CN116125074A - Method for detecting binding activity of IL-10-Fc fusion protein and receptor thereof - Google Patents
Method for detecting binding activity of IL-10-Fc fusion protein and receptor thereof Download PDFInfo
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Abstract
The invention relates to the field of biological medicine, and provides a detection method for the binding activity of IL-10-Fc fusion protein and a receptor thereof, which comprises the following steps: the sample to be tested and the biotin-marked IL-10-Fc fusion protein competitively perform a binding action with IL-10 RA; adding strepitavidin-HRP to react with IL-10-Fc fusion protein; chromogenic substrate was added and a four parameter curve fitted. The invention provides a detection method specially applicable to the binding activity of IL-10-Fc fusion protein and a receptor thereof, which does not need to culture cells, has low requirement on environment, has the advantages of specificity of the detection method, greatly shortened detection time, lower cost, simpler operation, greatly improved precision and detection accuracy, more stable experimental result and can be effectively used for detecting the binding activity of the IL-10-Fc fusion protein.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a detection method for the binding activity of IL-10-Fc fusion protein and a receptor thereof.
Background
Interleukin 10 (IL-10) is a multipotent cytokine secreted by a variety of cells, both immunosuppressive and immunostimulatory. IL-10R (IL-10 receptor) is a receptor for interleukin 10, and research shows that it can mediate the immunosuppressive signal of IL-10, thereby inhibiting the synthesis of pro-inflammatory cytokines. IL-10R belongs to the family of cytokines type 2, which are transmembrane glycoproteins with 21 amino acids in the extracellular domain and contain 2 tandem fibronectin type III domains. The receptor reportedly promotes bone marrow cell survival via the insulin receptor substrate-2/PI 3-kinase/AKT pathway.
Activated IL-10R is a signaling complex comprising 2 distinct receptor chains, an IL-10R1 (also known as IL-10 RA) subunit and an accessory IL-10R2 (also known as IL-10 RB) subunit, respectively, which bind to a ligand, wherein the IL-10R1 amino acid chain is relatively long and is the primary signaling component; IL-10R2 also known as type 2 cytokine receptor family member4 (cytokine-receptorfamily type-2 meber 4, CRF 2-4) is a short peptide chain with fewer transmembrane segments within the membrane. The formation of the IL-10R complex is in two steps: first, IL-10 binds to the high affinity receptor long chain IL-10R 1; second, it binds with low affinity to IL-10R 2. IL-10 interacts with cell surface IL-10R1 and IL-10R2 and exerts biological effects primarily by activating the JAK-STAT pathway, thereby affecting mRNA transcription and exerting IL-10 regulation. IL-10 biological activity is achieved by specific binding of IL-10 to receptors on the target cell membrane.
IL-10-Fc fusion proteins are designed to link IL-10 to the Fc region of human IgG1, primarily to increase the half-life of IL-10 by linking the Fc fragment. In the development of IL-10-Fc fusion proteins, detection of binding activity to the receptor is extremely important.
Currently, the detection methods for binding activity to the receptor are mainly MC/9 cell proliferation and flow cytometry. The flow cytometry is mainly used for monoclonal antibody screening in early research and development stages, does not perform relevant evaluation in various aspects such as specificity, repeatability and accuracy, and is high in price, unstable in method and not suitable for product release detection; the MC/9 cell proliferation method relates to cell culture, cells need to be cultured in advance, the cycle is long, the reagent cost is high, the method stability is uncertain, no detection method specially suitable for the binding activity of IL-10-Fc fusion protein exists in the existing detection methods, and therefore, in the research and development process aiming at the IL-10-Fc fusion protein, the detection method specially suitable for the binding activity of the IL-10-Fc fusion protein and a receptor thereof is urgently required.
Disclosure of Invention
In order to solve the problems that the detection period is long, the method has no specificity, the method is not stable enough and is not suitable for product release detection and the like in the existing detection method for the binding activity, the invention discloses a detection method specially suitable for the binding activity of IL-10-Fc fusion protein and a receptor thereof.
The specific technical scheme of the invention is as follows:
the invention provides a detection method for the binding activity of IL-10-Fc fusion protein and a receptor thereof, which comprises the following steps:
s1, competitively combining a sample to be tested with biotin-labeled IL-10-Fc fusion protein with IL-10RA arranged in an ELISA plate, wherein the sample to be tested is a drug sample containing the IL-10-Fc fusion protein, the amino acid sequence of the IL-10-Fc fusion protein is shown as SEQ ID NO. 1, and the amino acid sequence of the IL-10RA is shown as SEQ ID NO. 2;
s2, adding strepitavidin-HRP into the ELISA plate, and carrying out a binding reaction with the IL-10-Fc fusion protein marked by biotin;
s3, adding a chromogenic substrate of the strepitavidin-HRP, taking an OD450nm value as an ordinate, taking the protein concentration of a sample to be detected as an abscissa, and fitting a four-parameter curve to obtain the binding activity of the sample to be detected and the IL-10 RA.
Further, in step S1, the sample to be tested and the biotin-labeled IL-10-Fc fusion protein competitively bind to IL-10RA in the ELISA plate by the following steps:
s11, coating: diluting the IL-10RA by using a coating liquid, coating the IL-10RA into the ELISA plate for incubation, discarding the coating liquid, adding a plate washing liquid, and washing for later use;
s12, sealing: adding a sealing liquid into the ELISA plate for sealing treatment and incubation, discarding the sealing liquid in the hole, adding the plate washing liquid, and washing for later use;
s13, preparing the IL-10-Fc fusion protein marked by biotin;
s14, respectively diluting the sample to be tested and the IL-10-Fc fusion protein marked by biotin, mixing and incubating in a volume ratio of 1:1, and transferring the incubated sample to the ELISA plate for standby in the step S12 for standby.
Further, in step S13, preparing the IL-10-Fc fusion protein labeled with biotin comprises the following steps:
dissolving the IL-10-Fc fusion protein by deionized water, adding the solution into an ultrafiltration centrifuge tube of 30kDa, and changing the solution by PBS (phosphate buffered saline) with pH of 7.3;
calculating the protein concentration after liquid exchange, adding NHS-PEG4-Biotin for marking, incubating at room temperature for about 40min, then exchanging liquid with PBS again, and freezing and preserving to obtain the Biotin-marked IL-10-Fc fusion protein.
Preferably, the coating liquid is carbonate buffer with a pH value of 9.6.
Preferably, the sealing liquid is phosphate buffer solution containing 1% of skimmed milk powder by mass concentration.
Further, in step S14, the initial concentration of the gradient dilution of the sample to be tested is 1mg/ml, and the concentration of the biotin is 4000ng/ml.
Further, in step S2, strepitavidin-HRP is added to the ELISA plate to react with the biotin-labeled IL-10-Fc fusion protein in a binding way, and the method comprises the following steps: labeling the Streptavidin by the strepitavidin-HRP, diluting, adding into the ELISA plate, adding 100 μl/hole, incubating in a biochemical incubator, adding the plate washing liquid, and washing for later use.
Preferably, the adding ratio of the strepavidin-HRP is 1:10000 (v: v).
Preferably, the plate washing liquid is PBS phosphate buffer solution containing 0.5 per mill Tween-20, and the washing liquid dosage is 300 mu l/hole.
Further, in step S3, adding a chromogenic substrate of the strepavidin-HRP, taking an OD450nm value as an ordinate, taking the protein concentration of a sample to be detected as an abscissa, and fitting a four-parameter curve to obtain the binding activity between the sample to be detected and the IL-10RA, wherein the specific method is as follows:
and (3) taking the ELISA plate, adding 100 mu L/hole of the chromogenic substrate into the ELISA plate, performing light-shielding color development at room temperature, adding 100 mu L of 1M sulfuric acid into each hole to terminate the reaction, measuring the OD450nm value of each hole reaction liquid by using an ELISA instrument, taking the OD450nm value as an ordinate, taking the protein concentration of the sample to be measured as an abscissa, and automatically fitting a four-parameter curve by using software to measure the binding activity of the sample to be measured and the IL-10 RA.
The beneficial effects of the invention are as follows: compared with the prior art, the method does not need to culture cells, has low environmental requirements, has the special detection time, is lower in cost, simpler to operate, has more stable experimental results, is convenient for quality control, and can be effectively used for detecting the IL-10-Fc fusion protein synthesis activity.
Drawings
FIG. 1 is a graph of a binding activity curve fit of a reference sample and a test sample of the present invention;
FIG. 2 is a graph of a prior art method for fitting a binding activity curve to a sample to be tested;
FIG. 3 is a diagram showing the specificity evaluation of binding activity detection of a sample to be tested according to the present invention;
FIG. 4 is a graph of a reference repetitive binding activity curve fit of the present invention;
FIG. 5 is a graph showing the validation (linear and range) -linear fit of the binding activity method of the IL-10-Fc fusion proteins of the present invention.
Detailed Description
The invention will be described in further detail with reference to the following examples.
Example 1
The embodiment 1 of the invention provides a detection method for the binding activity of IL-10-Fc fusion protein and a receptor thereof, which comprises the following steps:
s1, carrying out competitive binding action on samples to be tested with different concentrations and biotin-labeled IL-10-Fc fusion proteins and IL-10RA arranged in an ELISA plate (manufacturer: corning, product number: 9018), wherein the IL-10RA is an IL-10RA extracellular region sequence protein, the higher the concentration of the samples to be tested is, the less the biotin-labeled IL-10-Fc fusion proteins are bound with the IL-10RA, the samples to be tested are drug samples containing the IL-10-Fc fusion proteins, the amino acid sequence of the IL-10-Fc fusion proteins is shown as SEQ ID NO:1, and the extracellular region amino acid sequence of the IL-10RA is shown as SEQ ID NO: 2;
s2, adding strepavidin-HRP, namely horseradish peroxidase labeled Streptavidin (manufacturer: jackson ImmunoResearch, product number: 016-030-084) into an ELISA plate, and carrying out a binding reaction with biotin-labeled IL-10-Fc fusion protein;
s3, adding a chromogenic substrate of strepavidin-HRP, wherein the chromogenic substrate is TMB substrate chromogenic kit (manufacturer: jiangsu kang is century biotechnology Co., ltd., product number: CW 0050S) and OD is used 450nm The value is the ordinate, the protein concentration of the sample to be measured is the abscissa, and the binding activity of the sample to be measured and IL-10RA can be measured by fitting a four-parameter curve.
SEQ ID NO. 1 (amino acid sequence of IL-10-Fc fusion protein);
SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKAYACAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。
SEQ ID NO. 2 (amino acid sequence of the extracellular region of IL-10 RA):
MLPCLVVLLAALLSLRLGSDAHGTELPSPPSVWFEAEFFHHILHWTPIPNQSESTCYEVALLRYGIESWNSISNCSQTLSYDLTAVTLDLYHSNGYRARVRAVDGSRHSNWTVTNTRFSVDEVTLTVGSVNLEIHNGFILGKIQLPRPKMAPANDTYESIFSHFREYEIAIRKVPGNFTFTHKKVKHENFSLLTSGEVGEFCVQVKPSVASRSNKGMWSKEECISLTRQYFTVTN。
example 2
Example 2 of the present invention further defines, based on example 1, that in step S1, the sample to be tested and the biotin-labeled IL-10-Fc fusion protein competitively bind to IL-10RA placed in the elisa plate by the following method:
s11, coating: diluting IL-10RA to 1-3 mu g/ml by coating liquid, coating 100 mu l/hole into a 96-hole ELISA plate, incubating overnight at 2-8 ℃, discarding Kong Nabao coating liquid, adding plate washing liquid, 300 mu l/hole, and washing for 3 times for later use;
s12, sealing: adding sealing liquid into the ELISA plate, placing 300 mu l/hole into a constant temperature incubator at 37 ℃, sealing and incubating, discarding the sealing liquid in the hole, adding plate washing liquid, 300 mu l/hole, and washing for 3 times for standby;
s13, preparing biotin-labeled IL-10-Fc fusion protein;
s14, respectively diluting a sample to be tested and the biotin-marked IL-10-Fc fusion protein, diluting the sample to be tested into a series of different protein concentrations, adding 120 mu l/hole into a 96-hole dilution plate (manufacturer: corning, product number: 3365), diluting the biotin-marked IL-10-Fc to 2000-8000ng/ml by using a diluent, mixing with the sample to be tested 1:1, incubating for 1-2h at 37 ℃, preferably for 1h, transferring 200 mu l/hole into an ELISA plate for standby in the step S12, incubating again at 37 ℃, discarding Kong Nabao quilt liquid, adding a plate washing liquid, 300 mu l/hole, and washing for 3 times for standby.
During the experiment, in order to allow the biotin-labeled IL-10-Fc fusion protein to be well mixed with the sample to be tested, an incubation of 1 hour in advance was required.
The pre-dilution concentration of the sample to be tested is 1mg/ml, and the sample to be tested is diluted in a 5-fold gradient from the A-H row in a 96-well dilution plate. The biotin-labeled IL-10-Fc was diluted to 4000ng/ml.
The coating liquid was a carbonate buffer solution (anhydrous sodium carbonate, manufacturer: national drug group; sodium bicarbonate, manufacturer: national drug group) having a pH of 9.6.
The blocking solution was phosphate buffer containing 1% by mass of skimmed milk powder (manufacturer: company, limited of the inner Mongolian illi group).
The plate washing solution was PBS phosphate buffer solution (manufacturer: ZLI-9061, product number: north China fir Biotechnology Co., ltd.) containing 0.5%Tween-20 (manufacturer: merck, product number: 9005-64-5), and the amount of the washing solution was 300. Mu.l/well.
The dilution was 1% BSA (manufacturer: beijing Soy Bao technology Co., ltd., product number: A8020) solution.
Further, in step S14, the initial concentration of the sample to be measured in gradient dilution is 1mg/ml, and the concentration of biotin after dilution is 4000ng/ml.
Example 3
Example 3 of the present invention further defines step S13 on the basis of example 2, the preparation of a biotin-labeled IL-10-Fc fusion protein comprising the following steps:
dissolving the IL-10-Fc fusion protein by deionized water, adding the solution into an ultrafiltration centrifuge tube of 30kDa, and changing the solution by PBS (phosphate buffered saline) with pH of 7.3;
calculating protein concentration after liquid exchange, adding NHS-PEG4-Biotin (manufacturer: thermo, product number: A39259) for marking, incubating at room temperature for about 40min, then exchanging liquid with PBS again, and freezing for preservation to obtain the Biotin-marked IL-10-Fc fusion protein.
Furthermore, the labeled solution needs to be changed several times to ensure that IL-10-Fc fusion protein which is not connected with Biotin in the sample to be tested is removed.
Example 4
Example 4 of the present invention further defines step S2 on the basis of example 1, wherein in step S2, strepitavidin-HRP is added to the ELISA plate to react with biotin-labeled IL-10-Fc fusion protein, comprising the following steps: streptavidin is marked by strepavidin-HRP, then diluted and added into an ELISA plate, 100 μl/hole is added into a biochemical incubator at the same time, after incubation at 37 ℃, the reaction solution in the hole is discarded, and the plate washing solution is added, 300 μl/hole is washed for 5 times for standby.
The addition ratio of strepavidin-HRP was 1:10000 (v: v).
The plate washing solution was PBS phosphate buffer solution (manufacturer: ZLI-9061, product number: north China fir Biotechnology Co., ltd.) containing 0.5%Tween-20 (manufacturer: merck, product number: 9005-64-5), and the amount of the washing solution was 300. Mu.l/well.
Example 5
In the invention, the embodiment 5 further defines the step S3 on the basis of the embodiment 1, wherein a chromogenic substrate of strepitavidin-HRP is added, the OD450nm value is taken as an ordinate, the protein concentration of a sample to be detected is taken as an abscissa, and the binding activity between the sample to be detected and IL-10RA can be detected by fitting a four-parameter curve, and the specific method is as follows:
and (3) taking an ELISA plate, adding a chromogenic substrate into the ELISA plate at 100 mu L/hole, using a TMB substrate chromogenic solution, performing light-proof color development at room temperature, adding 100 mu L of 1M sulfuric acid (manufacturer: beijing chemical plant) into each hole to terminate the reaction, measuring the OD450nm value of each hole reaction solution by using an ELISA instrument, using the OD450nm value as an ordinate, using the protein concentration of a sample to be measured as an abscissa, and automatically fitting a four-parameter curve by using software to measure the binding activity of the sample to be measured and IL-10 RA.
The four-parameter equation corresponding to the four-parameter curve is Y= (A-D)/(1+ (X/C) B) +D,
wherein X is the protein concentration of the sample to be detected, Y is the reaction liquid OD 450nm The value A is the estimated value of the asymptote on the four-parameter curve, D is the estimated value of the asymptote under the four-parameter curve, B is the slope of the curve, and C is the corresponding concentration at half maximum, i.e. EC 50 Values.
The EC50 values of the sample to be detected and the reference sample are detected respectively by the method, and the curve fitting constant R 2 The binding activity of the sample to be tested was calculated according to the following formula:
the binding activity (%) of the test sample=reference EC50 value/test sample EC50 value x 100%.
Experimental example 1 sample Activity detection method comparison
(1) The sample activity detection is carried out by the method provided in the embodiment 1 of the invention, and the method is concretely as follows:
sample to be measured: IL10-Fc fusion protein samples were provided by Biotechnology, inc., botto Biotech, beijing.
Reference: IL-10-Fc fusion protein has an amino acid sequence shown in SEQ ID NO. 1.
The detection method provided by the embodiments 1-5 of the present invention is used for detecting the binding activity of the sample to be detected, and the detection result is shown in fig. 1.
As can be seen from FIG. 1, the curve fitting condition of the reference and the sample to be tested is good, and the curve fitting constant R 2 >0.95, and the relative binding activity of the sample to be tested is calculated to be 95%.
(2) The activity of the sample is detected by the prior method, and the method is as follows:
sample to be measured: IL10-Fc fusion protein samples were provided by Biotechnology, inc., botto Biotech, beijing.
Reference: IL-10-Fc fusion protein has an amino acid sequence shown in SEQ ID NO. 1.
The existing method comprises the following steps:
1. the samples to be tested with different concentrations interact with IL-10RA placed in an ELISA plate (manufacturer: corning, cat# 9018), the higher the concentration of the samples to be tested is, the more the samples to be tested are combined with IL-10RA, the samples to be tested are drug samples containing IL-10-Fc fusion proteins, and the amino acid sequences of the IL-10-Fc fusion proteins and the IL-10RA are the same as those disclosed in the example 1;
2. adding goat anti-human IgG-HRP (manufacturer: china fir gold bridge, product number: ZB-2304) into the ELISA plate, and performing binding reaction with IL-10-Fc fusion protein;
3. adding chromogenic substrate as TMB substrate chromogenic kit (manufacturer: jiangsu kang is century biotechnology Co., ltd., product number: CW 0050S) and OD 450nm The value is the ordinate, the protein concentration of the sample to be detected is the abscissa, the four-parameter curve is automatically fitted by using software, and the binding activity between the sample to be detected and IL-10R can be detected, and the detection result is shown in figure 2.
As can be seen from fig. 2, the curve fitting S-shaped curve of the reference and the sample to be measured is not obvious, and the upper platform does not meet two platform points.
In summary, compared with the existing general detection method, the detection method provided by the invention is obviously not applicable to detection of IL10-FC fusion protein samples.
Experimental example 2 detection of IL-10-Fc fusion protein binding Activity (specificity evaluation)
the detection method provided by the embodiments 1-5 of the present invention is used for detecting the binding activity of the sample to be detected, and the detection result is shown in fig. 3.
As can be seen from fig. 3, antibodies at different targets did not specifically bind to IL-10RA even at the same dilution concentration as the reference, and the absorbance values were almost equal to the negative control; therefore, the detection method can specifically detect the binding activity of the IL-10-Fc fusion protein.
Experimental example 3IL-10-Fc fusion protein binding Activity detection (precision evaluation)
Sample to be measured: a medicine sample containing IL-10-Fc fusion protein is provided by Beijing Oriental Biotech Co., ltd. The amino acid sequence of the IL-10-Fc fusion protein is shown as SEQ ID NO. 1.
Reference: IL-10-Fc fusion protein has an amino acid sequence shown in SEQ ID NO. 1.
Firstly, carrying out parallel experiments on the same sample to be tested for 5 times by 1 tester by adopting the detection method provided by the invention, and examining the repeatability of the method; then, 2 testers adopt the method provided by the invention to detect the binding activity of the same sample to be detected, and examine the personnel error of the method; finally, 1 tester adopts the detection method provided by the invention to detect the binding activity of the same sample to be detected in 3 different working days, and the daytime difference of the method is investigated. The results are shown in tables 1, 2 and 3.
TABLE 1 IL-10-Fc fusion protein binding Activity assay reproducibility evaluation results summary table
TABLE 2 summary of results of operator error assessment of IL-10-Fc fusion protein binding Activity
TABLE 3 summary of results of evaluation of IL-10-Fc fusion protein binding Activity detection
As shown in Table 1 and FIG. 4, 1 tester uses the detection method provided by the invention to test the same sample to be tested in parallel for 5 times, and the detected RSD<Curve fitting parameter R of 10, 5 times sample to be measured 2 >0.95, demonstrating that the reproducibility of the method of the invention can meet the precision requirements.
As shown in Table 2, 2 testers adopt the detection method provided by the invention to detect the same sample to be detected in parallel for 1 time, and the detection result RSD<10%, curve fitting parameter R of twice to-be-measured sample 2 >0.95, the operator error of the method of the invention can meet the precision requirement.
As shown in Table 3, 1 tester detects the same sample to be tested 1 time a day in 3 different working days by adopting the detection method provided by the invention, and the detection result RSD<10%, curve fitting parameter R of twice to-be-measured sample 2 >0.95, which indicates that the daytime difference of the method of the invention can meet the precision requirement. Experimental example 4 detection of IL-10-Fc fusion protein binding Activity (evaluation of accuracy)
Sample to be measured: a pharmaceutical sample containing I L-10-Fc fusion protein has the amino acid sequence shown in SEQ ID NO. 1, which is provided by Beijing Oriental Biotech Co., ltd.
The samples to be tested were diluted with a diluent to 3 different concentration levels with relative activities of 80%, 100% and 120%, respectively (note: 100% means that the samples were diluted in the same manner in two parts, one part being the reference itself and the other part being the 100% sample), and the 3 samples to be tested were each tested 1 time by 2 test persons according to the test method provided by the present invention, and the statistical test results are shown in table 4.
TABLE 4 results of evaluation of accuracy of binding Activity of IL10-Fc fusion protein
The results are shown in Table 4, and for 3 samples to be tested whose predicted binding activities were 80%, 100% and 120%, each sample to be tested was detected 1 time by 2 analysts, respectively, and curve fitting parameters R of the reference and the samples to be tested were calculated 2 >0.95, RSD of 2 detection results of each sample to be tested<10% accuracy is 80% -120%, which shows that the method can meet the accuracy requirement.
Experimental example 5IL-10-Fc fusion protein binding Activity assay (Linear and Range evaluation)
Sample to be measured: a pharmaceutical sample containing I L-10-Fc fusion protein is provided by Beijing Oriental Biotech Co., ltd. And the amino acid sequence of IL-10-Fc fusion protein is shown as SEQ ID NO. 1.
The 5 samples to be tested were tested according to the test method provided in the present invention by diluting them with a diluent to 5 different activity levels of 50%, 75%, 100%, 125%, 150% respectively, as test samples, the results being shown in fig. 5.
As shown in FIG. 5, linear regression is performed in combination with the theoretical and actual values of activity titer levels, and the parameters R are linearly fitted 2 >0.95, indicating that the linearity of the process of the invention is good in the range of 50% -150% potency level.
In summary, according to the precision, accuracy and linear verification result, the detection range of the method is 80% -120%, so that the method for rapidly, simply and accurately detecting the IL-10Fc fusion protein binding activity is provided, the method can meet the requirements of specificity, accuracy including repeatability, experimenter operation error, daytime difference, accuracy, linearity and range in the process of verification, and has important significance for research and development of detecting the IL-10Fc fusion protein binding activity and quality control.
The present invention is not limited to the above-described preferred embodiments, and any person who can obtain other various products under the teaching of the present invention, however, any change in shape or structure of the product is within the scope of the present invention, and all the products having the same or similar technical solutions as the present application are included.
Claims (10)
1. A method for detecting the binding activity of an IL-10-Fc fusion protein to its receptor, comprising:
s1, competitively combining a sample to be tested with biotin-labeled IL-10-Fc fusion protein with IL-10RA arranged in an ELISA plate, wherein the sample to be tested is a drug sample containing the IL-10-Fc fusion protein, the amino acid sequence of the IL-10-Fc fusion protein is shown as SEQ ID NO. 1, and the amino acid sequence of the IL-10RA is shown as SEQ ID NO. 2;
s2, adding strepitavidin-HRP into the ELISA plate, and carrying out a binding reaction with the IL-10-Fc fusion protein marked by biotin;
s3, adding chromogenic substrate of the strepitavidin-HRP to OD 450nm And the value is an ordinate, the protein concentration of the sample to be detected is an abscissa, and the binding activity of the sample to be detected and the IL-10RA can be detected by fitting a four-parameter curve.
2. The method according to claim 1, wherein in step S1, the sample to be tested and the biotin-labeled IL-10-Fc fusion protein competitively bind to IL-10RA in an ELISA plate by:
s11, coating: diluting the IL-10RA by using a coating liquid, coating the IL-10RA into the ELISA plate for incubation, discarding the coating liquid, adding a plate washing liquid, and washing for later use;
s12, sealing: adding a sealing liquid into the ELISA plate for sealing treatment and incubation, discarding the sealing liquid in the hole, adding the plate washing liquid, and washing for later use;
s13, preparing the IL-10-Fc fusion protein marked by biotin;
s14, respectively diluting the sample to be tested and the IL-10-Fc fusion protein marked by biotin, mixing and incubating in a volume ratio of 1:1, and transferring the incubated sample to the ELISA plate for standby in the step S12 for standby.
3. The method of claim 2, wherein in step S13, preparing the IL-10-Fc fusion protein labeled with biotin comprises the following steps:
dissolving the IL-10-Fc fusion protein by deionized water, adding the solution into an ultrafiltration centrifuge tube of 30kDa, and changing the solution by PBS (phosphate buffered saline) with pH of 7.3;
calculating the protein concentration after liquid exchange, adding NHS-PEG4-Biotin for marking, incubating at room temperature for about 40min, then exchanging liquid with PBS again, and freezing and preserving to obtain the Biotin-marked IL-10-Fc fusion protein.
4. The method of claim 2, wherein the coating solution is a carbonate buffer having a pH of 9.6.
5. The method according to claim 2, wherein the blocking solution is a phosphate buffer solution containing skimmed milk powder at a mass concentration of 1%.
6. The method according to claim 2, wherein in step S14, the initial concentration of the sample to be tested is 1mg/mL and the concentration of biotin is 4000ng/mL.
7. The assay of claim 1, wherein in step S2, strepavidin-HRP is added to the elisa plate to react with the biotin-labeled IL-10-Fc fusion protein in a binding manner, comprising the following steps: and (3) diluting the Strepitavidin-HRP, adding the diluted Strepitavidin-HRP into the ELISA plate, adding 100 mu L/hole into a biochemical incubator for incubation, adding the plate washing liquid, and washing for later use.
8. The detection method according to claim 7, wherein the Strepitavidin-HRP is added at a ratio of 1:10000 (v: v).
9. The method according to claim 2 or 7, wherein the plate washing solution is PBS phosphate buffer containing 0.5%o Tween-20, and the washing solution is used in an amount of 300. Mu.L/well.
10. The detection method according to claim 1, wherein in step S3, the chromogenic substrate of Strepitavidin-HRP is added as OD 450nm The value is ordinate, the protein concentration of the sample to be detected is abscissa, and the binding activity of the sample to be detected and the IL-10RA can be detected by fitting a four-parameter curve, and the specific method is as follows:
taking the ELISA plate, adding the chromogenic substrate into the ELISA plate at 100 mu L/hole, developing at room temperature in a dark place, adding 100 mu L of 1M sulfuric acid into each hole to terminate the reaction, and measuring the OD of the reaction solution of each hole by using an ELISA instrument 450nm Value in OD 450nm And the value is an ordinate, the protein concentration of the sample to be detected is an abscissa, and the binding activity of the sample to be detected and the IL-10RA can be measured by automatically fitting a four-parameter curve by software.
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