 M Tris-HCl solution was added, and incubated at room temperature for 30 min.

[0085] 4. Magnetic fluorescence coded microsphere counting

[0086] After incubation, the microspheres were magnetically removed from the supernatant, washed with 1 mL of PBS, and the supernatant was magnetically removed. 1 mL of PBS was added to mix well. 2 μL of each type of microspheres were mixed, diluted to 200 μL with PBS, counted with a flow cytometer, and finally diluted to 1×10 4 Pieces / μL.

[0087] 2. Flow Cytometry Screening of Monoclonal Antibodies

[0088] 1. The labeled magnetic fluorescent coded microspheres bind to the capture antibody

[0089] The protein G-labeled microspheres and capture antibodies were added to an EP tube containing 500 μL PBS at a ratio of 1w:1 μg and incubated with shaking for 1 hour.

[0090] 2. After the microspheres bind to the capture antibody, the blank sites of the microspheres are blocked

[0091] After the microspheres were bound to the capture antibody, the supernatant was removed by magnetic aspiration, and 1.0 mL of a homemade blocking solution (3% FBS by volume) was added to each tube and incubated for 0.5 h.

[0092] 3. Add a certain concentration of antigen for incubation

[0093] The antigen was diluted to 0.5 μg / mL using a homemade diluent (0.5% FBS + 1% gelatin). After incubation of the microspheres, the supernatant was removed by magnetic aspiration, and the 9 microspheres in each group were mixed and divided into 2 groups. One group was added with 1 mL of 0.5 μg / mL antigen, and the other group was added with the same volume of diluent, and incubated at room temperature with shaking.

[0094] 4. Add detection antibody for incubation

[0095] After diluting the detection antibody with a homemade diluent (0.5% FBS + 1% gelatin), take 100 μL / well into a 96-well plate, magnetically remove the supernatant from the microspheres that have been incubated in step 3, add 270 μL of the diluent and shake to mix, then take 10 μL / well and add it to the 96-well plate, and incubate at room temperature for 30 minutes.

[0096] 5. Add fluorescent secondary antibody SA-PE for incubation

[0097] Remove the supernatant from the microspheres that have completed incubation in step 4 by magnetic aspiration, dilute the fluorescent secondary antibody SA-PE with PBS at a volume ratio of 1:6k, take 100 μL / well into a 96-well plate, and incubate at room temperature with shaking for 30 minutes. After the incubation is completed, remove the supernatant by magnetic aspiration, and add 100 μL / well PBS to the 96-well plate to mix.

[0098] 6. Flow cytometry test and result analysis.

[0099] 6.1 Comparative experimental results of different dilutions of H381 project

[0100] Table 1 Detection results of diluent 1% FBS + 1% gelatin

[0101]

[0102] Table 2 Test results of diluent 1% FBS + 0.5% gelatin

[0103]

[0104] Table 3 Detection results of diluent 0.5% FBS + 1% gelatin

[0105] Comparing the test results of different dilutions of the H381 project above, the dilution using 1% FBS + 1% gelatin had the best effect.

[0106] 6.2H173 project different dilution comparison test results

[0107] Table 4 Detection results of diluent 1% FBS + 1% gelatin

[0108]

[0109] Table 5 Detection results of diluent 1% FBS + 0.5% gelatin

[0110]

[0111]

[0112] Table 6 Detection results of diluent 0.5% FBS + 1% gelatin

[0113]

[0114] Comparing the test results of different dilutions of the H173 project above, the dilution using 1% FBS + 1% gelatin had the best effect.

[0115] Example 4: Comparison of flow cytometry and Elisa test results

[0116] 1. H198 (Human Galectin-3) Project

[0117] (1) Elisa chessboard screening of 15 monoclonal antibodies (experimental group-blank group)

[0118] Experimental steps:

[0119] Prepare biotinylated detection antibody, take 50 μg capture antibody and 0.5 μg biotin, mix, incubate overnight at 4°C, and prepare biotinylated detection antibody. Dilute the capture antibody to 2 μg / mL with CBS, add 100 μL / well to a 96-well ELISA plate (NEST manufacturer), incubate overnight at 4°C, and discard the supernatant; add 200 μL / well of blocking solution (2% FBS by volume), incubate at 37°C for 1h, discard the supernatant, and dry at 37°C for 30min; use diluent (1% BSA by volume) to gradient dilute the recombinant antigen H158-Ag to 100 ng / mL and 0 ng / mL, take 100 μL / well and add them to a 96-well ELISA plate, incubate at 37°C for 1.5h, and discard the supernatant; use diluent (1% BSA by volume) to gradient dilute the recombinant antigen H158-Ag to 100 ng / mL and 0 ng / mL, respectively, and add 100 μL / well .... The biotinylated antibody was diluted with BSA at a ratio of 1:4K, and 100 μL / well was added to a 96-well ELISA plate, incubated at 37°C for 1h, the supernatant was discarded, and the plate was washed 3 times with 0.02% PBST; the SA-labeled streptavidin was diluted with a diluent (1% BSA by volume) at a ratio of 1:4W, 100 μL / well was added to a 96-well ELISA plate, incubated at 37°C for 30min, the supernatant was discarded, and the plate was washed 5 times with 0.02% PBST; TMB color development solution was added, 90 μL / well, incubated at 37°C for 15min, 50 μL / well of stop solution was added, and OD450nm was read with an ELISA reader.

[0120] The test results are shown in Table 7. After the data were processed by experimental group-blank, the experimental group with OD value>3.30 was compared with the flow screening results. A total of 19 antibody pairs were screened by the Elisa method (15 monoclonal antibodies in the table were screened by flow cytometry).

[0121] Table 7 Elisa chessboard screening results of 15 monoclonal antibodies

[0122]

[0123] (2) Flow cytometry screening of 15 monoclonal antibodies (experimental group-blank group)

[0124] Experimental steps:

[0125] 1. Coat the labeled Protein G microspheres with the capture antibody at a ratio of 1 μL:1 μg, dilute with PBS, and incubate at room temperature with shaking for 1 hour.

[0126] 2. After the microspheres are combined with the coated antibodies, the supernatant is removed by magnetic aspiration, and a homemade blocking solution (2% FBS by volume) is added, 1 mL / group, and incubated at room temperature for 30 minutes;

[0127] 3. Remove the supernatant by magnetic absorption of the microspheres, dilute the standard (stock solution 2.00 mg / mL) at a concentration of 500 ng / mL with a homemade diluent (1% FBS + 1% gelatin), take 1 mL from each group and add it to the microspheres, and use the diluent (1% FBS + 1% gelatin) as a blank control, and incubate at room temperature with shaking for 1 hour;

[0128] 4. Remove the supernatant from the microspheres by magnetic absorption, dilute the biotinylated antibody (stock solution 200 μg / mL) with a homemade diluent at a volume ratio of 1:1k, take 100 μl / well and add it to the microspheres, and incubate at room temperature with shaking for 30 minutes;

[0129] 5. Remove the supernatant from the microspheres by magnetic aspiration, dilute the fluorescent secondary antibody SA-PE with PBS at a volume ratio of 1:5k, take 100 μL / well and add it to the microspheres, incubate at room temperature with shaking for 30 minutes, wash with PBS after incubation, remove the supernatant by magnetic aspiration, add 100 μL / well PBS to the 96-well plate and mix well.

[0130] 6. Flow cytometry test and result analysis.

[0131] The test results are shown in Table 8. After the data were processed by the experimental group-blank, the experimental group with a PE signal Flu value > 330 was compared with the flow screening results. A total of 23 antibody pair schemes were screened by the flow method (underlined data in the table).

[0132] Table 8 Results of flow cytometry screening of 15 monoclonal antibodies

[0133]

[0134]

[0135] Due to certain differences between the two methods, when comparing the screened antibody schemes, if a clear signal is detected by package anti-A-test anti-B or package anti-B-test anti-A, the two methods are considered to be consistent in the test results. Comparing the results of Elisa and flow screening of the H198 project, the consistency reached more than 85%. There were four groups of data (italics in the table) that were inconsistent with the flow, one group (6E7-5H2) analysis showed that the Elisa detection signal was low, one group (6E7-2A12) analysis showed that the flow detection signal was low, and the other two groups of data showed no signal in Elisa but had signal in flow. The subsequent natural sample test results showed that these two groups of data could not detect natural samples.

[0136] 2. H515 (human Caspase 4) project

[0137] (1) Elisa chessboard screening of 9 monoclonal antibodies (experimental group-blank group)

[0138] The experimental steps were different from those of the H198 (human Caspase 4) project in Example 4 in terms of the standard and biotinylated antibody. The standard was H515 (human Caspase 4) 100 ng / mL and 0 ng / mL, and the biotinylated antibody was 1 / 1000 (stock solution 200 μg / mL).

[0139] The test results are shown in Table 9. After the data were processed by experimental group-blank, the experimental group with OD value>3.00 was compared with the flow screening results. A total of 10 antibody pair schemes were screened by the Elisa method (underlined data in the table).

[0140] Table 99 monoclonal antibodies screened by Elisa chessboard method

[0141]

[0142] (2) Flow cytometry screening of 9 monoclonal antibodies (experimental group-blank group)

[0143] Experimental steps:

[0144] 1. Labeled Protein G microspheres and coated antibodies were coated at a ratio of 1 μL:1 μg, diluted with PBS, and incubated at room temperature with shaking for 1 hour.

[0145] 2. After the microspheres are combined with the coated antibodies, the supernatant is removed by magnetic aspiration, and a homemade blocking solution (2% FBS by volume) is added, 1 mL / group, and incubated at room temperature with shaking for 30 minutes;

[0146] 3. Remove the supernatant by magnetic absorption of the microspheres, dilute the standard (stock solution 1.20 mg / mL) at a concentration of 500 ng / mL with a homemade diluent (1% FBS + 1% gelatin), take 1 mL from each group and add it to the microspheres, and use the diluent (1% FBS + 1% gelatin) as a blank control, and incubate at room temperature with shaking for 1 hour;

[0147] 4. Remove the supernatant from the microspheres by magnetic absorption, dilute the biotinylated antibody (stock solution 200 μg / mL) with a homemade diluent at a volume ratio of 1:1k, take 100 μl / well and add it to the microspheres, and incubate at room temperature with shaking for 30 minutes;

[0148] 5. Remove the supernatant from the microspheres by magnetic aspiration, dilute the fluorescent secondary antibody SA-PE with PBS at a volume ratio of 1:5k, take 100 μL / well and add it to the microspheres, incubate at room temperature with shaking for 30 minutes, wash with PBS after incubation, remove the supernatant by magnetic aspiration, add 100 μL / well PBS to the 96-well plate and mix well.

[0149] 6. Flow cytometry test and result analysis.

[0150] The test results are shown in Table 10. After the data were processed by experimental group-blank, the experimental group with PE signal Flu value>5000 was compared with the flow screening results. A total of 17 antibody pair schemes were screened by the flow method (underlined data in the table).

[0151] Table 109 monoclonal antibody flow screening results

[0152]

[0153] Comparing the results of H515 project Elisa (Table 9) and flow cytometry (Table 10), the consistency reached more than 85%. There were 4 groups of data (see italics in Table 3) that were inconsistent with the flow cytometry, and all of them had low Elisa detection signals.

[0154] 3. Summary

[0155] After comparing and verifying Elisa and flow cytometry data from multiple projects, it was shown that the screening method for monoclonal antibody pairing by high-throughput flow cytometry can significantly improve work efficiency while ensuring reliable results.

[0156] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A screening method for monoclonal antibody pairing based on high-throughput flow cytometry, characterized in that: The following steps are involved: (1) The magnetic fluorescent coded microspheres labeled with Protein G are incubated and combined with the capture antibody; (2) After incubation, a blocking solution is added to block the blank sites of the microspheres; (3) After blocking, add antigen and incubate; (4) adding detection antibody after antigen incubation for incubation; (5) After incubation with the detection antibody, the fluorescent secondary antibody SA-PE was added for incubation, and then detected by flow cytometry.

2. The screening method according to claim 1, characterized in that The preparation steps of the magnetic fluorescent coded microspheres labeled with Protein G in step (1) are as follows: (1) Activation of magnetic fluorescent coded microspheres: Add magnetic fluorescent coded microspheres to MES solution and mix well. After magnetic suction to remove the supernatant, add to EDC / NHS mixed solution and incubate; (2) Magnetic fluorescent coded microsphere labeling: After the incubated microspheres are washed, Protein G is added for incubation to obtain magnetic fluorescent coded microspheres labeled with Protein G.

3. The screening method according to claim 2, characterized in that The EDC and NHS were mixed uniformly in a volume ratio of 1:

1.

4. The screening method according to claim 2, characterized in that 10 6 The amount of Protein G used in each of the magnetic fluorescent encoded microspheres is 200 μg.

5. The screening method according to claim 2, characterized in that The incubation time during the preparation of the magnetic fluorescent encoded microspheres labeled with Protein G is 20 minutes to 2 hours.

6. The screening method according to claim 1, characterized in that The mass volume ratio of the capture antibody to the magnetic fluorescent coding microspheres labeled with Protein G is 1:1-2.

7. The screening method according to claim 1, wherein The blocked microspheres in step (3) are divided into two groups, one of which is added with antigen, and the other is added with the same volume of diluent, and incubated at room temperature with shaking for 30 minutes to 1 hour.

8. The screening method according to claim 1, characterized in that In the step (2), the blocking solution is 2%-3% FBS by volume; in the step (4), the detection antibody is diluted with a mixture of 0.5%-1% FBS by volume and 0.5%-1% gelatin by volume.

9. The screening method according to claim 1, characterized in that In the step (5), the fluorescent secondary antibody SA-PE is diluted with PBS at a volume ratio of 1:5000-6000.

10. The screening method according to claim 1, wherein The incubation time in steps (1) to (5) is 30 min to 1 h.

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