CN111273019A - Duraluvian peptide ELISA detection method - Google Patents
Duraluvian peptide ELISA detection method Download PDFInfo
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- CN111273019A CN111273019A CN201811470486.XA CN201811470486A CN111273019A CN 111273019 A CN111273019 A CN 111273019A CN 201811470486 A CN201811470486 A CN 201811470486A CN 111273019 A CN111273019 A CN 111273019A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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Abstract
The invention relates to a method for detecting the concentration of dolabrus peptide in a blood sample, in particular to an enzyme-linked immunosorbent assay (ELISA) test method for rapidly determining the blood concentration of the dolabrus peptide. The method shortens detection time and improves detection sensitivity.
Description
Technical Field
The invention relates to a method for measuring the concentration of dolabrin in a blood sample, in particular to an enzyme-linked immunosorbent assay (ELISA) test method for rapidly measuring the blood concentration of the dolabrin.
Background
Diabetes is a chronic metabolic disease, mainly manifested by hyperglycemia, and persistent hyperglycemia is a main factor causing diabetic complications, such as retinal, renal, nervous system and microvascular complications. There are about 3.82 million diabetics worldwide, with a projected 2035 year patient reaching 6 million. Diabetes can be divided into insulin-dependent (type 1 diabetes) and non-insulin-dependent (type 2 diabetes). Type2diabetes patients account for approximately 90% of the total number, and novel drugs having a protective effect on islet B cells have become a research hotspot in this field. The patient's in vivo cell membrane receptor Glucagon-like peptide-1 (GLP-1) level is too low to cause pancreatic B cell damage, and is an important pathogenic factor of type2 diabetes.
GLP-1 is an incretin, which is secreted mainly by L cells distributed in jejunum, ileum and caecum and is transcribed, translated and processed from glucagon gene. GLP-1 consists of 30 or 31 amino acid residues, has two subtypes of GLP-1(7-36) and GLP-1(7-37), and can obtain a long-acting GLP-1R agonist after the structure of natural GLP-1 is modified. In recent years, the FDA in the united states has approved a series of GLP-1R agonists including dolaglutide (Dulaglutide) and the like to be marketed.
Dolabrin (trade name:
) The long-acting GLP-1R agonist is a novel long-acting GLP-1R agonist developed by Lily company in America, is obtained by fusing two GLP-1 analogs with DPP-4 inhibition effect and a human immunoglobulin heavy chain IgG4-Fc fragment, has activity similar to that of endogenous GLP-1 and half-life of 5d, and can effectively delay the clearance effect of kidney. The FDA approved dolabrus peptide subcutaneous injection for marketing in 9 months 2014, which is mainly suitable for improving the blood sugar control level of adult type2 diabetic patients on the basis of diet and physical exercise. The european union committee approved dolauda subcutaneous injection marketed in europe at 12 months 2014.
The dolaglu peptide belongs to fusion protein, two GLP-1(7-37) ends with dipeptidyl peptidase inhibition effects are connected with the N ends of human immunoglobulin IgG4-Fc crystallizable protein fragments through small peptide molecular chains, specific amino acid site modification is carried out, including that arginine (Arg36) at the 36 th position of GLP-1(7-37) and phenylalanine (Phe234) at the 234 th position, leucine (Leu235) at the 235 th position and serine (Ser228) at the 228 th position of IgG4-Fc fragment heavy chain are respectively replaced by glycine (Gly), alanine (Ala), Ala and proline (Pro), and lysine (Lys) at the carbon end of IgG4-Fc is removed. The modification of the amino acid site reduces the binding effect of lgG4-Fc and a receptor thereof, reduces cytotoxicity and improves the safety of the medicament. The dolaglutide activates related signal transduction pathways in cells by combining with GLP-1R on the surface of cell membranes, promotes the synthesis and secretion of insulin in a glucose-dependent mode, reduces the secretion of glucagon and further lowers the blood sugar. Dulaglutide delays gastric emptying, potentially reducing the rate of absorption of oral therapeutic drugs, and requires close enough monitoring when combined with oral diabetic drugs with narrow therapeutic indices.
However, the methods for measuring the blood concentration of the dolabrus peptide in the prior art are all radioactive immune methods (RIA), which have high requirements on laboratories and experimenters, are complex to operate and have long time; meanwhile, the sensitivity of the method is not high, such as the literature of Clinical pharmacy of Dulaglutide in Patients with Type2Diabetes, analysis of Data from Clinical Trials- -clinicokinet DOI10.1007, the detection range of the dolaglutide is 5-50ng/ml by adopting a Radioimmunoassay (RIA); (ii) literature; the document "A5-week study of the pharmacological and pharmacological assays of LY2189265, anovel, long-acting glucagon-like peptide-1analog, in Patients with type2 diabetes" also measures dolaud peptide by the Radioimmunoassay (RIA), in the range of 5-40 ng/ml; these methods fail to meet the needs of clinical testing in terms of sensitivity; a method for detecting the blood concentration of the dolabrus peptide with high sensitivity and simple operation is urgently needed in clinical and scientific research development to meet the requirement of the present-stage dolabrus peptide drug development.
Disclosure of Invention
Aiming at the defects in the prior art, the invention carries out in-depth research and provides a method for detecting the dolabrin, which is simple to operate and high in sensitivity; the method adopts an enzyme-linked immunosorbent assay (ELISA) technology, uses a GLP-1 capture antibody to capture dolaferin, then adds an anti-human FC end secondary antibody with HRP, and adds TMB for color development and detection; the anti-human FC-terminal secondary antibody with the HRP is a monkey serum adsorption secondary antibody.
The method for measuring the blood concentration of the dolabrus peptide by adopting an enzyme-linked immunosorbent assay (ELISA) technology is characterized in that the GLP-1 capture antibody is a mouse anti-human GLP-1 capture antibody.
The method for measuring the blood concentration of the dolabrus peptide by adopting an enzyme-linked immunosorbent assay (ELISA) technology is characterized in that the adopted antibody plating concentration is more than or equal to 2 mu g/ml, and preferably the adopted antibody plating concentration is 2 mu g/ml.
The method for measuring the blood concentration of the dolabrus peptide by adopting an enzyme-linked immunosorbent assay (ELISA) technology is characterized in that the collected biological sample is diluted by serum, the dilution multiple of the serum is more than or equal to 50 times, and preferably, the dilution multiple of the serum is 50 times.
The method for measuring the blood concentration of the dolabrin by adopting an enzyme-linked immunosorbent assay (ELISA) technology is characterized in that the monkey serum adsorbs a secondary antibody and is diluted by 1: 15000.
The method for measuring the blood concentration of the dolabruxin by adopting an enzyme-linked immunosorbent assay (ELISA) technology is characterized in that the antibody plating concentration adopted by the method is 2 mu g/ml, the dilution multiple of a biological sample by serum is 50 times, and a monkey serum adsorbs a secondary antibody and is diluted by 1: 15000.
The method for measuring the blood concentration of the dolabrus peptide by adopting the enzyme-linked immunosorbent assay (ELISA) technology can be used for detecting the content change of the dolabrus peptide in vivo and the application of metabolic research after the mammal uses the dolabrus peptide preparation.
The method for measuring the blood concentration of the dolabrus peptide by the enzyme-linked immunosorbent assay (ELISA) technology is a measuring method for measuring the blood concentration of the dolabrus peptide, which has the advantages of simple operation, accurate result, good specificity and high sensitivity.
The method adopts enzyme-linked immunosorbent assay (ELISA) technology, uses GLP-1 capture antibody to capture dolaferin, then adds anti-human FC end secondary antibody with HRP, adds TMB for color development and detection; the anti-human FC-terminal secondary antibody with the HRP is a monkey serum adsorption secondary antibody.
In the enzyme-linked immunosorbent assay (ELISA) technology, the selection of anti-human FC-terminal secondary antibody with HRP plays a very important role. The anti-human FC-terminal secondary antibody with the HRP has better and better chromatographic behavior when measuring the dolaglutide; the detection method provided by the invention is used for detecting trace amount of the dolabrin in a standard solution sample or detecting the dolabrin in a plasma sample, and the lowest detection limit can reach 0.4 ng/ml.
The invention is realized by the following technical scheme:
1. antibody-coated 96-well plates: preparing 2 mu g/ml of mouse anti-human GLP-1 antibody and PBS, adding 100 mu l/well of the antibody into a 96-well plate with high adsorption, and performing dressing at 4 ℃ overnight;
BSA blocking: PBST is used for preparing 1 percent BSA blocking solution, and the blocking solution is blocked for 1 hour at 37 ℃ in 200 mul/hole;
3. dulaglutide (3mg/ml) standard and quality-controlled dilution:
adding the prepared standard yeast and quality control sample into the plate at a volume of 100 mul/hole, and laying for 2 hours at 37 ℃;
4. dilution of the sample: the sample to be assayed is diluted to a quantitative range (dulaglutide immunized cynomolgus monkey, plasma thereof is extracted and processed to obtain an assay sample).
5. Secondary antibody: the monkey serum adsorbs the second antibody to be diluted by 1:15000 (the OD highest value of 1:20000 dilution is less than 1.5, the OD value of 1:10000 dilution exceeds 3, which exceeds the accurate measurement range of a SpectraMax190 enzyme-labeling instrument, the dilution ratio of 1:15000 is selected to meet the requirement in the experiment), 100 mul/hole is added into the plate, and the plate is incubated for 1 hour at 37 ℃;
6. color development: preparing TMB developing solution, mixing solution A and B at equal ratio, adding into plate at a ratio of 100 μ l/hole, and applying at 37 deg.C for 20 min;
7. adding a stop solution: add stop solution (1M H3PO4) in 100. mu.l/well
OD value read by SpectraMax190 microplate reader
The invention also relates to application of the method for determining the trace amount of the dolabrus peptide in detecting the content of the dolabrus peptide in the body of the mammal after the use of the dolabrus peptide preparation and the change process of the dolabrus peptide.
The method can successfully detect the trace amount of the dolabrus peptide in the sample, and realizes the detection of the content and the change process of the dolabrus peptide in the body after the dolabrus peptide preparation is used. The method provided by the invention is used as a trace amount dolabrin determination method, has the advantages of sensitivity and specificity superior to those of the existing detection technology, is short in detection time, can be used for detecting the trace amount dolabrin in a sample, and provides an effective and rapid detection means for detecting the trace amount dolabrin sample and determining the drug metabolism research of the dolabrin preparation in vivo. The method can be used for developing a dolabrus peptide preparation, the change of the concentration of the drug in a patient using the dolabrus peptide is clinically determined, and experimental basis can be provided for guiding the reasonable use of the clinical dolabrus peptide by continuously observing the dynamic change of the dolabrus peptide.
Drawings
FIG. 1 Standard Curve for the detection of dolastatin by ELISA method of example 4
Detailed Description
The invention will be better understood with reference to the following examples. However, it is to be understood that the following examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention in any way.
Example 1 antibody coating concentration and Duraluvian peptide dilution gradient selection
Four concentration-coated 96-well plates of 2 mug/ml, 4 mug/ml, 6 mug/ml and 8 mug/ml are selected, and the experimental result shows that 4 fitting curves are nearly coincident and have no obvious difference, so that the antibody plating concentration is 2 mug/ml which is the minimum concentration.
To obtain a complete sigmoidal curve, dolabrin was diluted 12 points starting from 10000ng/ml with a three-fold gradient. The experimental results show a complete curve and the curve has reached the upper plateau at 100ng/ml, thus the dilution method for the antibody was determined as follows: starting from 100ng/ml, three times the gradient dilution 8 points.
Example 2 determination of minimum dilution factor of serum
In order to determine the minimum dilution multiple of serum interference, 10, 20, 50, 100, 200 and 500 times and 6 serum dilution multiples are selected, 6 diluted sera are respectively used for diluting the dolabrin to obtain 6 fitting curves, the results show that platforms on the 6 curves are almost overlapped, but the lower platforms are different, the platforms under the 10 and 20 diluted serum curves are higher than other curves, which shows that the 10 and 20 diluted sera interfere with the experiment, and the minimum dilution multiple of the sera is determined to be 50 times.
Example 3 selection of quantitative Range and quality control Point
Based on the above results, a minimum serum dilution factor of 50 was selected for subsequent experiments, and the dulaglutide was diluted with pure serum starting at 5. mu.g/ml for 8 point triple gradient dilutions, and then diluted 50-fold with PBST and added to 96-well plates so that the final concentration added to the plates was 100ng/ml for 8 point triple gradient dilutions starting. The concentrations of 10 quality control sources are respectively as follows: 0.02, 0.025, 0.03, 0.04, 0.05, 0.06, 0.1, 0.3, 0.6, 0.75, 1, 1.5. mu.g/ml, and the results show that 0.02, 0.025, 0.03, 0.04, 0.05, 0.06, 0.1, 0.3, 0.6, 0.75, 1, 1.5. mu.g/ml all meet the quality control requirements.
Example 4 assay for detection of samples by ELISA
The main reagents of the experiment are as follows: monkey serum adsorbed Secondary antibody (Goat Anti-Human IgG, Monkey ads-HRP, 2049-05, southern Biotech) mouse Anti-Human GLP-1(05-0141B, Beijing Tiancheng Xinmai Biotechnology)
1. Antibody-coated 96-well plates: preparing 2 mu g/ml of mouse anti-human GLP-1 antibody and PBS, adding 100 mu l/well of the antibody into a 96-well plate with high adsorption, and performing dressing at 4 ℃ overnight;
2. blocking with BSA: PBST is used for preparing 1 percent BSA blocking solution, and the blocking solution is blocked for 1 hour at 37 ℃ in 200 mul/hole;
3. diluting Duraluvian peptide (3mg/ml) standard yeast and quality control, adding prepared standard yeast and quality control sample into plate at 100 μ l/hole, and applying at 37 deg.C for 2 hr;
4. dilution of the sample: the sample to be assayed is diluted to a quantitative range (dulaglutide immunized cynomolgus monkey, plasma thereof is extracted and processed to obtain an assay sample).
5. Secondary antibody: monkey serum-adsorbed secondary antibody was diluted at 1:15000, added to the plate at 100. mu.l/well, and incubated at 37 ℃ for 1 hour;
6. color development: preparing TMB developing solution, mixing solution A and B at equal ratio, adding into plate at a ratio of 100 μ l/hole, and applying at 37 deg.C for 20 min;
7. adding a stop solution: add stop solution (1M H3PO4) in 100. mu.l/well
8. OD value read by SpectraMax190 microplate reader
The detection results of the sample standard curve are shown in figure 1, the detection results of the quality control points are shown in the following table, and the curve fitting R2＝0.999。
Controls(ng/ml)
Claims (9)
1. A method for determining the blood concentration of dolabrus peptide is characterized in that an enzyme-linked immunosorbent assay (ELISA) technology is adopted, a GLP-1 capture antibody is used for capturing the dolabrus peptide, then an anti-human FC end secondary antibody with HRP is added, and TMB is added for color development and detection; the anti-human FC-terminal secondary antibody with the HRP is a monkey serum adsorption secondary antibody.
2. The method of claim 1, wherein said GLP-1 capturing antibody is a murine anti-human GLP-1 capturing antibody.
3. The method of claim 2, wherein the enzyme-linked immunosorbent assay (ELISA) is performed using an antibody plating concentration of 2 μ g/ml or more.
4. The method according to claim 3, characterized in that the enzyme-linked immunosorbent (ELISA) method is carried out with an antibody plating concentration of 2 μ g/ml.
5. The method according to claim 2, wherein the biological sample collected in the enzyme-linked immunosorbent assay (ELISA) is diluted with serum at a dilution rate of 50 or more.
6. The method according to claim 5, wherein the serum is diluted by a factor of 50.
7. The method of claim 2, wherein the monkey serum is adsorbed to the secondary antibody at a dilution of 1: 15000.
8. The method of claim 2, wherein the enzyme-linked immunosorbent assay (ELISA) method is performed using antibody plating concentration of 2. mu.g/ml, the biological sample is diluted 50-fold with serum, and the monkey serum-adsorbed secondary antibody is diluted 1: 15000.
9. The method according to any one of claims 1 to 8, for use in detecting alterations in the content of dolabrin and metabolic studies in a mammal after administration of the preparation of dolabrin.
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