CN111122880A - Competitive ELISA detection method for dolafectin and kit thereof - Google Patents
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Abstract
The invention relates to a detection method of competitive ELISA of dolaferin and a kit thereof, which have the advantages of good specificity, simple and quick operation and high-throughput detection. The method takes a mouse anti-human GLP-1 monoclonal antibody as a capture antibody, and performs quantitative analysis by competitive combination of the dolabrus peptide and the biotinylated dolabrus peptide, so that the method has the characteristics of small background signal and wide quantitative range, and can reduce errors caused by a sample dilution step.
Description
Technical Field
The invention relates to a detection method of competitive ELISA of dolabrus peptide and a kit thereof, develops and establishes a competitive enzyme-Linked immunosorbent assay (ELISA) for the concentration determination of the dolabrus peptide in the serum of rhesus monkey, and has the characteristics of high sensitivity and good specificity.
Technical Field
According to research data, the proportion of adults suffering from diabetes worldwide is increased from 4.7% in 1980 to 8.5% nowadays, more than 4.22 hundred million people suffer from diabetes nowadays, China is the first major country of diabetes worldwide, according to survey data published in journal of American medical society (JAMA) in 2017, the prevalence rate of adult diabetes in China is 10.9%, and according to the conclusion, the number of patients exceeds 1 hundred million people, diabetes seriously threatens the health and life quality of people, wherein the type 2 diabetes is originally named as adult-onset diabetes and accounts for more than 90% of the diabetes patients, and the type 2 diabetes is characterized by high blood sugar caused by impaired function of pancreatic islet β cells, insulin resistance and glucagon.
Glucagon-like peptide-1 (GLP-1) receptor agonists are part of the incretin mimetic diabetes treatment options. Endogenous GLP-1 is an incretin hormone that is released from the intestine in response to food intake. GLP-1 effects include increased insulin secretion, decreased glucagon release, increased satiety and slower gastric emptying. GLP-1 is degraded by dipeptidyl peptidase-IV (DPP-4) in the human body within minutes after release, resulting in limited replication of this incretin hormone.
The dolaglutide is a long-acting GLP-1 receptor agonist developed by American etiquette, is obtained by fusing two GLP-1 analogs with DPP-4 inhibition effect and a human immunoglobulin heavy chain IgG4-Fc fragment, has similar activity to endogenous GLP-1, has a half-life of 5d, and is mainly suitable for controlling blood sugar of a type 2 diabetic patient.
The dolastatin is fusion protein with molecular weight of about 60 KDa. The determination of drug concentration by means of double antibody sandwich ELISA, liquid chromatography-mass spectrometry (LC-MS/MS) and radioimmunoassay has been reported in the literature. In the literature, GLeasner W et al report the determination of drug concentrations in rat and monkey plasma by using antibodies recognizing the N-terminus and Fc domain of GLP-1-Fc antibodies as sandwich ELISA. Barrington P et al, however, use radioimmunoassay to determine changes in drug concentration in type 2 diabetic patients. However, the quantitative ranges of the two methods are narrow, and are respectively 0.9-80ng/mL and 5.0-40 ng/mL. LC-MS/MS is also receiving more and more attention as an emerging technology for measuring macromolecular drugs, but the complex sample pretreatment and the precise requirement on instruments make the method expensive and time-consuming to develop. On the basis, a competitive ELISA method is developed, the quantitative range can reach 5.0-1000ng/mL, and the method has good specificity, precision and accuracy.
Disclosure of Invention
The dolabrus peptide is a fusion protein and is obtained by fusing two GLP-1 analogues with DPP-4 inhibition effect and a human immunoglobulin heavy chain IgG4-Fc fragment. A mouse anti-human GLP-1 monoclonal antibody is used as a capture antibody, a GLP-1 end of the dolabrus peptide is specifically recognized, the dolabrus peptide and the biotinylated dolabrus peptide are used for competitive combination, the quantitative range is improved, the drug concentration is more accurately measured, the pharmacokinetics of the dolabrus peptide in a rhesus monkey body is analyzed, and errors caused by background interference and sample dilution are reduced.
A detection method of competitive ELISA of dolaferin is characterized by comprising the following steps:
(1) a mouse anti-human GLP-1 antibody is used as a capture antibody coated plate,
(2) adding dolabrus peptide and biotinylated dolabrus peptide for competitive combination,
(3) adding streptomycin-labeled horse radish peroxidase,
(4) the enzyme and the substrate are subjected to a color reaction, and the absorbance is measured.
Preferably, the chromogenic reaction of the enzyme and substrate is: adding substrate tetramethyl benzidine, changing into blue under the catalysis of HRP, and changing into yellow under the catalysis of acid, and/or detecting the concentration of the dolauda peptide in serum by the detection method.
Preferably, the plate concentration of the mouse anti-human GLP-1 antibody is 1-5 μ g/mL, and/or the dilution of serum is 1:1-10, and/or the dilution of streptomycin-labeled horseradish peroxidase is 1:500-5000, and/or the blocking solution is 0.5-5% BSA/PBS, and/or the dilution is 0.05-1% BSA/PBST, and/or the reaction time is 0.5-3h, and/or the reaction temperature is 30-40 ℃, and/or the rotation speed is 300-400 rpm.
Preferably, the mouse anti-human GLP-1 antibody is coated at a concentration of 2. mu.g/mL, and/or the serum is diluted at 1:5, and/or the streptomycin-labeled horseradish peroxidase is diluted at 1:1500, and/or the blocking solution is 1% BSA/PBS, and/or the blocking solution is 0.1% BSA/PBST, and/or the reaction time is 1h, and/or the reaction temperature is 37 ℃ and/or the rotation speed is 350 rpm.
Preferably, the serum is rhesus monkey serum.
Preferably, the biotinylated dulaglutide is prepared by:
(1) diluting dolafectin with PBS, and ultrafiltering the labeled antigen in sodium bicarbonate solution;
(2) weighing biotin to prepare a solution, and labeling the dolaglutide with biotin with 2-50 times of molar excess;
(3) ultrafiltering the dolacilin solution after the labeling reaction is completed, and fixing the volume; 1% -5% BSA in an amount of 5% -25% by volume of the final product and 20% -80% glycerol in an amount of the final product were added simultaneously and the concentrations were measured.
Preferably, the biotinylated dulaglutide is prepared by:
(1) diluting dolastatin to 2mg/mL by PBS, and placing 0.5mL on an ice box for later use; transferring the antigen to 10MW ultrafiltering tube, and ultrafiltering in 0.1M sodium bicarbonate solution for 3 times at 12000r/min for 10 min;
(2) precisely weighing 1mg of biotin, adding 180 mu L of ultrapure water to prepare a solution of 10mM, placing the solution in a dark place, and labeling the dolacilin with biotin with 10-fold molar excess; mixing, placing into a light-tight bag, marking on an ice box, and standing for 2 hr;
(3) ultrafiltering the dolastatin solution subjected to the labeling reaction for 3 times by using PBS (phosphate buffer solution) through a 10MW ultrafiltration tube, and fixing the volume of the antibody in the ultrafiltration tube to a proper volume through the PBS, wherein the volume accounts for 40% of the final volume; simultaneously adding 2% BSA (bovine serum albumin) accounting for 10% of the volume of the final product and 50% glycerol accounting for 50% of the volume of the final product, and uniformly mixing by vortex; and the concentration was determined using Nanodrop at 280 nm.
A kit for detecting dolabric peptide by competitive ELISA is characterized in that the kit contains biotinylated dolabric peptide.
Preferably, the preparation of the biotinylated dulaglutide is prepared by the preparation method described previously.
Preferably, the concentration of the biotinylated dulaglutide is 1-100 ng/mL.
And coating the monoclonal antibody on a 96-well plate by using a mouse anti-human GLP-1 monoclonal antibody as a capture antibody, and dividing the hole on the ELISA plate into a sample hole to be detected and a standard curve hole. Adding a series of diluted durauu peptide and a certain concentration of biotinylated durauu peptide into a standard curve hole, then adding streptomycin labeled horse radish peroxidase (SA-HRP), adding substrate tetramethyl benzidine (TMB), changing into blue under the catalysis of HRP, changing into yellow under the action of acid, finally measuring the absorbance value, and drawing a standard curve according to the absorbance value of the durauu peptide with known concentration.
And adding a monkey serum sample to be detected into a sample hole to be detected, and carrying out competitive combination on the dolabrus peptide in the serum and the added biotinylated dolabrus peptide and the coated mouse anti-human GLP-1 monoclonal antibody. Then adding SA-HRP, adding substrate TMB, changing into blue under the catalysis of HRP, changing into yellow under the action of acid, finally measuring absorbance value, and calculating the concentration of the dolabric in serum according to the standard curve.
The quantitative range can reach 5.0-1000 ng/mL. The absorbance values determined are those at 450nm of the microplate reader.
Advantageous effects
The method for detecting the dolabrus peptide has the characteristics of strong specificity, high sensitivity, simple sample treatment and high flux. Is not limited by specific reagents and special instruments. The linear range of the invention is 5.0-1000ng/mL, which can meet the requirement of the preclinical plasma drug concentration determination, and the method can also be applied to the analysis of clinical samples.
Drawings
Fig. 1 is a standard graph of dolabrin.
Fig. 2 is a graph of drug concentration versus time in serum after subcutaneous injection of dolabraglutide in rhesus monkeys (upper panel is a constant coordinate, and lower panel is a semilog coordinate).
Detailed Description
The present invention is further illustrated but is not to be limited by the following examples.
Example 1
Biotinylated duravide: the dulaglutide was first diluted to 2mg/mL with PBS and 0.5mL was placed on an ice-box until use. The surrogate labelled antigen was transferred to a 10MW ultrafiltration tube and ultrafiltered 3 times in 0.1M sodium bicarbonate solution at 12000r/min for 10 min.
Secondly, 1mg of biotin was precisely weighed and added to 180. mu.L of ultrapure water to prepare a 10mM solution, which was left in the dark and labeled with biotin in a 10-fold molar excess of dolaferin. Mixing, placing into a light-tight bag, marking on an ice box, and standing for 2 hr.
Finally, the solution of dolastatin, which has completed the labeling reaction, is ultrafiltered 3 times with PBS through a new ultrafiltration tube (10 MW), and the antibody in the ultrafiltration tube is made to a suitable volume, which is 40% of the final volume, by PBS. 2% BSA at 10% volume of final product and 50% glycerol at 50% volume of final product were added simultaneously and mixed by vortexing. And the concentration was determined using Nanodrop at 280 nm.
Example 2
Mouse anti-human GLP-1 monoclonal antibody was diluted in Phosphate Buffered Saline (PBS) and plated overnight at 4 ℃ at a concentration of 2. mu.g/mL in a volume of 100. mu.L/well. The 96 well plates were washed four times with PBST and the plate contents removed. Blocking was performed with 1% BSA/PBS in a blocking volume of 200. mu.L/well. The BSA PBS solution contained therein was a BSA/PBS solution with a mass fraction of 1%. The sealing time was 1h, the speed 350rpm and the temperature 37 ℃. And after the completion of blocking, the 96-well plate was washed four times with PBST and the liquid in the plate was removed.
Serum dilution was determined to be 5-fold. Blank monkey serum was used as a dilution of the standard curve and quality control samples, and the standard curve and quality control samples were diluted 5-fold with 0.1% BSA/PBST prior to the well-entry. The PBST solution containing BSA is BSA/PBST solution with the mass fraction of 0.1%. Serum samples were also analyzed by 5-fold dilution with 0.1% BSA/PBST solution.
The dolaglutide standard substance solution 5, 10, 20, 50, 100, 200, 500 and 1000ng/mL are respectively added into an enzyme label plate, and each dilution is provided with 2 parallel holes. Simultaneously preparing samples with lower limit of quantification (5 ng/mL), low, medium and high quality control samples (15ng/mL, 100ng/mL, 800ng/mL) and upper limit of quantification (1000 ng/mL), respectively adding the samples into the ELISA plate, wherein each dilution is provided with two parallel holes, and each analysis batch is provided with at least two sets of quality control samples.
The diluted standard curve, quality control and serum samples were added to the plate at 50. mu.L per well.
The competitive antigen was biotinylated dulaglutide, diluted with serum from a blank monkey to a concentration of 50ng/mL, and further diluted 5-fold to 10ng/mL with 0.1% BSA/PBST, 50. mu.L per well was added to the plate.
The competitive binding reaction was 1h at 350rpm and 37 ℃. And after binding was complete, the 96-well plate was washed four times with PBST and the plate liquid was removed. SA-HRP was diluted 1500-fold with PBS and 100. mu.L/well was added to the plate at 350rpm for 1 h. After washing the 96-well plate four times with PBST and removing the liquid in the plate, 100. mu.L of TMB was added to each well and reacted for 20 minutes in the absence of light. Add 100. mu.L/well of 1M H3PO4The reaction was terminated and the color changed from blue to yellow. And reading the absorbance value at 450nm on a microplate reader within 30 minutes, drawing a standard curve and calculating the drug concentration of the dolaglutide in the serum. The standard curve is shown in figure 1, and the standard curve equation is as follows: y =0.171+ (3.679-0.171)/(1+ (x/317.4)1.37)。
Preparing standard solutions with 5 concentrations, namely, a high quantitative upper limit (1000 ng/mL), a medium quantitative upper limit (800ng/mL), a medium quantitative lower limit (100ng/mL), a low quantitative lower limit (15ng/mL) and a low quantitative lower limit (5 ng/mL), measuring 6 holes at each concentration point in one experiment, and calculating the intra-batch variation coefficient. And 6 batches are continuously measured, and the inter-batch variation coefficient is calculated. The precision test result shows that the detection method has the advantages that the intra-batch precision is 1.25-16.74%, the inter-batch precision is 1.39-10.57%, the accuracy is less than 6%, and the requirement of biological analysis is met. The results are shown in Table 1.
TABLE 1 precision accuracy measurement results
Example 3
The evaluation data of Duraluvian peptide cynomolgus pharmacokinetic by biotinylation competition ELISA method:
the test drug, dolacilin (0.5 mL per injection pen), was administered subcutaneously at 0.3mg/kg to rhesus monkeys before and after administration and during the experiment without fasting of animals, with normal drinking water, and before and after administration for 1h, 4h, 6h, 8h, 12h, 1d,2d, 3d, 4d, 5d, 7d, 10d, and 14d in the saphenous vein of the lower extremities, and about 1 mL of blood was collected at each point, and serum was separated, frozen at-20 ℃ and stored for testing. The concentration of the durapeptide in the serum of the rhesus monkey is measured by a double-antibody sandwich ELISA method (a reported method, hereinafter referred to as a "simulated double-antibody sandwich method") and a biotinylation competition ELISA method of the patent. The double-antibody sandwich method comprises the following steps: the dolabrus peptide is a GLP-1(7-37) -FC fusion antibody, and is captured by a mouse anti-human GLP-1 capture antibody, then an anti-human FC end secondary antibody with HRP is added, and TMB is added for color development and detection. The concentration-time curve of the dolabrin in serum determined by the two methods is shown in figure 2. Pharmacokinetic parameters such as peak concentration (Cmax, about 2500 ng/mL), area under the curve of drug administration (AUC, about 133 hr. mu.g/mL) and elimination half-life (t 1/2, about 6 h) obtained by detection and analysis of two methods, namely a biotinylation competition ELISA method and a double antibody sandwich ELISA method, have good consistency. However, the quantitative range of the method can reach 5.0-1000ng/mL, and the reported quantitative ranges of the double-antibody sandwich ELISA determination method and the radioimmunoassay method are narrower, and are respectively 0.9-80ng/mL and 5.0-40ng/mL, so that the method of the patent is more convenient to apply, can save a large number of sample dilution steps, and is convenient to detect and easy to implement, and has reliable data.
Claims (10)
1. A detection method of competitive ELISA of dolaferin is characterized by comprising the following steps:
(1) a mouse anti-human GLP-1 antibody is used as a capture antibody coated plate,
(2) adding dolabrus peptide and biotinylated dolabrus peptide for competitive combination,
(3) adding streptomycin-labeled horse radish peroxidase,
(4) the enzyme and the substrate are subjected to a color reaction, and the absorbance is measured.
2. The detection method according to claim 1, wherein the color reaction between the enzyme and the substrate is: adding substrate tetramethyl benzidine, changing into blue under the catalysis of HRP, and changing into yellow under the catalysis of acid, and/or detecting the concentration of the dolauda peptide in serum by the detection method.
3. The detection method according to claim 2, wherein the plate concentration of the mouse anti-human GLP-1 antibody is 1-5 μ g/mL, and/or the dilution of serum is 1:1-10, and/or the dilution of streptavidin-labeled horseradish peroxidase is 1:500-5000, and/or the blocking solution is 0.5% -5% BSA/PBS, and/or the dilution is 0.05-1% BSA/PBST, and/or the reaction time is 0.5-3h, and/or the reaction temperature is 30-40 ℃, and/or the rotation speed is 300-400 rpm.
4. The assay of claim 3, wherein the mouse anti-human GLP-1 antibody is coated at a concentration of 2 μ g/mL, and/or the serum is diluted at 1:5, and/or the streptomycin-labeled horseradish peroxidase is diluted at 1:1500, and/or the blocking solution is 1% BSA/PBS, and/or the blocking solution is 0.1% BSA/PBST, and/or the reaction time is 1h, and/or the reaction temperature is 37 ℃, and/or the rotation speed is 350 rpm.
5. The method of claim 2, wherein the serum is rhesus monkey serum.
6. The detection method according to claim 1, wherein the biotinylated dulaglutide is prepared by:
(1) diluting dolafectin with PBS, and ultrafiltering the labeled antigen in sodium bicarbonate solution;
(2) weighing biotin to prepare a solution, and labeling the dolaglutide with biotin with 2-50 times of molar excess;
(3) ultrafiltering the dolacilin solution after the labeling reaction is completed, and fixing the volume; 1% -5% BSA in an amount of 5% -25% by volume of the final product and 20% -80% glycerol in an amount of the final product were added simultaneously and the concentrations were measured.
7. The detection method according to claim 6, wherein the biotinylated dulaglutide is prepared by:
(1) diluting dolastatin to 2mg/mL by PBS, and placing 0.5mL on an ice box for later use; transferring the antigen to 10MW ultrafiltering tube, and ultrafiltering in 0.1M sodium bicarbonate solution for 3 times at 12000r/min for 10 min;
(2) precisely weighing 1mg of biotin, adding 180 mu L of ultrapure water to prepare a solution of 10mM, placing the solution in a dark place, and labeling the dolacilin with biotin with 10-fold molar excess; mixing, placing into a light-tight bag, marking on an ice box, and standing for 2 hr;
(3) ultrafiltering the dolastatin solution subjected to the labeling reaction for 3 times by using PBS (phosphate buffer solution) through a 10MW ultrafiltration tube, and fixing the volume of the antibody in the ultrafiltration tube to a proper volume through the PBS, wherein the volume accounts for 40% of the final volume; simultaneously adding 2% BSA (bovine serum albumin) accounting for 10% of the volume of the final product and 50% glycerol accounting for 50% of the volume of the final product, and uniformly mixing by vortex; and the concentration was determined using Nanodrop at 280 nm.
8. A kit for detecting dolabric peptide by competitive ELISA is characterized in that the kit contains biotinylated dolabric peptide.
9. The kit of claim 8, wherein the preparation of biotinylated dulaglutide is prepared according to the preparation method of any one of claims 6 or 7.
10. The kit of claim 8, wherein the biotinylated dulaglutide is at a concentration of 1-100 ng/mL.
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| CN101458256A (en) * | 2007-12-14 | 2009-06-17 | 武汉大学 | Hepatitis C virus envelope antigen ELISA kit and detecting method |
| CN101672851A (en) * | 2009-07-31 | 2010-03-17 | 华中科技大学同济医学院附属同济医院 | Preparation of Human Tissue Kallikrein ELISA Kit |
| CN108570109A (en) * | 2017-03-14 | 2018-09-25 | 广东东阳光药业有限公司 | Include double target spot fusion proteins of Fc portion of immunoglobulin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101458256A (en) * | 2007-12-14 | 2009-06-17 | 武汉大学 | Hepatitis C virus envelope antigen ELISA kit and detecting method |
| CN101672851A (en) * | 2009-07-31 | 2010-03-17 | 华中科技大学同济医学院附属同济医院 | Preparation of Human Tissue Kallikrein ELISA Kit |
| CN108570109A (en) * | 2017-03-14 | 2018-09-25 | 广东东阳光药业有限公司 | Include double target spot fusion proteins of Fc portion of immunoglobulin |
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| 李田等: "植物启动子研究进展" * |
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