A screening method for monoclonal antibody pairing based on high-throughput flow cytometry

Abstract

The present invention proposes a screening method for detecting monoclonal antibody pairing based on high-throughput flow cytometry, comprising the steps of: (1) magnetic fluorescent coded microspheres labeled Protein G are incubated with capture antibodies; (2) blocking solution is added after incubation to block microsphere blank sites; (3) antigen is added after blocking and incubated; (4) detection antibody is added after antigen incubation and incubated; (5) fluorescent secondary antibody SA PE is added after detection antibody incubation and incubated, and flow cytometry is used after terminating. The screening method of the present invention can shorten the experimental process and reduce experimental consumables and labor costs, and the experimental verification results show that the matching rate of the primary 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 pair screening work of different projects, and the flow screening result can be consistent with the Elisa double antibody sandwich, with a high accuracy rate.

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 pairings. Background Art

[0002] The Elisa double antibody sandwich method is a relatively mature, low-difficulty, and widely applicable antibody pair screening method used by most laboratories. However, in actual operation, many projects screen a large number of monoclonal antibodies. When using the Elisa double antibody sandwich method for monoclonal antibody cross-pairing screening, the experimental plan has an exponential relationship with the number of antibodies, resulting in a very large workload and cumbersome operation steps.

[0003] Flow cytometry is also widely used in cytokine detection. Its principle is also a double-antibody sandwich assay. However, the raw antibody materials are usually obtained through the Elisa method. Currently, flow cytometry is not used for large-scale antibody pair screening. The reason is that the double-antibody sandwich assay requires fluorescent magnetic microspheres that resemble cells instead of ELISA plates as the matrix material, and the requirements for the type and performance of the matrix material are relatively high. Therefore, in order to solve the problems of cumbersome operation steps and high workload when screening monoclonal antibody pairs using the 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 monoclonal antibody pairing based on high-throughput flow cytometry. The method not only solves the problems of complicated operation steps and high workload when screening monoclonal antibody pairs using the Elisa double-antibody sandwich method, but also greatly improves detection efficiency.

[0005] The technical solution of the present invention is achieved as follows:

[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) Magnetic fluorescently encoded microspheres labeled with Protein G are incubated and combined with the capture antibody;

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

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

[0010] (4) After antigen incubation, detection antibody is added 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 and incubated 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 during the preparation of 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-to-volume ratio of the capture antibody to the magnetic fluorescent encoded microspheres labeled with Protein G is 1:1-2 (units are μg and μL, respectively).

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

[0020] On the basis of 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 at room temperature with shaking.

[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] On the basis of 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 carboxyl magnetic bead microspheres into different microspheres according to the level of their own fluorescence signal intensity. 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 is performed. 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 determined by flow detection to determine the result of the screening. The screening method of the present invention can shorten the experimental process and 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 pair 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 following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any 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 embodiments described 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 making creative efforts 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] 100 μL of each type of magnetic fluorescent-encoded microspheres (purchased from Weidu Biotechnology, catalog numbers: MFB100501A to MFB100519A, see the table below for details) were placed in a 2.0 mL EP tube. 1 mL of 100 mM MES was added to each tube, mixed thoroughly, and the supernatant was removed by magnetic aspiration. 50 mg / mL EDC and 50 mg / mL NHS were mixed at a V:V ratio of 1:1, and 100 μL of each mixture was added to the EP tubes. The mixture was incubated at room temperature for 20 min.

[0035] Types of fluorescent 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, washed with 1 mL of 50 mM MES, and 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, washed with 1 mL of PBS, the supernatant was magnetically aspirated to remove the supernatant, and 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 supernatant of the microspheres was removed by magnetic aspiration, and the microspheres were washed with 1 mL of PBS. The supernatant was removed by magnetic aspiration, and 1 mL of PBS was added to mix. 2 μL of each type of microspheres was mixed, diluted to 200 μL with PBS, and counted using a flow cytometer to obtain the content of different types of microspheres (pieces / mL). Finally, the microspheres were diluted with PBS to 1×10 4 / μL, store at 4℃ in the dark.

[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 at 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 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 aspiration, and the 27 microspheres bound to the capture antibodies were divided into three groups: the first group contained capture antibodies 1-9, the second group contained capture antibodies 10-18, and the third group contained capture antibodies 19-27. The nine microspheres from each group were mixed separately and then divided equally into two groups. The antigen was diluted to 0.5 μg / mL using a homemade diluent (1% FBS + 1% gelatin). 1 mL of the 0.5 μg / mL antigen was added to one group, and the same volume of diluent (1% FBS + 1% gelatin) was added to the other group. The cells were incubated at room temperature with shaking.

[0049] As can be seen from the above scheme, in terms of experimental operation, flow cytometry combines 27 groups of capture antibodies into 3 groups, which greatly reduces the experimental content (compared to Elisa). In the final detection, the specific fluorescence differences of magnetic fluorescent coding microspheres are used to separate them into 9 groups for analysis (see Figure 1 ). Subsequently, data from two projects will be presented to compare the actual detection differences between ELISA and flow cytometry.

[0050] 4. Add detection antibody and incubate

[0051] After diluting the 27 groups of detection antibodies with homemade diluent (1% FBS + 1% gelatin), take 100 μL / well of each into a 96-well plate. After magnetically aspirating the supernatant from the microspheres incubated in step 3, 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] After the incubation in step 4, remove the supernatant by magnetic aspiration, dilute the fluorescent secondary antibody SA-PE with PBS at a volume ratio of 1:5000, and add 100 μL / well to a 96-well plate. Incubate at room temperature with shaking for 30 minutes. After the incubation is complete, remove the supernatant by magnetic aspiration, and add 100 μL / well PBS to the 96-well plate and mix well.

[0054] 6. Flow cytometer 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] 100 μL of each magnetic fluorescent encoded microsphere was placed in a 2.0 mL EP tube, and 1 mL of 100 mM MES was added to each tube. After mixing, the supernatant was removed by magnetic aspiration. EDC and NHS were mixed at a ratio of V:V = 1:1, and 100 μL was added to each EP tube and incubated 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, washed with 1 mL of 50 mM MES, the supernatant was magnetically aspirated to remove, and 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 incubated microspheres were magnetically aspirated to remove the supernatant, washed with 1 mL of PBS, magnetically aspirated to remove the supernatant, and 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 supernatant of the microspheres was removed by magnetic aspiration, and the microspheres were washed with 1 mL of PBS. The supernatant was removed by magnetic aspiration, and 1 mL of PBS was added to mix. 2 μL of each type of microspheres was mixed, diluted to 200 μL with PBS, and counted using a flow cytometer. Finally, the microspheres were diluted to 1×10 4 pcs / μL.

[0065] 2. Flow Cytometry Screening of Monoclonal Antibodies

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

[0067] 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 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] Antigen was diluted to 0.5 μg / mL using a homemade diluent (1% FBS + 0.5% gelatin). After incubation, the supernatant was magnetically aspirated and the nine microspheres from each group were mixed and divided equally into two groups. 1 mL of 0.5 μg / mL antigen was added to one group, and the same volume of diluent was added to the other group. The cells were incubated at room temperature with shaking.

[0072] 4. Add detection antibody and incubate

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

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

[0075] After the incubation in step 4, the supernatant was removed by magnetic aspiration. The fluorescent secondary antibody SA-PE was diluted with PBS at a volume ratio of 1:5k, and 100 μL / well was added to a 96-well plate. The plate was incubated at room temperature with shaking for 30 minutes. After the incubation was completed, the supernatant was removed by magnetic aspiration and 100 μL / well PBS was added to the 96-well plate and mixed.

[0076] 6. Flow cytometer 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] 100 μL of magnetic fluorescent encoded microspheres was placed in a 2.0 mL EP tube, and 1 mL of 100 mM MES was added to each tube. After mixing, the supernatant was removed by magnetic aspiration. EDC and NHS were mixed at a ratio of V:V = 1:1, and 100 μL was added to each EP tube and incubated 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, washed with 1 mL of 50 mM MES, the supernatant was magnetically aspirated to remove, and 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 incubated microspheres were magnetically aspirated to remove the supernatant, washed with 1 mL of PBS, magnetically aspirated to remove the supernatant, and 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 supernatant of the microspheres was removed by magnetic aspiration, and the microspheres were washed with 1 mL of PBS. The supernatant was removed by magnetic aspiration, and 1 mL of PBS was added to mix. 2 μL of each type of microspheres was mixed, diluted to 200 μL with PBS, and counted using a flow cytometer. Finally, the microspheres were diluted to 1×10 4 pcs / μL.

[0087] 2. Flow Cytometry Screening of Monoclonal Antibodies

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

[0089] 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 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] Antigen was diluted to 0.5 μg / mL using a homemade diluent (0.5% FBS + 1% gelatin). After incubation, the supernatant was magnetically aspirated and the nine microspheres from each group were mixed and divided equally into two groups. 1 mL of 0.5 μg / mL antigen was added to one group, and the same volume of diluent was added to the other group. The cells were incubated at room temperature with shaking.

[0094] 4. Add detection antibody and incubate

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

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

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

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

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

[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 Test results of diluent 0.5% FBS + 1% gelatin

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

[0106] 6.2H173 Project Comparison of Different Dilution Results

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

[0108]

[0109] Table 5: Test 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 for the H173 project above, the dilution solution of 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 mix with 0.5 μg biotin, incubate overnight at 4°C to prepare biotinylated detection antibody. Dilute the capture antibody to 2 μg / mL with CBS, add 100 μL / well to a 96-well enzyme-labeled plate (NEST manufacturer), incubate overnight at 4°C, and discard the supernatant; add 200 μL / well 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 enzyme-labeled plate, incubate at 37°C for 1.5h, and discard the supernatant .... Biotinylated antibody was diluted with BSA at a ratio of 1:4K, 100 μL / well was added to a 96-well ELISA plate, incubated at 37°C for 1 h, the supernatant was discarded, and the plate was washed three times with 0.02% PBST; SA-labeled streptavidin was diluted with 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 30 min, the supernatant was discarded, and the plate was washed five times with 0.02% PBST; TMB color development solution was added at 90 μL / well, incubated at 37°C for 15 min, 50 μL / well of stop solution was added, and OD450 nm was read on a microplate 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 cytometry screening results. A total of 19 antibody pair solutions were screened by the ELISA method (15 monoclonal antibodies in the table were screened by flow cytometry).

[0121] Table 7 Elisa chessboard screening results for 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 bind to the coated antibody, remove the supernatant by magnetic aspiration and add 1 mL / group of homemade blocking solution (2% FBS) and incubate at room temperature with shaking for 30 min.

[0127] 3. Remove the supernatant by magnetic aspiration of the microspheres, dilute the standard (stock solution 2.00 mg / mL) to 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. The diluent (1% FBS + 1% gelatin) is used as a blank control, and incubate at room temperature with shaking for 1 hour.

[0128] 4. Remove the supernatant from the microspheres by magnetic aspiration, 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 cytometer test and result analysis.

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

[0132] Table 8 Flow cytometry screening results of 15 monoclonal antibodies

[0133]

[0134]

[0135] Due to certain differences between the two methods, when comparing the screened antibody solutions, if a significant 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 their test results. Comparing the results of the H198 project ELISA and flow cytometry screening, the consistency reached over 85%. Four sets of data (italicized in the table) were inconsistent with the flow cytometry results. One set (6E7-5H2) showed a low ELISA detection signal, one set (6E7-2A12) showed a low flow cytometry signal, and two sets of data showed no ELISA signal but a flow cytometry signal. Subsequent natural sample testing results showed that these two sets 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 differed from those for the H198 (human Caspase 4) project in Example 4 in terms of the standards and biotinylated antibody. The standards were 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 solutions 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. Coat the labeled Protein G microspheres with the antibody at a ratio of 1 μL:1 μg, dilute with PBS, and incubate at room temperature with shaking for 1 hour.

[0145] 2. After the microspheres bind to the coated antibody, remove the supernatant by magnetic aspiration and add 1 mL / group of homemade blocking solution (2% FBS) and incubate at room temperature with shaking for 30 min.

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

[0147] 4. Remove the supernatant from the microspheres by magnetic aspiration, 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 cytometer test and result analysis.

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

[0151] Table 109 monoclonal antibody flow cytometry 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, all of which were due to low Elisa detection signals.

[0154] 3. Summary

[0155] After comparing and verifying the Elisa and flow cytometry data of multiple projects, it was shown that the screening method of monoclonal antibody pairing detected by high-throughput flow cytometry can significantly improve work efficiency while ensuring the reliability of the 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 scope of protection 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) Magnetic fluorescent microspheres with different fluorescence intensities labeled with Protein G are incubated and combined with the capture antibody; (2) After incubation, blocking solution is added to block the blank sites of the microspheres; (3) After blocking, add antigen and incubate; (4) After antigen incubation, detection antibody is added for incubation; (5) After incubation with the detection antibody, the fluorescent secondary antibody SA-PE is added for incubation. If the antibody pair is successfully paired, the fluorescent secondary antibody SA-PE will bind to the detection antibody and detect the PE signal. After the incubation, the APC signal and PE signal channels are detected by flow cytometry. The screening results are determined based on the final PE fluorescence signal intensity of the microspheres with different APC fluorescence intensities. The APC signal is the fluorescent signal of the magnetic fluorescent coding microspheres themselves.

2. The screening method according to claim 1, wherein 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 aspiration, remove the supernatant and add to EDC / NHS mixed solution for incubation. (2) Magnetic fluorescent coded microsphere labeling: After the incubated microspheres are washed, Protein G is added and incubated to obtain magnetic fluorescent coded microspheres labeled with Protein G.

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

1.

4. The screening method according to claim 2, wherein 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, wherein The incubation time during the preparation of the magnetic fluorescent coding microspheres labeled with Protein G is 20 minutes to 2 hours.

6. The screening method according to claim 1, wherein The mass-to-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, wherein In step (2), the blocking solution is 2%-3% FBS by volume; in 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, wherein 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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