CN108205063B - Detection method (filter membrane plate ELISA) for anti-CD 20 monoclonal antibody and antigen binding activity - Google Patents

Detection method (filter membrane plate ELISA) for anti-CD 20 monoclonal antibody and antigen binding activity Download PDF

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CN108205063B
CN108205063B CN201611186609.8A CN201611186609A CN108205063B CN 108205063 B CN108205063 B CN 108205063B CN 201611186609 A CN201611186609 A CN 201611186609A CN 108205063 B CN108205063 B CN 108205063B
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plate
filter membrane
sample
detection method
detected
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CN108205063A (en
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张玉杰
金佳驿
梅彩英
吴幼玲
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Zhejiang Teruisi Pharmaceutical Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Abstract

The invention relates to a detection method (filter membrane plate ELISA) for the binding activity of an anti-CD 20 monoclonal antibody and an antigen. Specifically, the detection method of the present invention comprises steps (1) to (9). The detection method provided by the invention meets the verification requirements of specificity, precision, linearity and range, accuracy, durability and the like, and can be effectively applied to detection of the binding activity of the anti-CD 20 monoclonal antibody.

Description

Detection method (filter membrane plate ELISA) for anti-CD 20 monoclonal antibody and antigen binding activity
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a detection method (filter membrane plate ELISA) for the binding activity of an anti-CD 20 monoclonal antibody and an antigen.
Background
In recent years, significant progress has been made in the study of monoclonal antibodies and clinical trials directed to the treatment of non-hodgkin's lymphomas, among which a widely used and fruitful preparation of monoclonal antibodies against CD 20. The research shows that when the affinity of the anti-CD 20 monoclonal antibody and the antigen is improved, the specificity of the antibody is enhanced, and the killing effect on tumor cells is also obviously improved. Therefore, in the development process of the anti-CD 20 monoclonal antibody, how to establish a detection method for evaluating the binding activity of the strong and weak affinity for screening the anti-CD 20 monoclonal antibody with high affinity is very important, and the method has important significance in research and development, quality control and even clinical application of the anti-CD 20 monoclonal antibody.
The CD20 protein has four transmembrane regions (TM 1-4), wherein only the amino acid sequence between TM3 and TM4 is outside the cell membrane, TM1 and TM2 are connected, and the region between TM2 and TM3, the N end and the C end are all in cytoplasm, so that the CD20 molecule is exposed to the outside of the cell in a short way, and the epitope in the extracellular region of CD20 is relatively few. Ala at position 170 and Pro at position 172 determine whether the CD20 molecule is recognized by anti-CD 20 antibodies. The activity of the recombinant expressed CD20 protein is lost, and the recombinant expressed CD20 protein cannot be combined with an antibody, so that the antigen-antibody combination capacity of CD20 is generally measured by using a living cell combination test.
From the current literature reports, there are two main methods for detecting the binding activity of the anti-CD 20 monoclonal antibody:
1. the flow cytometry detection method comprises the following steps: one is competitive binding detection method, which adopts fluorescent labeled anti-CD 20 monoclonal antibody and the sample to be detected to compete with CD20 on the cell surface, and the affinity between the sample to be detected and the antigen is judged according to the fluorescence intensity of the labeled antibody. The disadvantages of this method are: the fluorescent labeling efficiency is difficult to standardize, if the labeling is incomplete, the unlabeled sample to be detected and the labeled anti-CD 20 monoclonal antibody compete to bind with the cell surface antigen, and the antigen-antibody binding efficiency cannot be really reflected; the method is a competitive binding experiment, can only reflect the strength of the competitive binding capacity of a sample to be tested and a reference product to cell surface CD20, lacks systematic research on stability, repeatability and reliability, and cannot truly evaluate the binding activity of the sample to be tested. The other method is an indirect detection method, although a commercial fluorescence labeled FITC anti-human IgG antibody is also used for detection, the method is not standardized, and is mostly used for monoclonal antibody screening in the development stage, namely, negative and positive results are identified, the method lacks of relevant evaluation in the aspects of specificity, accuracy, repeatability and the like, the evaluation of experimental results is different, and the accuracy and reliability of the detection results are difficult to guarantee.
2. Enzyme-linked immunosorbent assay (ELISA) is carried out by directly coating cells with high expression of CD20, fixing and carrying out ELISA detection. The method has the defects that the CD20 membrane protein is easily damaged by cell fixing liquid, so that the binding force of the antigen and the antibody is reduced, and the binding activity of the antigen and the antibody cannot be truly reflected.
Therefore, there is still a need in the art to develop a method for rapidly, easily, accurately and efficiently detecting the binding activity of the anti-CD 20 monoclonal antibody.
Disclosure of Invention
Aiming at the defects of the methods reported in the prior documents, the invention aims to provide a method for quickly, simply, accurately and efficiently detecting the binding activity of an anti-CD 20 monoclonal antibody,
the invention provides a method for detecting the binding activity of an anti-CD 20 monoclonal antibody and an antigen, which comprises the following steps:
(1) washing the plate: stacking the filter membrane plate paved with the cells on an empty pore plate, adding washing liquid, centrifuging to wash the plate, and taking down the filter membrane plate; the cells express CD 20;
(2) sample adding: adding a reference substance with a series of gradient concentrations and a sample to be detected into the filter membrane plate treated in the step (1) for incubation; the reference substance and the sample to be detected are anti-human CD20 monoclonal antibodies;
(3) washing the plate: after incubation is finished, stacking the filter membrane plate treated in the step (2) on an empty pore plate, adding washing liquid, centrifuging to wash the plate, and taking down the filter membrane plate;
(4) adding a secondary antibody: adding an HRP-labeled anti-human IgG Fc specific antibody solution into the filter membrane plate treated in the step (3) for incubation;
(5) washing the plate: after incubation is finished, stacking the filter membrane plate treated in the step (4) on an empty pore plate, adding washing liquid, centrifuging to wash the plate, and taking down the filter membrane plate;
(6) color development: adding the chromogenic substrate solution into the filter membrane plate treated in the step (5), and terminating the reaction after developing color at room temperature;
(7) transferring: stacking the filter membrane plate treated in the step (6) on an empty pore plate, centrifuging, and filtering the liquid into the pore plate;
(8) and (3) enzyme-labeling instrument determination: reading the plate processed in the step (7) by using an enzyme-labeling instrument, and recording the plate reading result; and
(9) and fitting a curve to the concentration and the read plate result to determine the binding activity of the sample to be detected.
In another preferred embodiment, the incubation is performed at 37 ℃ in an incubator with 5% carbon dioxide.
In another preferred embodiment, the incubation time is 0.1-1 hour; preferably, 0.2 to 0.8 hour; more preferably 0.5 hour.
In another preferred embodiment, the well plate is a 96-well plate.
In another preferred embodiment, in steps (1), (3) and (5), the well plate on which the filter membrane plate is stacked is centrifuged before the washing solution is added.
In another preferred embodiment, the steps(1) The method also comprises the following cell culture steps: taking cells in logarithmic growth phase, and preparing viable cell density of 5-10x10 with complete culture medium5Cell suspension per mL, with which filter plates are plated, ready for use.
In another preferred embodiment, after plating, DPBS is added to the edge holes.
In another preferred embodiment, the viable cell density is 8x105one/mL.
In another preferred example, the filter membrane plate is a 96-well filter membrane plate.
In another preferred embodiment, the cell is a human Burkitt's lymphoma cell.
In another preferred embodiment, the cells are Raji cells.
In another preferred example, the complete medium is RPMI1640 complete medium containing 10% FBS and 1% streptomycin and penicillin.
In another preferred example, before the step (1), the method further comprises the steps of preparing a reference substance and a sample to be tested: and taking a reference product and a sample to be detected, and preparing a series of reference products and samples to be detected with gradient concentrations.
In another preferred embodiment, the preparation steps of the reference substance and the sample to be tested are as follows: taking a reference product and a sample to be detected, diluting the reference product and the sample to be detected to pre-dilution concentration by using DPBS, and then carrying out serial gradient dilution to obtain a plurality of (such as 10-20) dilutions.
In another preferred embodiment, the pre-dilution concentration is 0.15 mg/mL.
In another preferred embodiment, the serial gradient dilution is a3, 4, 5, 6, 7, 8, 9 or 10 fold serial gradient dilution.
In another preferred embodiment, the washing solution is a PBS solution.
In another preferred embodiment, the washing solution is a PBS solution containing 0.05% to 0.1% tween 20.
In another preferred embodiment, the amount of the washing solution is 100-300 microliters/well; preferably 150-; more preferably 200. mu.l/well.
In another preferred embodiment, the centrifugation is performed by a plate centrifuge.
In another preferred example, the rotation speed of the centrifugation is 600-1200 rpm; preferably 700 and 1100 rpm; more preferably 800 or 1000 rpm.
In another preferred embodiment, the time of centrifugation is 0.5-4 minutes; preferably 0.5-2 minutes; more preferably 1 minute.
In another preferred embodiment, the centrifugation may be performed once, or may be repeated two, three or more times.
In another preferred example, in step (8), the plate reading is performed at 600nm and 450 nm.
In another preferred example, the reference wavelength is 600 nm.
In another preferred example, in the step (9), a four-parameter fitting curve is performed by taking the concentration as an abscissa and taking the difference value between the plate reading result under 450nm and the plate reading result under 600nm as an ordinate, so as to obtain the EC of the sample to be measured and the EC of the reference product respectively50Comparison of EC of both50And determining the binding activity of the sample to be tested.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 is a graph of a curve fit of the binding activity of a reference sample and a sample to be tested.
FIG. 2 is a graph of a curve fit of the binding activity of a reference sample and a test sample.
FIG. 3 is a graph of the method validation (linear and range) -linear fit of the binding activity of the anti-CD 20 monoclonal antibody.
FIG. 4 shows a schematic flow chart of the detection method of the present invention.
Detailed Description
The inventor of the invention has found out a detection method for rapidly, simply, accurately and efficiently screening anti-CD 20 monoclonal antibody drugs and analyzing the binding activity through extensive and intensive research, and the method can meet the requirements of specificity, precision including repeatability, daytime difference, personnel operation error, linearity and range, accuracy, durability and the like in the verification process, and has important significance for research and development, quality control and even clinical application of anti-CD 20 monoclonal antibody. On this basis, the inventors have completed the present invention.
Detection method
The detection method of the invention can comprise the following steps:
(1) washing the plate: stacking the filter membrane plate paved with the cells on an empty pore plate, adding washing liquid, centrifuging to wash the plate, and taking down the filter membrane plate; the cells express the CD20 antigen;
in another preferred embodiment, the washing solution is a PBS solution.
In another preferred embodiment, the washing solution is a PBS solution containing 0.05% to 0.1% tween 20.
In another preferred embodiment, the amount of the washing solution is 100-300 microliters/well; preferably 150-; more preferably 200. mu.l/well.
In another preferred embodiment, the well plate on which the filter membrane plate is stacked is centrifuged before the wash solution is added.
In another preferred embodiment, the centrifugation is performed by a plate centrifuge.
In another preferred example, the rotation speed of the centrifugation is 600-1200 rpm; preferably 700 and 1100 rpm; more preferably 800 or 1000 rpm.
In another preferred embodiment, the time of centrifugation is 0.5-4 minutes; preferably 0.5-2 minutes; more preferably 1 minute.
In another preferred embodiment, the centrifugation may be performed once, or may be repeated two, three or more times.
In another preferred example, step (1) is preceded by a cell culture step: taking cells in logarithmic growth phase, and preparing viable cell density of 5-10x10 with complete culture medium5Cell suspension per mL, with which filter plates are plated, ready for use.
In another preferred example, the complete medium is RPMI1640 complete medium containing FBS and streptomycin and penicillin.
In another preferred example, the complete medium is RPMI1640 complete medium containing 10% FBS and 1% streptomycin and penicillin.
In another preferred embodiment, after plating, DPBS is added to the edge holes.
In another preferred embodiment, the viable cell density is 8x105one/mL.
In another preferred embodiment, the cell is a human Burkitt's lymphoma cell.
In another preferred embodiment, the cells are Raji cells.
In another preferred example, before the step (1), the method further comprises the steps of preparing a reference substance and a sample to be tested: and taking a reference product and a sample to be detected, and preparing a series of reference products and samples to be detected with gradient concentrations.
In another preferred embodiment, the preparation steps of the reference substance and the sample to be tested are as follows: taking a reference product and a sample to be detected, diluting the reference product and the sample to be detected to pre-dilution concentration by using DPBS, and then carrying out serial gradient dilution to obtain a plurality of (such as 10-20) dilutions.
In another preferred embodiment, the pre-dilution concentration is 0.15 mg/mL.
In another preferred embodiment, the serial gradient dilution is a3, 4, 5, 6, 7, 8, 9 or 10 fold serial gradient dilution.
In another preferred example, the reference is rituximab sold on the market or rituximab prepared by the inventor according to the production method provided by the rituximab single antigen research patent.
In another preferred example, the sample to be tested is rituximab sold in the market or rituximab prepared by the inventor according to the production method provided by the rituximab single antigen research patent.
(2) Sample adding: and (3) adding the reference substance and the sample to be tested with the series of gradient concentrations into the filter membrane plate treated in the step (1) for incubation.
In another preferred embodiment, the incubation is performed at 37 ℃ in an incubator with 5% carbon dioxide.
In another preferred embodiment, the incubation time is 0.1-1 hour; preferably, 0.2 to 0.8 hour; more preferably 0.5 hour.
(3) Washing the plate: and (3) after the incubation is finished, stacking the filter membrane plate treated in the step (2) on an empty pore plate, adding washing liquor, centrifuging to wash the plate, and then taking down the filter membrane plate.
In another preferred embodiment, the washing solution is a PBS solution.
In another preferred embodiment, the washing solution is a PBS solution containing 0.05% to 0.1% tween 20.
In another preferred embodiment, the amount of the washing solution is 100-300 microliters/well; preferably 150-; more preferably 200. mu.l/well.
In another preferred embodiment, the well plate on which the filter membrane plate is stacked is centrifuged before the wash solution is added.
In another preferred embodiment, the centrifugation is performed by a plate centrifuge.
In another preferred example, the rotation speed of the centrifugation is 600-1200 rpm; preferably 700 and 1100 rpm; more preferably 800 or 1000 rpm.
In another preferred embodiment, the time of centrifugation is 0.5-4 minutes; preferably 0.5-2 minutes; more preferably 1 minute.
In another preferred embodiment, the centrifugation may be performed once, or may be repeated two, three or more times.
(4) Adding a secondary antibody: adding an HRP-labeled anti-human IgG Fc specific antibody solution to the filter membrane plate treated in the step (3) for incubation.
In another preferred embodiment, the incubation is performed at 37 ℃ in an incubator with 5% carbon dioxide.
In another preferred embodiment, the incubation time is 0.1-1 hour; preferably, 0.2 to 0.8 hour; more preferably 0.5 hour.
(5) Washing the plate: and (3) after the incubation is finished, stacking the filter membrane plate treated in the step (4) on an empty pore plate, adding washing liquor, centrifuging to wash the plate, and then taking down the filter membrane plate.
In another preferred embodiment, the washing solution is a PBS solution.
In another preferred embodiment, the washing solution is a PBS solution containing 0.05% to 0.1% tween 20.
In another preferred embodiment, the amount of the washing solution is 100-300 microliters/well; preferably 150-; more preferably 200. mu.l/well.
In another preferred embodiment, the well plate on which the filter membrane plate is stacked is centrifuged before the wash solution is added.
In another preferred embodiment, the centrifugation is performed by a plate centrifuge.
In another preferred example, the rotation speed of the centrifugation is 600-1200 rpm; preferably 700 and 1100 rpm; more preferably 800 or 1000 rpm.
In another preferred embodiment, the time of centrifugation is 0.5-4 minutes; preferably 0.5-2 minutes; more preferably 1 minute.
In another preferred embodiment, the centrifugation may be performed once, or may be repeated two, three or more times.
(6) Color development: adding the chromogenic substrate solution into the filter membrane plate treated in the step (5), and terminating the reaction after developing color at room temperature.
In another preferred embodiment, the chromogenic substrate is TMB.
(7) Transferring: and (4) stacking the filter membrane plate treated in the step (6) on an empty pore plate, centrifuging, and filtering the liquid into the pore plate.
In another preferred embodiment, the centrifugation is performed by a plate centrifuge.
In another preferred example, the rotation speed of the centrifugation is 600-1200 rpm; preferably 700 and 1100 rpm; more preferably 800 or 1000 rpm.
In another preferred embodiment, the time of centrifugation is 0.5-4 minutes; preferably 0.5-2 minutes; more preferably 1 minute.
In another preferred embodiment, the centrifugation may be performed once, or may be repeated two, three or more times.
(8) And (3) enzyme-labeling instrument determination: and (4) reading the plate processed in the step (7) by using a microplate reader, and recording the plate reading result.
In another preferred example, the plate reading is performed at 600nm (as a reference wavelength) and 450nm for the well plate treated in step (7).
(9) And fitting a curve to the concentration and the read plate result to determine the binding activity of the sample to be detected.
In another preferred example, the concentration of the sample to be measured or the reference substance is taken as the abscissa, the difference value between the plate reading result under 450nm and the plate reading result under 600nm is taken as the ordinate, a four-parameter equation is used for fitting,
the four parameter equation is Y ═ A-D)/(1+ (X/C)B)+D，
Wherein the content of the first and second substances,
a represents the asymptote estimate on the curve;
b represents the slope of the curve;
d represents the asymptote estimate under the curve;
the C value represents the dose corresponding to half of the maximal binding, i.e., the corresponding EC50Value, respectively obtaining EC of the sample to be detected and reference substance50Value, EC for comparison of both50And determining the binding activity of the sample to be tested.
Binding activity (%) of the sample to be tested to reference sample EC50Value/sample to be tested EC50The value x 100%
The well plate used in the present invention is preferably a 96-well plate. The filter membrane plate adopted by the invention is preferably a 96-hole filter membrane plate, and the filter membrane hole diameter of the filter membrane plate is smaller than the cell diameter. The pore plate and the filter membrane plate are matched. Can be obtained commercially.
The detection method of the present invention may be performed as the flow shown in fig. 4.
The main advantages of the invention include:
1. the invention provides a method for detecting the binding activity of an anti-CD 20 monoclonal antibody and an antigen, which has the advantages of good specificity, strong specificity, high precision, repeatability, daytime difference and personnel operation error RSD less than 15%. Therefore, the method of the invention can rapidly, accurately and efficiently analyze the binding activity of the anti-CD 20 monoclonal antibody, and has important significance for research and development, quality control and even clinical application of the anti-CD 20 monoclonal antibody.
2. According to the method, a traditional ELISA plate washing mode is replaced by a centrifugal plate washing mode, damage of glutaraldehyde, paraformaldehyde and other reagents used for fixing cells on a solid phase carrier in the traditional method to cell surface protein is avoided, and the combination of the anti-CD 20 monoclonal antibody and the CD20 protein is favorably reflected.
The invention is further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
Materials and reagents
Materials:
rituxan (also known as Rituximab, Rituximab): product of Roche company, lot number: h0194;
TSR 001: manufactured by Tereis pharmaceutical industries, Inc.;
bevacizumab (Bevacizumab): product of Roche company, lot number: H0144B 02;
96-well plates with filter plates (from PALL, batch 8029).
Reagent:
RPMI1640 medium (purchased from Gibco, Lot: 11875-093);
streptomycin/penicillin double antibody (available from Hyclone, lot: SV 30010);
FBS (ex Cel lMax, lot number: SA 212.02);
DPBS (Dulbecco's phosphate buffered saline) available from Gibco under cat # 14190-;
anti-human IgG Fc specific antibody solution (purchased from Sigma, lot: A0170-1 ML);
TMB solution (available from Cygnus, Lot: F1100027);
tween 20 (from Chinese medicine, lot number: 30189328).
Cell lines:
raji cell line (cell bank of China academy of sciences type culture Collection, catalog number: TCTU 44);
p10 represents Raji cell passage 10;
p30 represents Raji cell passage 30.
II, TRS001 preparation
The TRS001 uses CHO-K1 domesticated by suspension as a host cell (ATCC from the United states) and utilizes an expression vector based on pcDNA3.0 to construct a monoclonal antibody Light Chain (LC) and Heavy Chain (HC) double expression plasmid, pcDNA3-RX-Neo and pcDNA 3-RX-GS. Both expression plasmids carry the TRS001 light and heavy chain (the TRS001 light and heavy chains have completely identical sequences with the commercial rituximab light and heavy chains) genes but carry different selection markers. The two expression plasmids co-transfect host cells, the stable transfer cells are screened by double screening marks, then a plurality of candidate monoclonal cell strains are screened step by using a semisolid culture medium cloning method, and the best monoclonal cell strain is screened by shaking a flask and a reactor.
The inventors prepared recombinant anti-human CD20 monoclonal antibody TSR001 by referring to the production method provided in the Rituximab monoantigen research patent (CN93121424.6) using the optimal monoclonal cell line selected in the above procedure as the final production cell line.
Third, anti-CD 20 monoclonal antibody binding activity detection
The general detection process is as follows:
a, cell culture:
raji cells highly expressing CD20 in logarithmic growth phase are taken, and prepared into cell suspension by using complete culture medium (namely RPMI1640 basic culture medium added with 10% FBS and 1% double antibody (streptomycin and penicillin)) containing 10% FBS and 1% double antibody (streptomycin and penicillin), and cell density and survival rate are counted and calculated.
Adjust viable cell density to about 8x105cells/mL, the cell suspension at this density was plated on 96-well filter plates and DPBS was added to the marginal wells to avoid bias due to marginal well effects.
b, diluting a reference substance and a sample to be tested:
taking a reference product and a sample to be detected, diluting the reference product and the sample to be detected to 0.15mg/mL by using DPBS according to the marked concentration, and then carrying out serial gradient dilution to obtain 10 dilutions which are 150000.00ng/mL, 50000.00ng/mL, 16666.67ng/mL, 5555.56ng/mL, 1851.85ng/mL, 617.28ng/mL, 205.76ng/mL, 68.59ng/mL, 22.86ng/mL and 7.62mg/mL respectively;
c, washing the plate:
the filter membrane plate with the well-paved cells is overlapped on an empty 96-well plate for fixation, the filter membrane plate is placed in a plate centrifuge for centrifugation at 800rpm for 1 minute, PBS solution containing 0.005% -1% Tween 20 is added into the cell plate, 200 mu L of the filter membrane plate is washed in each hole, and the filter membrane plate is centrifuged at 800rpm for 1 minute.
d, loading sample:
and sequentially adding the diluted reference substance and the to-be-detected substance with the series of gradient concentrations into the cell culture plate with the well-paved cells by 100 mu L/hole, and then placing the cell culture plate at 37 ℃ in a 5% carbon dioxide incubator for incubation for 0.5 h.
e, washing the plate:
and (3) stacking the filter membrane plates after incubation on an empty 96-well plate for fixation, placing the filter membrane plates into a plate centrifuge for centrifugation at 800rpm for 3 minutes, adding a PBS solution containing 0.005-1% Tween 20 into the cell plates, washing the plates by 200 mu L per hole, and centrifuging the plates at 800rpm for 1 minute. The plate washing operation was repeated 2 times.
f secondary antibody:
the HRP-labeled anti-human IgG Fc specific antibody solution is diluted 1:15000 by using PBS solution containing 0.005% -1% Tween 20, and added into the filter membrane plate in sequence at 100 mu L/hole, and then the filter membrane plate is placed at 37 ℃ and incubated for 0.5h in a 5% carbon dioxide incubator.
g, washing the plate:
and (3) overlapping the filter membrane plates after the incubation on an empty 96-well plate for fixation, placing the filter membrane plates into a plate centrifuge for centrifugation at 800rpm for 3 minutes, adding 200 mu L of PBS solution containing 0.005% -1% of Tween 20 into each well of the cell plate for plate washing, and centrifuging at 800rpm for 1 minute. The plate washing operation was repeated 3 times.
h, color development:
the chromogenic substrate TMB solution is added into the filter membrane plate in sequence at 100 mu L/hole, the color development is carried out for 5-15 minutes at room temperature, and 2M sulfuric acid is added to stop the reaction at 100 mu L/hole.
i transfer:
the filter membrane plate after termination of the reaction was stacked on an empty 96-well plate and fixed, and centrifuged in a plate centrifuge at 1000rpm for 3 minutes to completely filter the reacted liquid into a new 96-well plate.
j enzyme labeling instrument determination:
and (3) putting the 96-well plate into a microplate reader, and reading the plate by taking 450nm as a plate reading wavelength and 650nm as a reference wavelength. And taking the concentration of the sample as an abscissa, and taking the difference value obtained by subtracting the absorbance value of 650nm from the absorbance value of 450nm as an ordinate to perform four-parameter fitting.
The four parameter equation is Y ═ A-D)/(1+ (X/C)B) + D, wherein the value of C is the corresponding EC50The value is obtained.
Obtaining EC of reference substance and sample to be tested50Value, curve fitting constant R2Calculating the binding activity of the sample to be tested according to the following formula:
binding activity (%) of the sample to be tested to reference sample EC50Value/sample to be tested EC50The value x 100%
Example 1 detection of the binding Activity of an anti-CD 20 monoclonal antibody
And (3) detecting the binding activity of the polypeptide by using TSR001 as a sample to be detected and Rituximab as a reference substance by adopting the method.
The results are shown in FIG. 1, the curve fitting conditions of the reference product and the sample to be tested are good, and the curve fitting constant R is2>0.95。
EXAMPLE 2 detection of the binding Activity of an anti-CD 20 monoclonal antibody (specificity evaluation)
The binding activity of Human IgG1 (Bevacizumab, Bevacizumab and isotype control Human IgG1) which does not aim at a CD20 target point is detected by using the method as a sample to be detected and Rituximab as a reference substance.
The results are shown in fig. 2, and even under the condition of the same dilution concentration as the reference substance, the monoclonal antibody (Bevacizumab) not aiming at the CD20 target point still has no specific binding with the cells highly expressing CD20, and the detected binding activity approaches to 0%; the binding activity of the anti-human CD20 monoclonal antibody (Rituximab) against CD20 was detected to be 101%.
Therefore, the detection method can specifically detect the binding activity of the monoclonal antibody aiming at the CD20 target.
Example 3 detection of the binding Activity of an anti-CD 20 monoclonal antibody (precision evaluation)
The precision of the method was evaluated using anti-human CD20 monoclonal antibody Rituximab as a sample.
Firstly, 1 experimenter adopts the method to carry out parallel experiments on the same sample to be tested for 5 times, and inspects the repeatability of the method; then, 2 experimenters adopt the method to carry out the detection of the binding activity of the same sample to be detected, and investigate the personnel error of the method; finally, 1 experimenter carries out the detection of the binding activity of the same sample to be detected by adopting the method in 3 different working days, and the day difference of the method is investigated. The results are shown in tables 1, 2 and 3.
As shown in Table 1, 1 experimenter repeated the experiment 5 times on the same sample to be tested, and the RSD of the detection result<15%, five times curve fitting parameter R of sample to be measured2>0.95 (curve fitting), which shows that the repeatability of the method can meet the precision requirement.
As shown in Table 2, 2 examiners performed parallel detection on the same sample for 1 time, and the RSD of the detection result<15% of curve fitting parameter R of samples to be measured twice2>0.95 (curve fitting is omitted), which shows that the operator operation error of the method can reach the precision requirement.
As shown in Table 3, 1 experimenter tested the same sample for 1 time per day in 3 different working days, and the RSD of the test result<15% of curve fitting parameter R of three samples to be measured2>0.95 (curve fitting, not shown), which shows that the daytime difference of the method of the invention can reach the precision requirement.
TABLE 1 summary of the results of repeated evaluation of the binding activity of anti-human CD20 monoclonal antibody
Figure BDA0001185840480000121
TABLE 2 summary of the evaluation results of the human CD20 monoclonal antibody binding activity test personnel's operation error
Figure BDA0001185840480000122
TABLE 3 summary of the results of the evaluation of the daytime difference of the binding activity of the monoclonal antibody against human CD20
Figure BDA0001185840480000131
Example 4 detection of the binding Activity of an anti-CD 20 monoclonal antibody (evaluation of accuracy)
Rituximab is used as a material, Rituximab diluted by RPMI1640 complete culture medium to reach the titer levels of 80%, 100% and 120% is used as a sample to be detected, the reference substance and the sample to be detected with the titer levels of 3 are detected by 3 experimenters respectively for 1 time according to the experimental method, and the statistical experimental results are shown in Table 4.
The results are shown in Table 4, and for 3 samples to be tested having titer levels of 80%, 100%, and 120%, each sample was tested 1 time by 3 analysts, and the curve fitting parameter R of each sample to be tested having titer level was determined2>0.95 (curve fitting; omitted), RSD of 3 measurements per sample to be tested<15% standard deviation SD of theoretical and true values of titer levels<10%, which shows that the method of the invention can meet the accuracy requirement.
TABLE 4 summary of the results of the evaluation of the accuracy of the detection of the binding activity of the anti-CD 20 monoclonal antibody
Figure BDA0001185840480000132
Example 5 anti-CD 20 monoclonal antibody binding Activity assay (Linear and Range evaluation)
Rituximab is used as a reference substance, Rituximab with binding activities of 50%, 75%, 100%, 125% and 150% respectively, which are diluted by an RPMI1640 complete culture medium, is used as a sample to be detected, and the 5 samples to be detected and the reference substance with the binding activity level are detected by adopting the method.
The results are shown in FIG. 3, and a linear regression is performed by combining the theoretical value and the actual value of the activity titer level to fit the parameter R linearly2>0.95, indicating that the binding activity level was in the range of 50% to 150% and the process of the invention was linear.
According to precision, accuracy and linear verification results, the detection range of the method provided by the invention is 80% -120%.
Example 6 anti-CD 20 monoclonal antibody binding Activity assay (durability evaluation)
Rituximab was used as a sample, and the detection was performed by the method described above. And placing the sample plate after the color development is ended at room temperature, and detecting the sample plate by an enzyme-labeling instrument at 0min, 15min, 30min, 60min and 90min respectively to investigate the durability of the method.
Rituximab was used as a sample, and the detection was performed by the method described above. Experiments were carried out with the number of cell plates changed to 40000 cells/well and 120000 cells/well, respectively, to examine the durability of the method of the present invention.
Rituximab was used as a sample, and the detection was performed by the method described above. Experiments were performed using two generations of cells, P10 and P30, to examine the durability of the present method to the cell generations.
As shown in Table 5, after the color development of the sample is terminated, the sample is placed at room temperature, the microplate reader is used for detecting the sample plate at 0min, 15min, 30min, 60min and 90min respectively, and the curve fitting parameter R of each time point is2>0.95 (curve fitting outline), RSD satisfies<10 percent, and experimental data show that the light absorption value reflected by the sample to be detected and the reference substance combined with Raji is stable within 90min at room temperature, which indicates that the method can meet the requirement of durability on the stability of the light absorption value.
As shown in Table 6, the number of cell plates in the method was changed to 40000 cells/well and 120000 cells/well, respectively, and the experiment was performed, and the curve fitting parameter R was determined for each cell density2>0.95 (curve fitting outline), RSD satisfies<10%, the experimental data show that the results are stable in the range of 40000 cells/well to 120000 cells/well after varying the number of cell plating, indicating that the present invention is stableThe method can meet the requirement of durability on the cell plating number.
As shown in Table 7, experiments were performed using two generations of cells, P10 and P30, the curve fitting parameter R of the two generations of cells2>0.95 (curve fitting outline), RSD satisfies<10%, the experimental data show that the experimental results are stable in the range from generation 10 to generation 30 after changing the generation of the experimental cells, which indicates that the method can meet the requirement of durability on the generation of the cells.
TABLE 5 evaluation results of the durability (stability) of the anti-CD 20 monoclonal antibody binding Activity assay
Figure BDA0001185840480000141
TABLE 6 evaluation results of the durability (stability) of the anti-CD 20 monoclonal antibody binding activity assay
Figure BDA0001185840480000151
TABLE 7 evaluation results of the durability (stability) of the anti-CD 20 monoclonal antibody binding activity assay
Figure BDA0001185840480000152
In conclusion, the invention establishes an ELISA detection method for detecting the binding activity of the anti-CD 20 monoclonal antibody by using a 96-well plate with a filter membrane, the method can meet the requirements on specificity, precision, linearity and range, accuracy, durability and the like in the process of method verification, is a rapid, accurate, precise and stable semi-quantitative detection method for the binding activity of the monoclonal antibody of the CD20 target spot, can simply, conveniently and efficiently carry out drug screening and activity detection, and has important significance for research and development, quality control and even clinical application of the anti-CD 20 monoclonal antibody.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (18)

1. A method for detecting the binding activity of an anti-CD 20 monoclonal antibody to an antigen, comprising the steps of:
(1) washing the plate: stacking the filter membrane plate paved with the cells on an empty pore plate, adding washing liquid, centrifuging to wash the plate, and taking down the filter membrane plate; the cells express CD 20;
(2) sample adding: adding a reference substance with a series of gradient concentrations and a sample to be detected into the filter membrane plate treated in the step (1) for incubation; the reference substance and the sample to be detected are anti-human CD20 monoclonal antibodies;
(3) washing the plate: after incubation is finished, stacking the filter membrane plate treated in the step (2) on an empty pore plate, adding washing liquid, centrifuging to wash the plate, and taking down the filter membrane plate;
(4) adding a secondary antibody: adding an HRP-labeled anti-human IgG Fc specific antibody solution into the filter membrane plate treated in the step (3) for incubation;
(5) washing the plate: after incubation is finished, stacking the filter membrane plate treated in the step (4) on an empty pore plate, adding washing liquid, centrifuging to wash the plate, and taking down the filter membrane plate;
(6) color development: adding the chromogenic substrate solution into the filter membrane plate treated in the step (5), and terminating the reaction after developing color at room temperature;
(7) transferring: stacking the filter membrane plate treated in the step (6) on an empty pore plate, centrifuging, and filtering the liquid into the pore plate;
(8) and (3) enzyme-labeling instrument determination: reading the plate processed in the step (7) by using an enzyme-labeling instrument, and recording the plate reading result; and
(9) fitting a curve to the concentration and the read plate result to determine the binding activity of the sample to be detected;
and, step (1) is preceded by a cell culture step: logarithmic growth phaseCells, viable cell density 5-10x10 with complete medium5Plating a filter membrane plate with the cell suspension for later use;
wherein, the centrifugation is carried out by a plate centrifuge; the rotation speed of the centrifugation is 600-1200rpm and the time of the centrifugation is 0.5-4 minutes.
2. The detection method of claim 1, wherein DPBS is added to the marginal wells after plating.
3. The assay of claim 1, wherein the viable cell density is 8x105one/mL.
4. The assay of claim 1 wherein the filter membrane plate is a 96-well filter membrane plate.
5. The assay of claim 1, wherein the cell is a human Burkitt's lymphoma cell.
6. The detection method according to claim 1, wherein the cell is a Raji cell.
7. The assay of claim 1 wherein the complete medium is RPMI1640 complete medium containing 10% FBS and 1% streptomycin and penicillin.
8. The detection method according to claim 1, further comprising, before the step (1), a step of preparing a reference substance and a sample to be tested: and taking a reference product and a sample to be detected, and preparing a series of reference products and samples to be detected with gradient concentrations.
9. The detection method according to claim 8, wherein the preparation steps of the reference substance and the sample to be detected are: and taking a reference product and a sample to be detected, diluting the reference product and the sample to be detected to pre-diluted concentration by using DPBS (double-stranded sequencing batch), and then performing serial gradient dilution to obtain a plurality of dilutions.
10. The assay of claim 9, wherein the pre-dilution concentration is 0.15 mg/mL.
11. The assay of claim 8 wherein the serial gradient dilution is a3, 4, 5, 6, 7, 8, 9 or 10 fold serial gradient dilution.
12. The detection method according to claim 1, wherein the washing solution is a PBS solution.
13. The assay of claim 1 wherein the wash solution is a PBS solution containing 0.05% to 0.1% tween 20.
14. The assay of claim 11 wherein the amount of wash solution used is 100-.
15. The detection method according to claim 1, wherein the centrifugation is performed once or repeated two, three or more times.
16. The detection method according to claim 1, wherein in the step (8), plate reading is performed at 600nm and 450 nm.
17. The detection method according to claim 1, wherein the reference wavelength is 600 nm.
18. The detection method according to claim 1, wherein in the step (9), a four-parameter fitting curve is performed by taking the concentration as an abscissa and the difference between the plate reading result at 450nm and the plate reading result at 600nm as an ordinate, thereby obtaining the EC of the sample to be detected and the EC of the reference substance respectively50Comparison of EC of both50And determining the binding activity of the sample to be tested.
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