CN109682963B - Method for analyzing binding rate of recombinant human insulin and analogues or conjugates thereof and insulin receptor - Google Patents
Method for analyzing binding rate of recombinant human insulin and analogues or conjugates thereof and insulin receptor Download PDFInfo
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Abstract
The invention discloses a method for analyzing the binding rate of human insulin and analogues or conjugates thereof and an insulin receptor, which comprises the steps of firstly transfecting the insulin receptor onto HEK-293 cells; the antibody of the human insulin receptor is coated on the enzyme label plate, the insulin receptor antibody is combined with the insulin receptor, the human insulin and the analogue or the conjugate are combined with the insulin receptor by competition with the biotin-labeled insulin, the avidin is combined with the biotin, and the receptor combination rate is determined by HRP color development. The invention can accurately determine the change of the insulin receptor binding rate by utilizing the principles of antigen-antibody binding, ligand-receptor binding and biotin-binding avidin, and has the requirements of simple and convenient operation steps, no pollution, low cost, high accuracy and the like of the biological product receptor binding experimental determination method.
Description
Technical Field
The invention relates to the technical field of in vitro pharmacodynamic experimental detection of Insulin receptor binding rate, in particular to the technical field of in vitro pharmacodynamic experimental detection of recombinant human Insulin analogue or conjugate receptor binding rate, which comprises rapid-acting insulins such as Insulin aspart (Insulin aspart), Insulin Lispro (Insulin Lispro), Insulin Glulisine (Insulin Glulisine), long-acting insulins such as Insulin glargine (Insulin glargine), Insulin Detemir (Insulin Detemir), Insulin degluudedec and modified conjugates such as pegylated Insulin (such as PEG-Lispro, PEG-Insulin) and PEG-Insulin (PEG conjugated des B30-Insulin) and human Insulin receptor binding rate determination methods.
Background
Insulin is a double-chain (A, B chain) protein hormone consisting of 51 amino acids, and has a molecular weight of 5374, an isoelectric point of 5.35, an A chain containing 21 amino acids, and a B chain containing 30 amino acids. The two peptide chains are connected by two disulfide bonds, and the 6 th and 11 th amino acid residues of the A chain are also connected by a disulfide bond. Insulin exerts the function of reducing blood sugar through an insulin receptor, the insulin receptor is mainly distributed on cell membranes of liver cells, muscle cells and fat cells, the insulin receptor mainly promotes the target cells to take in and utilize glucose, a target cell membrane carrier is promoted to transfer the glucose in blood into the cells, the glucose is converted into fatty acid to be used as glycogen for storage, and the glucose is accelerated to be oxidized into a high-energy phosphate compound to be used as an energy source, so that the blood sugar level is reduced, and the synthesis of glycogen, fat and protein is promoted. Insulin is used as a main medicine for treating diabetes and can effectively regulate the blood sugar level of a human body.
The insulin receptor is a transmembrane glycoprotein and is a heterogeneous tetramer composed of a pair of alpha subunits and a pair of beta subunits. Two alpha subunits and two beta subunits are connected by a plurality of disulfide bonds (S-S), and the structure is a homodimer. The molecular masses of the alpha and beta subunits were l35000 and 95000, respectively. The single chain insulin receptor plus precursor consists of 1382 amino acids (154 kDa). The alpha subunit consists of 735(84kDa) amino acid residues, is located on the outer surface of cells, and can be combined with insulin molecules, and the combination site is mainly in a cystine-rich domain; the β subunit consists of 620(70kDa) amino acid residues, and is divided into three structural regions, spanning the membrane: the N-terminal region consisting of 194 amino acid residues is exposed on the cell surface, is connected with the a subunit through a disulfide bond, and comprises a transmembrane region consisting of 23-26 amino acid residues with strong hydrophobicity and a C-terminal region consisting of 403 amino acid residues. The beta subunit crosses the cell membrane, a small part is outside the cell membrane, and a large part is inside the cell membrane, and mainly plays a role in biological effect. The signal peptide of the single-chain insulin receptor consists of 27 amino acids. The full-length single-chain sequence of the insulin receptor is (1-1382 amino acids).
Insulin receptors are divided into 2 subtypes, IR-A and IR-B. The insulin receptor has two isoforms (isofom) in IR due to differences in splicing patterns of the 11 th exon of mRNA. Exon11 encodes 12 amino acids (717-728) and is inserted upstream of the penultimate 3 amino acid residue of the extramembranous alpha subunit of IR-B type. While IR-A (Exon11-) deleted these 12 amino acids. With the peptide encoded by exon11, receptors are formed with different chemical properties. The two subtypes have similar affinities for insulin, and also have different kinase activities, different rates of internalization and circulation. These two subtypes localize to different regions of the plasma membrane and regulate insulin and β -glucokinase gene expression by activating different types of PI 3K.
It is known that biological activity of biological drugs is measured by measuring biological effects such as proliferation, apoptosis, second messenger cAMP, phosphorylation and glucose transport, etc. generated after ligand and receptor binding, but biological activity is on the level of living cells, and the study is relatively objective, and in order to more intuitively study the in vitro pharmacodynamics of biological drugs, biological drugs need to measure the binding rate of receptor and ligand to reflect the primary efficacy of drugs.
The domestic and foreign literature reports that the commonly adopted method for the existing receptor binding rate of insulin is a method for extracting insulin-containing receptor membrane protein by using insulin-labeled isotope and adopting competition combination of human insulin, analogues or conjugates and isotope-labeled insulin with rat liver cells, and the principle is as follows: human insulin and analogs or conjugates thereof and125i labelling of insulin to compete for binding to insulin receptor, washing free125I labelling of insulin, determining competition for insulin receptor125I labelling of insulin complexes125I, measuring the receptor binding rate. The method needs to adopt isotopes, causes harm to the bodies and the environment of experimenters, and has the disadvantages of careful operation and complicated steps. In order to evaluate the pharmacodynamics more accurately and simply, a method for analyzing the binding of recombinant human insulin and analogs or conjugates to insulin receptors, which is simple and convenient to operate, free of pollution and high in accuracy, is needed.
Disclosure of Invention
In order to achieve the above objects, the present invention provides a method for analyzing the binding rate of recombinant human insulin and analogs or conjugates thereof to insulin receptor. The method is practical, high in accuracy and free of radioactive hazards, and solves the problems that the existing method has radiation and complicated steps.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for analyzing the binding rate of recombinant human insulin and analogues or conjugates thereof to insulin receptors, comprising the steps of:
s1: preparing recombinant human insulin receptor cells;
s2: adding cell lysis solution into the recombinant human insulin receptor cell of the step S1 for lysis, and collecting insulin receptor;
s3: combining insulin receptor in the lysate collected in step S2 by enzyme-linked immunosorbent assay;
s4: preparing recombinant human insulin and analogues thereof or recombinant human insulin conjugates with different concentrations as samples to be detected, and preparing recombinant human insulin standard substances with different concentrations as reference substances; dividing the result obtained in the step S3 into two parts, respectively adding the sample to be detected and the standard substance, adding insulin marked by biotin with a certain concentration, and determining the OD values of the sample to be detected and the standard substance after the binding of the receptors by a competitive binding method of biotin-avidin enzyme-linked immunity;
s5: and calculating the receptor binding rate of the recombinant human insulin and the analogues or conjugates thereof according to the OD value after receptor binding measured in the step S4.
Further, the human insulin and the analogues comprise common insulin, insulin aspart, insulin lispro, insulin glulisine, insulin glargine and mixed recombinant human insulin injection.
Further, the human insulin conjugate comprises fatty acid chains with different lengths and recombinant human insulin modified by glutamylated fatty acid chains with different lengths.
Further, the human insulin conjugate comprises insulin detemir and insulin deglutition.
Further, the human insulin conjugate comprises the recombinant human insulin modified by polyethylene glycol with different molecular weights.
Further, the human insulin conjugate comprises 20kd pegylated insulin lispro and 20kd pegylated common insulin with deletion of threonine at position 30 of the B chain.
Further, step S3 includes: coating of insulin receptor antibody, blocking, insulin receptor antibody binding insulin receptor.
Further, step S4 includes: insulin and biotin insulin compete for binding to insulin receptor, avidin binds to biotin, and a color reaction occurs.
Further, the cell lysate described in step S2 is: 20-50mM HEPES, 80-150mM NaCl, 1-3mM MgCl25-20% of glycerol, 0.5-1.5% of Triton X-100 and 1-2mM of EGTA or a combination of more of the above components.
Further, the concentration range of the recombinant human insulin and the analogues or conjugates thereof can be detected to be 1pg/ml-100 mu g/ml.
The invention discloses an application of an experimental determination method for the receptor binding rate of recombinant human insulin and analogues or conjugates in the in vitro drug effect determination of recombinant human insulin and analogues or conjugates. The receptor binding rate of the recombinant product is an important detection index of in vitro pharmacodynamics so as to ensure that the product has reliable pharmacodynamics.
The invention relates to a method for calculating the receptor binding rate of recombinant human insulin and analogues or conjugates, which is characterized in that the antibodies of the human insulin receptor can bind to the human insulin receptor, recombinant human insulin and analogues or conjugates with different concentrations and insulin marked by biotin with a certain concentration can compete to bind to the insulin receptor, and the OD value measured by an enzyme-linked immunosorbent assay is in inverse proportion to the concentration of a sample to be detected.
Compared with the prior art, the invention has the following beneficial effects:
firstly, the scheme of using isotope labeling in the prior art is changed, and the method for measuring the receptor binding rate of the recombinant human insulin and the analogues or the conjugates by adopting the enzyme-linked immunosorbent assay has the advantages of simplicity, convenience, no radioactive pollution and good safety.
Secondly, by using the standard substance as a control and measuring the OD value after the receptor is combined, the method simplifies the experimental steps and has high accuracy; the method is stable and easy to popularize.
Drawings
FIG. 1 is a graph of the binding rate of recombinant human insulin receptor measured by enzyme-linked immunosorbent assay (ELISA method) in example 1;
FIG. 2 is a graph of the binding rate of the recombinant human insulin receptor measured by enzyme-linked immunosorbent assay (ELISA) in example 2;
FIG. 3 in example 2125I, measuring a recombinant human insulin receptor binding rate graph by a marked insulin radiation method;
FIG. 4 is a graph showing the binding rate of the receptor of recombinant human insulin, insulin aspart, insulin deglutamide, insulin Lispro, insulin glargine, insulin detemir, PEG and modified conjugate (PEG-Lispro) measured by the ELISA method in example 3.
Detailed Description
The present invention will be described in further detail below by way of specific embodiments:
the human insulin receptor cell comprises various combinations of easily transfected cells and recombinant human insulin receptor plasmids, and the invention preferably adopts HEK-293-IR-A, HEK-293-IR-B, HEK-293-IGF1, and further preferably adopts HEK-293-IR-A, HEK-293-IR-B.
An alternative cell lysate of the present invention comprises 20-50mM HEPES, 80-150mM NaCl, 1-3mM MgCl25-20% of glycerol, 0.5-1.5% of Triton X-100 and 1-2mM of EGTA or a combination of more of the above components; among them, 0.5 to 1.5% Triton X-100 is preferable. When the cell lysate is in various combinations, the concentration needs to be adjusted according to actual conditions. The pH value of the cell lysate is 6.5-8.0.
The final concentration of the recombinant human insulin and the analogues or conjugates thereof which can be detected by the invention is 1pg/ml-100 mug/ml.
The method can be used for detecting the recombinant human insulin and the analogues or conjugates thereof, wherein the human insulin and the analogues comprise common insulin, insulin aspart, insulin lispro, insulin glulisine, insulin glargine and mixed recombinant human insulin injection. The human insulin conjugate comprises insulin detemir and insulin degludec, polyethylene glycol modified recombinant human insulin with different molecular weights, 20kd polyethylene glycol insulin lispro and 20kd polyethylene glycol common insulin with 30-bit threonine of B chain deleted.
A method for analyzing the binding rate of recombinant human insulin and analogues or conjugates thereof to insulin receptors, comprising the steps of:
s1: preparing recombinant human insulin receptor cells;
s2: adding cell lysis solution into the recombinant human insulin receptor cell of the step S1 for lysis, and collecting insulin receptor; insulin, blocking, insulin receptor antibody binding to insulin receptor
S3: coating the insulin receptor antibody on an enzyme label plate, then sealing, adding the insulin receptor in the lysate collected in the step S2 into the insulin receptor antibody, and combining the insulin receptor by an enzyme-linked immunosorbent assay;
s4: preparing recombinant human insulin and analogues thereof or recombinant human insulin conjugates with different concentrations as samples to be detected, and preparing recombinant human insulin standard substances with different concentrations as reference substances; dividing the result obtained in the step S3 into two parts, respectively adding the sample to be detected and the standard substance, adding insulin marked by biotin with a certain concentration, and determining the OD values of the sample to be detected and the standard substance after the binding of the receptors by a competitive binding method of biotin-avidin enzyme-linked immunity;
s5: and calculating the receptor binding rate of the recombinant human insulin and the analogues or conjugates thereof according to the OD value after receptor binding measured in the step S4.
Example 1 determination of the binding Rate of recombinant human insulin receptor by enzyme-Linked immunosorbent assay (ELISA method)
The recombinant human Insulin receptor plasmid is constructed by Shanghai Jikai gene technology Co., Ltd, the used HEK-293 cell is presented by Chongqing science and engineering university college of chemistry and biological engineering, the HEK-293-IR-B cell is transfected and screened by the company, Biotin is purchased from Shanghai Dongliren chemical technology Co., Ltd, Insulin-Biotin is marked by the company, recombinant human Insulin standard substances (from China food and drug testing institute, the content of each marker is 20 mg/28.3 IU/mg) are prepared by the company. The method for measuring the binding rate of the recombinant human insulin receptor comprises the following steps:
(1) after cell transfection, HEK-293-IR-B cells are cultured in a complete culture medium in a logarithmic growth phase, when the cell confluence rate reaches 70%, the cells are washed for 2 times by PBS to remove the culture medium containing serum, a proper amount of cell lysate is added to crack the HEK-293-IR-B cells, and the cells are placed for 1 hour at 4 ℃; and the lysate was placed in a centrifuge tube at 4 ℃ and centrifuged at 3500rpm for 10 minutes while maintaining 4 ℃. And (4) taking the centrifugal supernatant, and preserving at-20 ℃ to obtain the insulin membrane protein containing the insulin receptor.
(2) Anti-Insulin Receptor alpha antibody [83-7] was coated with coating buffer at 0.5-2ug/ml in 96-well microplate, 100 ul/well, maintained at 4 ℃ and incubated overnight in the dark.
(3) The microplate incubated overnight at 4 ℃ was removed, the coating buffer was discarded, washed 3 times with 1 XPBS, 200 ul/well blocking solution was added, and incubation was carried out at 37 ℃ for 1-3 hours.
(4) Washed 3 times with 1 XPBS, added with 100 ul/well of insulin membrane protein, shaken 1-3 hours at room temperature on a shaker at 300 rpm.
(5) Using PBS as a sample diluent, pre-diluting the recombinant human insulin standard product and the recombinant human insulin to 10-50 ug, and then diluting by 3-8 times, wherein each sample has 6-10 dilution gradients; adding a standard substance with a certain dilution gradient and 100 ul/hole of a sample to be detected, then adding Insulin-biotin with the concentration of about 0.05-1 ng/hole and 100 ul/hole, and reacting for 12-16 hours at the temperature of 4 ℃.
(6) Washing with 1 × PBS for 3 times, adding 1: 2000-1: 5000 Avidin-HRP 100 ul/well, and then adding 300rpm at room temperature and shaking for 1-3 hours.
(7) Washing with 1 × PBS for 4 times, adding TMB color development solution 100 ul/well, developing in dark for 30min, and reading by 450nm enzyme label.
(8) And (4) calculating a result: the binding rate of recombinant human insulin receptor was calculated from the measured OD value.
It is preferable that the cells in the logarithmic growth phase in step (1) are seeded in a T75 cell flask with the amount of seeding adjusted to 2.5X 105Each/ml, 10 ml/bottle, placing the cell bottle at 37 deg.C and 5% CO2Culturing in incubator overnight, removing cell culture supernatant, adding appropriate amount of PBS to wash cells for 2 times, addingAdding a proper amount of cell lysate to lyse HEK-293-IR-B cells, standing at 4 ℃ for 1 hour, taking the lysate in a centrifuge tube at 4 ℃, and centrifuging at 3500rpm for 10 minutes at 4 ℃. The centrifuged supernatant was stored at-20 ℃.
Further preferably, in the step (5), PBS is used as a sample diluent, and when the recombinant human insulin standard and the recombinant human insulin to be detected are pre-diluted to 10-50 ug, the samples are diluted by 3-8 times, and each sample has 6-10 dilution gradients; adding a certain dilution gradient of standard substance and 100 ul/hole of sample to be detected, adding Insulin-biotin with the concentration of about 0.05-1 ng/hole and 100 ul/hole, and reacting at 4 ℃ overnight.
Further preferably, the enzyme label plate is used for washing for 3 times in the step (6), Avidin-HRP 100 ul/hole with the ratio of 1: 2000-1: 5000 is added, and then the shaking table with the speed of 300rpm at room temperature is added for shaking for 1-3 hours.
More preferably, the step (8) comprises performing four-parameter (Logistic) calculation by ORIGIN software to obtain receptor competition IC of recombinant human insulin and standard substance50，IC50Values were compared for receptor binding rates (see figure 1).
Method for calculating receptor binding rate: default recombinant human insulin standard is 100% binding, and IC is competed with receptor of recombinant human insulin standard50Value divided by the receptor competition IC for recombinant human insulin produced by the company50The value is multiplied by 100% to calculate the receptor binding rate of the recombinant human insulin produced by the company corresponding to the recombinant human insulin standard, and the receptor binding rate is used for expressing the receptor affinity.
TABLE 1 statistics of results of measuring the binding rate of recombinant human insulin receptor by enzyme-linked immunosorbent assay (ELISA method)
The experimental results are shown in fig. 1, the calculated receptor binding rate of the recombinant human insulin is 104.791%, which is equivalent to the receptor affinity of the recombinant human insulin standard, and the method is proved to be feasible.
Example 2 determination of insulin receptor binding Rate by enzyme-Linked immunosorbent assay (ELISA)125Comparison of the binding Rate of recombinant human insulin receptor measured by I-labeled insulin Radioactive method
The recombinant human insulin receptor binding rate is measured by two methods at the same time, one is an enzyme-linked immunosorbent assay (ELISA), and the other is an ELISA125I labeled insulin irradiation.
Determination of insulin receptor binding rate by enzyme-linked immunosorbent assay (ELISA): the binding rate of recombinant human insulin receptor was determined by the method of example 1 (see FIG. 2)
125I, measuring the binding rate of the recombinant human insulin receptor by a labeled insulin radiation method:
firstly, after adult Wistar rats are cut off the neck and blood is taken, immediately dissecting and taking the liver, removing connective tissues, cutting into pieces, rinsing with ice-cold normal saline until no blood exists, draining, adding a proper amount of homogenate buffer solution, and homogenizing by a Dounce homogenizer for 20 times in an ice bath.
② pouring the homogenate into 200mL of homogenate buffer solution under ice bath, subpackaging in 4 centrifuge tubes with 250mL, centrifuging for 10min at 2500rpm at 4 ℃, and removing precipitates (unbroken cells and cell nucleuses).
③ filtering the supernatant with a screen, centrifuging the filtrate at 4 ℃ and 5000rpm for 30min, and discarding the precipitate (mitochondria). The supernatant was centrifuged at 13000rpm for 50min at 4 ℃ and the supernatant was removed.
Fourthly, collecting the precipitate, adding TBS with 5 times volume to homogenate in ice bath, centrifuging for 50min at 13000rpm at 4 ℃, and collecting the precipitate; the pellet was suspended in 1mL ice-cold TBS solution and stored at-70 ℃ in a freezer for further use.
Adding 500 mug membrane protein 50 mul into one group of reaction tubes for standby; adding 50 mu L of diluted INS activity standard substance into each dilution gradient; will be marked again125I-Ins was dissolved in TBS and diluted 153-fold, added and125150 μ L of TBS solution I-Ins, 300 μ L of the reaction system was made up with TBS solution, incubated at 4 ℃ and shaken for 16h, centrifuged at 5000rpm for 30min, the supernatant was discarded, the precipitate was washed twice with TBS solution, centrifuged in the same manner, resuspended in 200 μ L of TBS solution and counted in a gamma counter (measurement time 0.5 min). Two replicates were made at each point and the average was taken (results are shown in Table 2,Fig. 2, 3).
Table 2: enzyme linked immunosorbent assay (ELISA) method for determining insulin receptor binding rate125I comparison of the binding Rate of recombinant human insulin receptor by radiolabeling with insulin (three times)
The experimental results are shown in fig. 2 and 3. The results of the enzyme-linked immunosorbent assay and the radiation assay for measuring the binding rate of the recombinant human insulin receptor show that: receptor competition IC obtained by two detection methods50And (5) the consistency is achieved. Compared with the radiation method, the enzyme-linked immunosorbent assay has the advantages of simple and convenient steps, rapidness, high accuracy, good safety, less waste, easy treatment and the like, and is a research and development trend of in vitro pharmacodynamics.
Example 3 determination of the binding rates of recombinant human insulin, insulin aspart, insulin Lispro, insulin glargine, insulin detemir, PEG and modified conjugate (PEG-Lispro) receptor by enzyme-linked immunosorbent assay (ELISA)
Insulin receptor binding rates of insulin aspart (from Nodekanerjie 300 units/3 ml/lot), insulin Lispro (from Li Lai Youxile 300 units/3 ml/lot), insulin degu (from Nodekanerjie, trade name: Tresiba 300 units/3 ml/lot), recombinant human insulin (from China food and drug assay institute, each indicated at 28.3IU/mg, 20 mg/lot), insulin glargine (from Senaofen-Anwan medicine, 300 units/3 ml/lot), insulin detemir (from Nonah and Nonaddc 300 units/3 ml/lot), PEG-Lispro (from EP2476430A1, homemade by companies) were determined by the enzyme-linked immunosorbent assay (ELISA) method of example 1, performing four-parameter (Logistic) operation by using ORIGIN software in 5 times of repeated experiments to obtain receptor competition IC50The data are shown in Table 3, and the results are shown in FIG. 4.
TABLE 3 measurement of the binding rate of recombinant human insulin, insulin aspart, insulin deglutamide, insulin Lispro, insulin glargine, insulin detemir, PEG and modified conjugate (PEG-Lispro) receptor by enzyme-linked immunosorbent assay (ELISA)
Receptor competition IC by measuring receptor binding rate of recombinant human insulin titer standard product50The value is 81.128ng/ml, according to the standard of recombinant human insulin international standard, the default is that the recombinant human insulin activity standard is 100% combined, and the receptor of the recombinant human insulin activity standard is used for competing IC50Value divided by receptor competition IC of the sample to be tested50The value is multiplied by 100% to calculate the receptor affinity of the sample corresponding to recombinant human insulin. Table 3 is the receptor competition IC tested50And (4) data.
The statistical analysis of the above test data determined that the receptor binding rates (%) of insulin aspart, insulin Lispro, insulin deglutamide, insulin glargine, insulin detemir, and PEG-Lispro were 102.98%, 97.32%, 18.81%, 88.47%, 21.51%, and 22.42%, respectively.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (10)
1. A method for analyzing the binding rate of recombinant human insulin and analogues or conjugates thereof to insulin receptors, which is characterized by comprising the following steps: comprises the following steps:
s1: preparing recombinant human insulin receptor cells;
s2: adding cell lysis solution into the recombinant human insulin receptor cell of the step S1 for lysis, and collecting insulin receptor;
s3: combining insulin receptor in the lysate collected in step S2 by enzyme-linked immunosorbent assay;
s4: preparing recombinant human insulin and analogues thereof or recombinant human insulin conjugates with different concentrations as samples to be detected, and preparing recombinant human insulin standard substances with different concentrations as reference substances; dividing the result obtained in the step S3 into two parts, respectively adding the sample to be detected and the standard substance, adding insulin marked by biotin with a certain concentration, and determining the OD values of the sample to be detected and the standard substance after the binding of the receptors by a competitive binding method of biotin-avidin enzyme-linked immunity;
s5: and calculating the receptor binding rate of the recombinant human insulin and the analogues or conjugates thereof according to the OD value after receptor binding measured in the step S4.
2. The method for analyzing the binding rate of recombinant human insulin and analogues or conjugates thereof to insulin receptor according to claim 1, wherein said binding rate is selected from the group consisting of: the human insulin and the analogues comprise common insulin, insulin aspart, insulin lispro, insulin glulisine, insulin glargine and mixed recombinant human insulin injection.
3. The method for analyzing the binding rate of recombinant human insulin and analogues or conjugates thereof to insulin receptor according to claim 2, wherein said binding rate is selected from the group consisting of: the human insulin conjugate comprises fatty acid chains with different lengths and recombinant human insulin modified by glutamylated fatty acid chains with different lengths.
4. The method for analyzing the binding rate of recombinant human insulin and analogues or conjugates thereof to insulin receptor according to claim 2, wherein said binding rate is selected from the group consisting of: the human insulin conjugate comprises insulin detemir and insulin deglutition.
5. The method for analyzing the binding rate of recombinant human insulin and analogues or conjugates thereof to insulin receptor according to claim 2, wherein said binding rate is selected from the group consisting of: the human insulin conjugate comprises polyethylene glycol modified recombinant human insulin with different molecular weights.
6. The method for analyzing the binding rate of recombinant human insulin and analogues or conjugates thereof to insulin receptor according to claim 2, wherein said binding rate is selected from the group consisting of: the human insulin conjugate comprises 20kd pegylated insulin lispro and 20kd pegylated common insulin with deletion of threonine at position 30 of B chain.
7. The method for analyzing the binding rate of recombinant human insulin and analogues or conjugates thereof to insulin receptor according to claim 1, wherein said binding rate is selected from the group consisting of: step S3 includes: coating of insulin receptor antibody, blocking, insulin receptor antibody binding insulin receptor.
8. The method for analyzing the binding rate of recombinant human insulin and analogues or conjugates thereof to insulin receptor according to claim 7, wherein said binding rate is selected from the group consisting of: step S4 includes: insulin and biotin insulin compete for binding to insulin receptor, avidin binds to biotin, and a color reaction occurs.
9. The method for analyzing the binding rate of recombinant human insulin and analogues or conjugates thereof to insulin receptor according to any one of claims 1-8, wherein: the cell lysate described in step S2 is: 20-50mM HEPES, 80-150mM NaCl, 1-3mM MgCl25-20% of glycerol, 0.5-1.5% of Triton X-100 and 1-2mM of EGTA or a combination of more of the above components.
10. The method for analyzing the binding rate of recombinant human insulin and analogues or conjugates thereof to insulin receptor according to claim 9, wherein said binding rate is selected from the group consisting of: the concentration range of the recombinant human insulin and the analogues or conjugates thereof can be detected to be 1pg/ml-100 mu g/ml.
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