CN112255415A - Detection method of FGF-21 concentration in cynomolgus monkey serum - Google Patents
Detection method of FGF-21 concentration in cynomolgus monkey serum Download PDFInfo
- Publication number
- CN112255415A CN112255415A CN202010948663.1A CN202010948663A CN112255415A CN 112255415 A CN112255415 A CN 112255415A CN 202010948663 A CN202010948663 A CN 202010948663A CN 112255415 A CN112255415 A CN 112255415A
- Authority
- CN
- China
- Prior art keywords
- fgf
- concentration
- serum
- cynomolgus monkey
- incubation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
- G01N2333/50—Fibroblast growth factors [FGF]
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Endocrinology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种食蟹猴血清中FGF‑21浓度的检测方法，包括以下步骤：(1)在预包被兔抗人FGF‑21多抗的96微孔板上加入待测血清样品进行孵育；(2)洗板再加入检测抗体进行孵育；(3)洗板后加入酶标抗体进行孵育；(4)洗板后加入HRP底物四甲基联苯胺进行避光孵育，然后加入终止液终止酶反应，测定450nm处的吸光度；(5)根据吸光度的值计算得到血清中的FGF‑21浓度。该检测方法具有较高的准确度和精确度，并且受到干扰小，可以对成纤维细胞生长因子21的作用机制进行更好地研究。The invention discloses a method for detecting the concentration of FGF-21 in cynomolgus monkey serum, comprising the following steps: (1) adding a serum sample to be tested on a 96-well microplate pre-coated with rabbit anti-human FGF-21 polyclonal antibodies to carry out (2) After washing the plate, add detection antibody for incubation; (3) After washing the plate, add enzyme-labeled antibody for incubation; (4) After washing the plate, add HRP substrate tetramethylbenzidine to incubate in the dark, and then add to stop The enzyme reaction was terminated in the liquid, and the absorbance at 450 nm was measured; (5) the FGF-21 concentration in the serum was calculated according to the absorbance value. The detection method has high accuracy and precision, and suffers little interference, so the mechanism of action of fibroblast growth factor 21 can be better studied.
Description
Technical Field
The invention belongs to the field of biochemical detection, and particularly relates to a method for detecting the concentration of FGF-21 in serum.
Background
Fibroblast growth factor 21 (FGF 21) is a new blood sugar regulating factor discovered in recent years, and the development of a biologically active method thereof has been difficult because of the absence of specific glucose-absorbing cells.
In the course of research on fibroblast growth factor 21, it is often necessary to know the exact amount of FGF21 in serum in order to further study its metabolic processes and effects on related conditions. At present, researchers often select cynomolgus monkeys for the pharmacokinetics of FGF21, but a method for rapidly determining the content of FGF21 in cynomolgus monkey serum is not reported in a patent.
Disclosure of Invention
The invention provides a method for detecting the concentration of FGF-21 in cynomolgus monkey serum, which has higher accuracy and sensitivity and small interference.
A method for detecting the concentration of FGF-21 in serum comprises the following steps:
(1) adding a serum sample to be tested on a 96 micro-porous plate pre-coated with a rabbit anti-human FGF-21 polyclonal antibody for incubation;
(2) adding a detection antibody into the washed plate for incubation;
(3) adding an enzyme-labeled antibody for incubation after washing the plate;
(4) after washing the plate, adding HRP substrate tetramethyl benzidine for dark incubation, then adding stop solution to terminate the enzyme reaction, and measuring the absorbance at 450 nm;
(5) the FGF-21 concentration in the serum was calculated from the values of absorbance.
The detection method has the following determination principle:
the kit is used for quantitatively detecting the concentration of the cynomolgus monkey serum FGF-21 based on a double-antibody sandwich ELISA method. A standard solution or a sample containing FGF-21 is added to a 96 micro-well plate pre-coated with a rabbit anti-human FGF-21 polyclonal antibody, so that the standard solution or the sample is combined with the solid-phase antibody. After washing away unbound material, a detection antibody (biotinylated human FGF-21 polyclonal antibody) is added. After washing away unbound antibody, horseradish peroxidase-labeled streptavidin (STP-HRP) was added. After washing, an HRP substrate Tetramethylbenzidine (TMB) was added to develop a color, and after the enzyme reaction was terminated, the absorbance (OD value) at 450nm was measured. The absorbance intensity is proportional to the concentration of FGF-21 contained in the sample.
Preferably, the serum to be detected is cynomolgus monkey serum.
Preferably, in the step (1), the dosage of the serum sample to be detected is 100 mu L/hole;
the incubation temperature is 25-30 ℃, and the incubation time is 1-1.5 h.
Preferably, in the step (2), the detection antibody is biotinylated human FGF-21 polyclonal antibody, and the dosage is 100 mu L/hole;
the incubation temperature is 25-30 ℃, and the incubation time is 1-1.5 h.
Preferably, in the step (3), the enzyme label is horseradish peroxidase labeled streptavidin, and the dosage is 100 mu L/hole;
the incubation temperature is 25-30 ℃, and the incubation time is 20-30 min.
Preferably, in the step (4), the amount of the HRP substrate tetramethylbenzidine is 100 μ L/hole;
the incubation temperature is 25-30 ℃, and the incubation time is 15-20 min.
Preferably, in the step (5), the value of the absorbance of the sample to be tested is compared with a prepared standard curve method, so as to obtain the concentration of FGF-21 in serum.
Preferably, the standard curve fitting method is as follows: preparing a standard curve by taking the concentration logarithm as an abscissa and the absorbance (OD value) as an ordinate, fitting by adopting a four-parameter Log-Logistic (4-PL) model in Origin 7.5 data statistical software, and forming the standard curve into a Sigmoidal curve
Preferably, in the step (5), the biological matrix used for preparing the standard curve is 10% cynomolgus monkey blank serum;
in the step (1), the serum to be detected is diluted by a certain multiple with blank serum and then diluted by 10 times with Assay buffer.
Preferably, in the step (1), the content of the serum sample to be tested is 31.25 pg/mL-4000 pg/mL.
Compared with the prior art, the invention has the beneficial effects that:
(1) the accuracy (RE%) of the back-calculated concentration of each concentration point of the average standard curve of FGF-21 is in the range of-2.0%, and the relative standard deviation (RSD%) is in the range of 1.1-6.0%;
(2) the detection method has a wider detection range, and the quantitative range is between 31.25pg/mL and 4000 pg/mL;
(3) the detection method has higher specificity, and the insulin-like growth factor 1(IGF-1), the Epidermal Growth Factor (EGF), the recombinant human interleukin-11 (rhIL-11), the recombinant human granulocyte colony stimulating factor (rhG-CSF), the recombinant human interferon gamma for injection (rhIFN-gamma), the recombinant human granulocyte macrophage stimulating factor for injection (rhGM-CSF) and the recombinant human II-type tumor necrosis factor receptor-antibody fusion protein (rhTNFR: Fc) do not interfere with the determination result of FGF-21;
(4) the detection method has higher selectivity, and other irrelevant substances in the serum matrix have less interference on the determination of the target protein during determination;
(5) the detection method has higher precision and accuracy, and meets the technical requirements on ligand binding analysis in the verification and guidance principle of the quantitative analysis method of the biological sample;
drawings
FIG. 1 is a comparison of the parallelism of standard curves for FGF-21 prepared with different matrices in example 1;
FIG. 2 is a graph showing the average standard curve of FGF-21 in example 2
FIG. 3 is a comparison of the parallelism of the FGF-21 standard curve of example 7 with the serial dilution preparation curve of a serum sample from a test animal;
Detailed Description
The invention is further described with reference to the following figures and specific embodiments.
The reagent Kit used in the present invention was developed by hong Kong university of Human FGF-21 Immunass-ay Kit (product No. 31180).
The general detection method is as follows:
(1) sample adding: respectively adding a standard solution, a quality control sample or a sample to be detected on a 96 micro-porous plate, incubating for 1h at room temperature (25 ℃) in 100 mu L/hole; (2) washing the plate: discarding liquid in the hole, washing the plate for 3 times, 1 min/time, and draining; (3) adding a detection antibody: 100 μ L/well, incubation for 1h at room temperature (25 ℃); (4) washing the plate: discarding liquid in the hole, washing the plate for 3 times, 1 min/time, and draining; (5) enzyme-labeled antibody (STP-HRP): 100 μ L/well, incubation at room temperature (25 ℃) for 20 min; (6) washing the plate: the liquid in the wells is discarded, the plate is washed 4 times for 1 min/time, and the plate is drained. (7) Color development: adding substrate solution TMB, incubating at 100 μ L/well and keeping away from light at room temperature (25 deg.C) for 15 min; (8) and (4) terminating: adding stop solution into the mixture, and adding 100 mu L of stop solution into each hole; (9) and (3) determination: reading the absorbance (OD value) of each hole on a microplate reader, and measuring the wavelength of 450 nm; (10) data processing: and (3) preparing a standard curve by taking the concentration logarithm as an abscissa and the absorbance (OD value) as an ordinate, and fitting by adopting a four-parameter Log-Logistic (4-PL) model in Origin 7.5 data statistical software, wherein the standard curve is a Sigmoidal curve.
Example 1 Minimum Required Dilution (MRD) test
Preparing an FGF-21 standard curve by respectively adopting three different biological matrixes, namely (1): assay buffer (sample dilution supplied by Kit); (2) 50% cynomolgus monkey blank Serum (Monky Serum. times.2): firstly, preparing a series of high-concentration (2-fold concentration) serum samples by using cynomolgus monkey blank serum, and then diluting the serum samples by using an Assay buffer multiple ratio for determination; (3) 10% cynomolgus monkey blank Serum (Monky Serum. times.10): serial high-concentration (10-fold concentration) serum samples were prepared from cynomolgus monkey blank serum, and then diluted 10-fold with Assay buffer for determination. The three standard curves of FGF-21 are prepared according to the method, so that the final concentration of the standard curves is 4000, 2000, 1000, 500, 250, 125, 62.5 and 31.25 pg/mL. Within the determined concentration range (31.25-4000 pg/mL), the drug concentration (C) and the absorbance (Abs) are fitted into a Sigmoidal curve by a four-parameter Log-Logistic (4-PL) model, and the weight is 1/Abs2. Each standard curve was fitted separately to investigate the effect of serum matrix on the accuracy of the sample measurements, the results are shown in table 1. The test result shows that under the conditions designed by the test, the correlation coefficient R of the FGF-21 three standard curves2Are all larger than 0.9980, and the accuracy (RE%) of the sample is between-7.4% and 5.9%. However, the measured OD value of the FGF-21 standard sample containing the cynomolgus monkey blank serum matrix with the same concentration is obviously lower than that of the FGF-21 sample prepared by the Assay buffer, which indicates that the cynomolgus monkey blank serum used as the matrix has certain influence on the sample measurement. Within the range of the concentration to be determined,the Log FGF-21 concentration (Log C) and Log absorbance (Log abs) for each of the three biomatrix formulations were linearly related by the equations LogAbs-2.593 +0.915 × LogC (R-0.9928), LogAbs-2.529 +0.840 × LogC (R-0.9993) and LogAbs-2.592 +0.882 × LogC (R-0.9985), respectively. The three standard curves are tested to be parallel to each other by standard curve parallelism determination method (slow-to-stay, benzyl like Lian, aging, etc.. pharmacological experimental methodology [ M)]Third edition, national health press, P217-218.), suggesting that the measured OD of FGF-21 samples formulated with 50% or 10% cynomolgus monkey blank serum is reduced in proportion to the OD of the same concentration drug formulated with Assay buffer, and can be used for sample determination (see table 2 and fig. 1 for results). As the OD value of the 50% cynomolgus monkey blank serum sample is about 0.29, the matrix interference is obvious, and the sensitivity of sample detection is considered, 10% cynomolgus monkey blank serum (the OD value is lower than 0.15) is selected as a biological matrix for carrying out methodology research and analysis of the drug concentration of a real serum sample in the test. In order to reduce the influence of the cynomolgus monkey serum matrix on the accuracy of FGF-21 determination to the maximum extent, all serum sample stock solutions are diluted by a certain multiple by using cynomolgus monkey blank serum and then diluted by 10 times by using an Assay buffer, so as to ensure that a blood sample to be analyzed contains 10% of the cynomolgus monkey serum matrix and is consistent with a biological matrix for preparing a standard curve.
TABLE 1 Minimum Required Dilution (MRD) assay for the determination of FGF-21 concentration by ELISA
TABLE 2 parallel analysis of FGF-21 standard curves prepared with different matrices
Remarking: b: a slope; r: a correlation coefficient; n: correcting the number of standard samples; sb: standard error of slope; a: a FGF-21 standard curve formulated for Assay buffer, with the equation LogAbs ═ -2.593+0.915 × LogC (R ═ 0.9928); b: is 50% of edible crabA standard curve of FGF-21 prepared from monkey blank serum, with the equation LogAbs ═ -2.529+0.840 × LogC (R ═ 0.9993); c: FGF-21 standard curve formulated for 10% cynomolgus monkey blank serum, equation LogAbs ═ -2.592+0.882 × LogC (R ═ 0.9985).
Example 2 Standard Curve and quantitative Range
Firstly, preparing a series of FGF-21 standard solutions with high concentration (10-fold concentration) by using cynomolgus monkey blank serum, and then respectively diluting the solutions by 10-fold by using an Assay buffer to ensure that the final concentration is as follows: 4000. absorbance was measured at 2000, 1000, 500, 250, 125, 62.5, 31.25pg/mL as "under 2.8" and averaged for each concentration duplicate. Within the determined concentration range (31.25-4000 pg/mL), the drug concentration (C) and the absorbance (Abs) are fitted into a Sigmoidal curve by a four-parameter Log-Logistic (4-PL) model, and the weight is 1/Abs2. Table 3 is the standard curve mean OD value for FGF-21(n ═ 9) according to the equation: abs 5.800-5.786/[1+ (C/3681.309)1.129]，R20.9998. Fig. 2 is a graph of the average standard for FGF-21 (n-9). The individual data for the 9 standard curves are shown in tables 4-7. The accuracy (RE%) of the back-calculated concentration of each concentration point of the average standard curve of FGF-21 is in the range of-2.0% to 2.0%, and the relative standard deviation (RSD%) is in the range of 1.1% to 6.0%. The quantitative range of the standard curve is a concentration range between the lower limit of quantitation and the upper limit of quantitation, namely 31.25 pg/mL-4000 pg/mL.
TABLE 3 mean standard curve for FGF-21 concentration determination by ELISA: (
n＝9)
TABLE 4 Individual data (OD) of 9 standard curves for the determination of FGF-21 concentration by ELISA450nm)
TABLE 5 Individual data (background, OD) of 9 standard curves for the determination of FGF-21 concentration by ELISA450nm-0)
TABLE 6 Individual data of 9 standard curves for the determination of FGF-21 concentration by ELISA (Back-calculated concentration)
TABLE 7 Individual data (relative error, RE%)
Example 3 specificity test
7 endogenous cytokines or related proteins such as insulin-like growth factor 1(IGF-1), Epidermal Growth Factor (EGF), recombinant human interleukin-11 (rhIL-11), recombinant human granulocyte colony stimulating factor (rhG-CSF), recombinant human interferon gamma for injection (rhIFN-gamma), recombinant human granulocyte macrophage stimulating factor for injection (rhGM-CSF), recombinant human type II tumor necrosis factor receptor-antibody fusion protein (rhTNFR: Fc) and the like are selected, and the specificity of the method is examined. The results of the measurement using the ELISA method established in this experiment are shown in Table 8, where the final concentration of each of the proteins at higher concentrations was 100ng/mL, which is equivalent to 25 times the highest concentration of the FGF-21 standard curve. The test result shows that the OD values of other 6 proteins except EGF are all lower than the OD value of the lowest quantitative limit (LLOQ) of the test, which indicates that the tested substance (EGF can generate certain interference) does not interfere the measurement result of the target protein (FGF-21). The EGF added in the test has higher concentration (100ng/mL), and can generate certain interference on the measurement; however, considering that the background of 10% cynomolgus monkey blank serum is low (OD value <0.150), it indicates that the healthy cynomolgus monkey blank serum contains low concentration of EGF, and thus has little influence on the accuracy of the assay. The above results show that the presence of the 6 interfering substances in the cynomolgus monkey blank serum did not affect the measurement results of FGF-21. In addition, the kit has no cross reaction with mouse FGF-21, human sheep adipocyte fatty acid binding protein gene (FABP4), human lipocalin 2(LCN2), human Adiponectin (Adiponectin) and the like.
TABLE 8 determination of FGF-21 specificity by ELISA (n ═ 2)
nd: not detected, the detected concentration was below LLOQ.
Example 4 Selectivity test
Blank sera of 11 cynomolgus monkeys (namely blank sera before administration of 11 cynomolgus monkeys randomly selected from 18 healthy cynomolgus monkeys of the FGF-21 administration group) were selected, FGF-21 quality control samples (LQC) with a concentration of 80pg/mL were prepared, and the ELISA method established in this experiment was used to evaluate the accuracy of the samples, and the results are shown in Table 9. The results show that FGF-21(80pg/mL) formulated with blank serum from 11 different individual cynomolgus monkeys with an accuracy (RE%) between-11.7% and 12.5%. The ELISA method in the test is used for quantitatively determining the concentration of FGF-21 in the serum of the cynomolgus monkey, is not interfered by other non-related substances in a serum matrix, and has good selectivity on the determination of the target protein (FGF-21).
TABLE 9 results of ELISA assay for FGF-21(LQC) selectivity in 11 individual cynomolgus monkey blank sera
Example 5 precision and accuracy
In the standard curve range, firstly, blank cynomolgus monkey serum is used for preparing a series of FGF-21 standard solutions with high concentration (10 times concentration), then the solutions are respectively diluted by 10 times by an Assay buffer, and 5 Quality Control (QC) samples in the standard curve range are prepared, wherein the samples are respectively the lowest quantitative limit (LLOQ), the low quantitative Limit (LQC), the medium quantitative limit (MQC), the high quantitative limit (HQC) and the highest quantitative limit (ULOQ), namely 31.25, 80, 800, 3200 and 4000 pg/mL. The precision and accuracy of the batch are determined by repeating the determination on the same enzyme label plate for 5 times for each quality control sample, and taking the average value of each QC sample in duplicate wells. Precision is the percentage ratio of standard deviation to mean of 5 determinations, expressed as the relative standard deviation RSD%; the accuracy is the average of 5 determinations as a percentage of the addition, i.e. the relative error between the measured and theoretical values, expressed in RE%. The precision and accuracy between batches are measured by respectively measuring the OD value of each quality control sample on different enzyme label plates by 6 analysis batches, measuring double-hole (taking the average value) of each QC sample each time, and examining the precision (RSD%) and accuracy (RE%) between batches. The total error of the method (TE%) is the sum of the absolute value of the relative deviation and the relative standard deviation. The results are shown in tables 10 to 12. The results show that: the RSD% of FGF-21 in batches and among batches within the determined concentration range (31.25-4000 pg/mL) is respectively between 3.4% -17.1% and 4.2% -7.1%; the RE% in and between batches is between-11.9% and-0.7% and-7.0% and-0.7%, respectively. The TE% in and between batches was between 8.8% and 29.0% and 7.8% and 11.2%, respectively. In summary, the precision and accuracy of FGF-21QC samples are within 20%, the total error of the method is within 30%, and the method meets the technical requirements on ligand binding analysis in the validation guidance of quantitative analysis methods of biological samples (Guideline on biochemical analysis. EMEA, 2011.6.).
TABLE 10 precision and accuracy of the ELISA assay for FGF-21 concentration
TABLE 11 in-batch precision and accuracy of the ELISA determination of FGF-21 concentration
TABLE 12 batch-to-batch precision and accuracy of the ELISA determination of FGF-21 concentration
Example 6 dilution Linear test
Since the drug concentration in serum at some time points in this test is outside the quantitative range of the standard curve, i.e. the range of the standard curve does not cover the expected sample concentration, dilution linearity examination of the method was performed using QC samples to assess whether the sample was linear after dilution, whether a "front band" or "hook" effect existed, and the effect of sample dilution on the accuracy of the assay results. QC samples containing FGF-21(128ng/mL) at high concentrations (above the upper limit of quantitation) were prepared using cynomolgus monkey blank serum. And sequentially diluting the blank serum of the cynomolgus monkey by multiple ratios, and diluting each dilution before measurement by 10 times by using an Assay Buffer so as to ensure that the final dilutions of the FGF-21 are 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280 respectively. The assay was repeated 5 times for each dilution and the precision and accuracy of the back-calculated concentration was calculated. The back calculation concentration is calculated by adopting the following formula: the calculated concentration is the measured concentration multiplied by the dilution factor. The test result shows that: (1) when the dilution is not more than 1:1280 (1: 40-1: 1280), the average accuracy RE% of the calculated concentration of the sample measured after 10% of cynomolgus monkey blank serum is diluted by FGF-21 is between-3.8% and 2.5%, and the precision RSD% of the calculated concentration for 5 times is between 1.4% and 5.4% respectively. The results are shown in tables 13 and 14. The above results suggest that within the maximum dilution range set by the present experiment, FGF-21 is diluted with 10% cynomolgus monkey blank serum, and then the accuracy of the test results is not greatly affected by the determination, i.e. the high-concentration QC sample is diluted to have a calculated concentration within ± 20% of the expected concentration, and the precision of the calculated concentration of all the determined samples is not more than 20%, which meets the technical requirements of ligand binding analysis in the validation guidelines of the quantitative analysis method of biological samples (guidelines, emea, 2011.6.).
TABLE 13 FGF-21 dilution assay by ELISA (n ═ 5)
TABLE 14 dilution line assay for the ELISA method for FGF-21 concentration (individual data)
Example 7 parallelism test
Selecting a high concentration serum sample (C) from the test animalmax) After serial dilution in serum matrix, a regression curve established by OD values measured by 10-fold dilution of Assay buffer is compared with an FGF-21 standard curve, and a parallelism test of two straight lines is carried out to investigate the influence of possible matrix effect and the like on the measurement accuracy of the sample. 3 subjects selected from the 300. mu.g/kg dose groupC after administration of the substancemaxSerum samples, No.3(2h), No.8(3h) and No.16(2h), were mixed in equal amounts and diluted 30-fold with cynomolgus monkey blank serum according to the measured concentration, and 10-fold with Assay buffer at dilutions of 1:300, 1:600, 1:1200 and 1:2400 respectively, and the measurements were repeated 5 times at each dilution. And performing linear fitting on the measured concentration mean logarithm (Log C) and absorbance mean logarithm (Log Abs) to obtain a serial diluted serum sample curve. And compared to a standard curve established with the analytical batch calibration standards. Testing the parallelism of the serially diluted serum sample curves and the standard curve prepared from calibration standard by "Standard Curve parallelism assay" (Xu Tertiary clouds, benzyl as curtain, aging, etc.. pharmacological test methodology [ M]Third edition, people health Press, P217-218). The results are shown in tables 15 to 18 and FIG. 3. The results show that the standard curve of FGF-21 and the curves of serially diluted serum samples of test animals are parallel to each other. The regression equations established for the FGF-21 standard curve and the serially diluted serum samples were: log Abs ═ 3.087+1.014 × Log C, R ═ 0.9963(n ═ 8); log Abs ═ 3.249+1.089 × Log C, R ═ 0.9993(n ═ 4). The relative standard deviation (RSD%) of FGF-21 after dilution of the serum of the tested animal is between 0.8% and 9.4%.
TABLE 15 FGF-21 parallelism assay by ELISA: (
n＝5)
TABLE 16 parallel assay for FGF-21 by ELISA (Individual data)
TABLE 17 comparison of OD values of curves prepared from FGF-21 standard and serially diluted serum samples
TABLE 18 comparison of the parallelism of the Standard Curve prepared from FGF-21 Standard sample and serially diluted serum samples
Note: b: a slope; r: a correlation coefficient; n: correcting the number of standard samples; sb: standard error of slope; FGF-21 Standard Curve equation: log Abs ═ 3.087+1.014 × Log C, R ═ 0.9963(n ═ 8); standard curve equation for serial dilutions of serum samples: log Abs ═ 3.249+1.089 × Log C, R ═ 0.9993(n ═ 4).
Example 8 stability test
(1) Short-term storage stability of samples under different conditions: low and high concentration QC samples, namely 0.8 and 32ng/mL, were prepared from cynomolgus monkey blank serum, stored at-20 ℃ for 1 day, 4 ℃ for 1 day, and room temperature (22-26 ℃) for 3h, and subjected to three C freeze-thaw cycles [ d1 cryopreservation (-C20 ℃) → d5 thawing (room temperature), frozen C cryopreservation (-20 ℃) → d17 thawing (room temperature), frozen storage (-20 ℃)2 → d8 post-thaw sample treatment, assay 3 ] and the like under 4 different conditions. And (3) determining the instant day, diluting the corresponding concentrations by 10 times respectively by adopting an Assay buffer, preparing quality control samples with the concentrations of 80pg/mL (LQC) and 3200pg/mL (HQC), comparing the quality control samples with the quality control samples prepared freshly on the day, and repeating the determination for 5 times on each sample, wherein the accuracy (RE%) of the determined QC sample is used as an evaluation index.
(2) Long-term storage stability of the sample: the low-concentration and high-concentration QC samples of FGF-21, namely 0.8 and 32ng/mL, are prepared from the cynomolgus monkey blank serum and are stored for 60 days at the temperature of minus 20 ℃. And (3) measuring the instantaneous day, diluting the corresponding concentrations by 10 times respectively by adopting an Assay buffer, preparing quality control samples with the concentrations of 80pg/mL (LQC) and 3200pg/mL (HQC), measuring each sample for 5 times, and taking the accuracy (RE%) of the measured QC sample as an evaluation index.
The results show that FGF-21 is stable when stored at-20 ℃ for 1 or 60 days, at 4 ℃ for 1 day, at room temperature for 3h or after three freeze-thaw cycles, and that the RE% of the low-concentration QC sample (80pg/mL) is between-2.2% and 8.1% and the RE% of the high-concentration QC sample (3200pg/mL) is between-10.3% and-2.4%. The results are shown in tables 19 to 21. It is suggested that the measured concentration of the low and high concentration quality control samples is within + -20% of the formulated concentration, and meets the technical requirements of ligand binding analysis in the validation guidelines of the quantitative analysis methods of biological samples (Guideline on bioanalytical method evaluation. EMEA, 2011.6.).
TABLE 19 stability assay for FGF-21 samples by ELISA method under different storage conditions (
n＝5)
TABLE 20 stability assay for FGF-21LQC (80pg/mL) samples at different storage conditions by ELISA
TABLE 21 stability assay of FGF-21HQC (3200pg/mL) samples at different storage conditions by ELISA
The results show that the tests such as the Minimum Required Dilution (MRD), the standard curve and the quantitative range, the specificity, the selectivity, the precision and the accuracy, the dilution linearity, the parallelism, the stability and the like all accord with the technical requirements of the ligand binding analysis in the verification guiding principle of the quantitative analysis method of the biological sample through the methodological verification.
Claims (10)
1. A method for detecting the concentration of FGF-21 in cynomolgus monkey serum is characterized by comprising the following steps:
(1) adding a serum sample to be tested on a 96 micro-porous plate pre-coated with a rabbit anti-human FGF-21 polyclonal antibody for incubation;
(2) adding a detection antibody into the washed plate for incubation;
(3) adding an enzyme-labeled antibody for incubation after washing the plate;
(4) after washing the plate, adding HRP substrate tetramethyl benzidine for dark incubation, then adding stop solution to terminate the enzyme reaction, and measuring the absorbance at 450 nm;
(5) the FGF-21 concentration in the serum was calculated from the values of absorbance.
2. The method of claim 1, wherein the serum to be tested is cynomolgus monkey serum.
3. The method for detecting the concentration of FGF-21 in cynomolgus monkey serum according to claim 1, wherein in the step (1), the amount of the serum sample to be tested is 100. mu.L/well;
the incubation temperature is 25-30 ℃, and the incubation time is 1-1.5 h.
4. The method for detecting the concentration of FGF-21 in cynomolgus monkey serum according to claim 1, wherein in the step (2), the detection antibody is biotinylated human FGF-21 polyclonal antibody, and the dosage is 100 μ L/well;
the incubation temperature is 25-30 ℃, and the incubation time is 1-1.5 h.
5. The method for detecting the concentration of FGF-21 in cynomolgus monkey serum according to claim 1, wherein in the step (3), the enzyme label is horseradish peroxidase labeled streptavidin, and the dosage is 100 μ L/hole;
the incubation temperature is 25-30 ℃, and the incubation time is 20-30 min.
6. The method for detecting the concentration of FGF-21 in cynomolgus monkey serum according to claim 1, wherein in the step (4), the amount of the HRP substrate tetramethylbenzidine is 100 μ L/well;
the incubation temperature is 25-30 ℃, and the incubation time is 15-20 min.
7. The method for detecting the concentration of FGF-21 in cynomolgus monkey serum according to claim 1, wherein in the step (5), the concentration of FGF-21 in the serum is obtained by comparing the absorbance value of the sample to be tested with a prepared standard curve method.
8. The method for detecting the concentration of FGF-21 in cynomolgus monkey serum according to claim 7, wherein the standard curve fitting method comprises the following steps: and (3) preparing a standard curve by taking the concentration logarithm as an abscissa and the absorbance (OD value) as an ordinate, and fitting by adopting a four-parameter Log-Logistic (4-PL) model in Origin 7.5 data statistical software, wherein the standard curve is a Sigmoidal curve.
9. The method for detecting the concentration of FGF-21 in cynomolgus monkey serum according to claim 7, wherein in the step (5), the biological matrix used for preparing the standard curve is 10% cynomolgus monkey blank serum;
in the step (1), the serum to be detected is diluted by a certain multiple with blank serum and then diluted by 10 times with Assay buffer.
10. The method for detecting the concentration of FGF-21 in cynomolgus monkey serum according to claim 1, wherein in step (1), the content to be detected of the serum sample to be detected is 31.25 pg/mL-4000 pg/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010948663.1A CN112255415A (en) | 2020-09-10 | 2020-09-10 | Detection method of FGF-21 concentration in cynomolgus monkey serum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010948663.1A CN112255415A (en) | 2020-09-10 | 2020-09-10 | Detection method of FGF-21 concentration in cynomolgus monkey serum |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112255415A true CN112255415A (en) | 2021-01-22 |
Family
ID=74232203
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010948663.1A Pending CN112255415A (en) | 2020-09-10 | 2020-09-10 | Detection method of FGF-21 concentration in cynomolgus monkey serum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112255415A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113092392A (en) * | 2021-03-05 | 2021-07-09 | 苏州西山中科药物研究开发有限公司 | Universal method for detecting total concentration of monoclonal antibody drug targeting soluble protein in monkey serum |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108350072A (en) * | 2015-08-03 | 2018-07-31 | 诺华股份有限公司 | The method for treating FGF21 associated diseases |
| WO2019195514A1 (en) * | 2018-04-04 | 2019-10-10 | Genentech, Inc. | Methods for detecting and quantifying fgf21 |
| CN111474371A (en) * | 2020-03-30 | 2020-07-31 | 广州瑞博奥生物科技有限公司 | A marker combination for evaluating the quality of isolated blood samples and its application |
-
2020
- 2020-09-10 CN CN202010948663.1A patent/CN112255415A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108350072A (en) * | 2015-08-03 | 2018-07-31 | 诺华股份有限公司 | The method for treating FGF21 associated diseases |
| WO2019195514A1 (en) * | 2018-04-04 | 2019-10-10 | Genentech, Inc. | Methods for detecting and quantifying fgf21 |
| CN111474371A (en) * | 2020-03-30 | 2020-07-31 | 广州瑞博奥生物科技有限公司 | A marker combination for evaluating the quality of isolated blood samples and its application |
Non-Patent Citations (1)
| Title |
|---|
| 胡晓丞等: "注射用重组人成纤维细胞生长因子-21 食蟹猴药动学研究", 《药物评价研究》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113092392A (en) * | 2021-03-05 | 2021-07-09 | 苏州西山中科药物研究开发有限公司 | Universal method for detecting total concentration of monoclonal antibody drug targeting soluble protein in monkey serum |
| CN113092392B (en) * | 2021-03-05 | 2022-11-08 | 苏州西山中科药物研究开发有限公司 | Universal method for detecting total concentration of monoclonal antibody drug targeting soluble protein in monkey serum |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Platat et al. | Relationships of physical activity with metabolic syndrome features and low-grade inflammation in adolescents | |
| Takahashi et al. | Comparison of glycated albumin (GA) and glycated hemoglobin (HbA1c) in type 2 diabetic patients: usefulness of GA for evaluation of short-term changes in glycemic control | |
| Behiry et al. | Evaluation of TG-HDL ratio instead of HOMA ratio as insulin resistance marker in overweight and children with obesity | |
| Rubin et al. | The association between insulin resistance and cytokines in adolescents: the role of weight status and exercise | |
| CN110488025A (en) | A kind of chemiluminescence quantitative detection excrement calprotectin and its detection method and its intestinal health detection purposes | |
| Oncul et al. | Polycystic ovary syndrome and endothelial dysfunction: A potential role for soluble lectin-like oxidized low density lipoprotein receptor-1 | |
| Pedersen et al. | Body size, but neither age nor asymptomatic mitral regurgitation, influences plasma concentrations of dimethylarginines in dogs | |
| Oflazoglu et al. | The relationship between sarcopenia detected in newly diagnosed colorectal cancer patients and FGF21, irisin and CRP levels | |
| CN112255415A (en) | Detection method of FGF-21 concentration in cynomolgus monkey serum | |
| CN108362892B (en) | A kind of procalcitonin colloidal gold immunoturbidimetric detection reagent | |
| Li et al. | Concentration-dependent differences in urinary iodine measurements between inductively coupled plasma mass spectrometry and the Sandell-Kolthoff method | |
| CN112014572A (en) | Preparation method and application of latex particles for detecting KL-6 | |
| Manolov et al. | Serum hepcidin levels in Bulgarian population | |
| CN112255419B (en) | Midkine bioanalysis method and detection kit | |
| CN112424604A (en) | Method and kit for detecting 11-dehydro-thromboxane B2 | |
| Corey | The anion gap (AG): studies in the nephrotic syndrome and diabetic ketoacidosis (DKA) | |
| CN114527190A (en) | Glycosylated hemoglobin detection kit and detection method thereof | |
| CN114878825A (en) | C peptide determination kit and method for detecting content of C peptide in human serum | |
| CN117607459B (en) | Neutrophil gelatinase-associated lipocalin assay kit, and preparation method and application thereof | |
| CN109613232A (en) | A kind of detection kit and detection method of complete homogeneous determination adiponectin | |
| CN109425736B (en) | Method and kit for detecting blood concentration of PD-1antibody | |
| Li et al. | A simple dilute and shoot approach incorporated with pentafluorophenyl (PFP) column based LC-MS/MS assay for the simultaneous determination of trimethylamine N-oxide and trimethylamine in spot urine samples with high throughput | |
| WO2020167411A1 (en) | Calibrators and controls for the determination of percent glycated hemoglobin in a patient's liquid test sample | |
| CN109254145A (en) | For improving the dilution of matrix effect between fresh serum and third party's Quality Control | |
| Gonzalez-Rivera et al. | Matrix effect evaluation and validation of the 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation scavenging assay, as well as its application using a tejate, an ancient beverage in Mexico |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |