CN110736843A - quantitative detection kit for insulin-like growth factor binding protein 3 - Google Patents
quantitative detection kit for insulin-like growth factor binding protein 3 Download PDFInfo
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- CN110736843A CN110736843A CN201911182975.XA CN201911182975A CN110736843A CN 110736843 A CN110736843 A CN 110736843A CN 201911182975 A CN201911182975 A CN 201911182975A CN 110736843 A CN110736843 A CN 110736843A
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
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- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- 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)
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- 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
- G01N2333/575—Hormones
- G01N2333/65—Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2
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Abstract
The invention relates to the technical field of medical detection, in particular to a quantitative detection kit for insulin-like growth factor binding protein 3, which comprises magnetic particles coated with IGFBP-3 monoclonal antibodies, enzyme-labeled IGFBP-3 monoclonal antibodies and sample diluent, wherein the sample diluent is any of hydrochloric acid, citric acid, malic acid, phosphoric acid and sulfosalicylic acid.
Description
Technical Field
The invention relates to the technical field of medical detection, in particular to a quantitative detection kit for insulin-like growth factor binding protein 3.
Background
Insulin-like growth factor binding proteins (IGFBPs) are regulatory proteins capable of binding to insulin-like growth factors (IGFs), and mainly regulate the binding capacity of IGFs to their receptors, and influence the signal intensity in signal transduction pathways downstream of the insulin-like growth factor receptors (IGFR), thereby regulating the growth and proliferation of target cells. More and more researches show that IGFBP-3 can directly promote apoptosis and inhibit proliferation of various cells including breast cancer cells, lung cancer cells, colorectal cancer cells and the like
At present, the detection of insulin-like growth factor binding protein 3 is mostly carried out by enzyme-linked immunosorbent assay in the early stage, wherein antibodies are bound to the surface of a solid phase carrier, and other antibodies are labeled by enzyme, during the determination, the solid phase antibody, the antigen to be detected and the enzyme-labeled antibody are combined to form a compound with a sandwich structure, a chromogenic substrate under the action of enzyme is added, the absorbance is determined under the OD value determined by , the absorbance is in direct proportion to the content of the antigen to be detected, and the content of the antigen to be detected is calculated by using a standard curve.
Disclosure of Invention
In view of the above, the invention aims to provide a quantitative detection kit for insulin-like growth factor binding protein 3, which has high sensitivity, high precision and good accuracy.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a quantitative detection kit for insulin-like growth factor binding protein 3, which comprises magnetic particles coated with an IGFBP-3 monoclonal antibody, an enzyme-labeled IGFBP-3 monoclonal antibody and a sample diluent;
the sample diluent is of hydrochloric acid, citric acid, malic acid, phosphoric acid and sulfosalicylic acid.
The invention adopts the principle of a double-antibody sandwich method, namely, a magnetic particle coated antibody, an antigen to be detected and an enzyme-labeled antibody are combined to form a compound with a sandwich structure, and an enzymatic chemiluminescence substrate is added. The substrate is catalytically cracked under the action of enzyme to form an unstable excited state intermediate, when the excited state intermediate reaches the ground state, photons are emitted to form a luminescence reaction, photon energy is recorded through a photon reading system, the light energy intensity is converted into the concentration of the antigen to be detected on a standard curve through a computer processing system, and the result is reported.
The invention adopts specific sample diluent, thus improving the clinical compliance rate of the kit. Under the action of the sample diluent, the IGF-1, ALS and IGFBP-3 in the sample are dissociated, so that the binding capacity of an IGFBP-3 antibody and an antigen is effectively improved, and the clinical compliance rate is improved.
in some embodiments, the sample diluent is aqueous citric acid hydrate in some embodiments, the sample diluent is aqueous citric acid hydrate with a mass fraction of 1%.
in some embodiments, the magnetic particles have a particle size of 1.00-3 μm and a magnetic content of 40%.
in some embodiments, the enzyme-labeled IGFBP-3 monoclonal antibody is a horseradish peroxidase-labeled IGFBP-3 monoclonal antibody.
in some embodiments, the horseradish peroxidase-labeled IGFBP-3 monoclonal antibody is prepared from horseradish peroxidase, IGFBP-3 monoclonal antibody and an enzyme diluent, wherein the enzyme diluent consists of a buffer solution, a protective protein and a preservative, bovine serum albumin, and the pH of the enzyme diluent is 6.0-8.0, wherein:
the buffer solution is any of Bis-Tris, Tris-NaCl and PBS.
The protective protein is or two of bovine serum albumin, bovine serum and casein.
The antiseptic is any or more of ProClin300, BRONIDOX (5-bromo-5 nitro-1, 3-dioxane), MIT (2-methyl-4-isothiazoline-3-one hydrochloride), and NaN3 (sodium azide).
in some embodiments, the kit further comprises a chemiluminescent substrate which is luminol or isoluminol.
in some embodiments, the kit of the invention further comprises an IGFBP-3 antigen calibrator made from IGFBP-3 antigen and a calibrator diluent that is Tris-NaCl buffer containing 2% bovine serum albumin.
And , the concentration of the IGFBP-3 antigen calibrator is 0-16 mu g/mL.
The kit for quantitatively detecting the insulin-like growth factor binding protein 3 comprises magnetic particles coated with IGFBP-3 monoclonal antibodies and enzyme-labeled IGFBP-3 monoclonal antibodies, wherein a sample diluent is of hydrochloric acid, citric acid, malic acid, phosphoric acid and sulfosalicylic acid.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a diagram showing the correlation analysis between the kit of the present invention and the Western sub-kit;
FIG. 2 shows a graph of the correlation analysis between the control kit 1 and the Western sub-kit.
Detailed Description
The present invention discloses quantitative detection kits for insulin-like growth factor binding protein 3, which can be adapted by persons skilled in the art with reference to the disclosure herein, and which are particularly adapted to process parameters, it is noted that all such similar substitutes and modifications which are obvious to those skilled in the art are deemed to be included within the invention the methods and uses of the invention have been described by way of example only, and it will be apparent to persons skilled in the art that modifications or appropriate variations and combinations of the methods and uses described herein can be made to implement and use the techniques of the invention without departing from the spirit, scope, or concept of the invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention.
In the detection test of the quantitative detection kits for the insulin-like growth factor binding protein 3, the kit disclosed by the invention is matched with a full-automatic detection instrument and matched consumables (cleaning solution and chemiluminescent substrates) of AnTu bioengineering GmbH for detection.
The invention is further illustrated at with reference to the following examples:
EXAMPLE 1 preparation of the kit of the invention
1. Preparation of magnetic microparticles coated with IGFBP-3 monoclonal antibody
Fully and uniformly mixing magnetic particle stock solution with the particle size of 1.00-3 mu m and the magnetic content of 40%, taking out 30 mu l, adding 300 mu l of phosphate buffer solution for washing for 5 times, washing for 5min each time, and adsorbing and fixing the magnetic particles by using a magnet during washing to discard supernatant; after washing, adding 100 mu l of EDC (carbodiimide) and NHS (succinimide ester) activators dissolved in sodium acetate buffer solution, wherein the concentration of the activators is 20mg/ml, uniformly mixing, vibrating and activating for 1 h; and after activation, adding 300 mu l of sodium acetate buffer solution for washing for 2 times, washing for 5min each time, then adding 0.3 mu g/part of insulin-like growth factor binding protein 3 antibody, uniformly mixing, shaking and coating for 2h, after coating, adding 30 mu l of phosphate buffer solution containing 2% bovine serum albumin, sealing for 4 times, finally using the phosphate buffer solution containing 2% bovine serum albumin to fix the volume to 3ml, and storing at 2-8 ℃.
2. Preparation of enzyme-labeled IGFBP-3 monoclonal antibody
Adding 2% bovine serum albumin into the prepared Tris-NaCl buffer solution, mixing to obtain an enzyme diluent, adding an HRP (horse radish peroxidase) -labeled antibody according to the proportion of 1:5000, uniformly mixing, and storing at 2-8 ℃.
3. Preparation of IGFBP-3 antigen calibrator
Adding 2% bovine serum albumin into the prepared Tris-NaCl buffer solution, uniformly mixing to obtain a calibrator diluent, diluting the IGFBP-3 antigen into 6 gradients of 0 mu g/mL, 1 mu g/mL, 2 mu g/mL, 4 mu g/mL, 8 mu g/mL and 16 mu g/mL by using the calibrator diluent, subpackaging into 1 mL/bottle, and storing at 2-8 ℃.
4. Preparation of sample dilutions
Adding hydrated citric acid into purified water according to the proportion of 1%, mixing to obtain a sample diluent, and storing at 2-8 ℃.
Example 2 detection method of the kit of the present invention
1. Collecting whole blood sample by correct medical technique, centrifuging (rotation speed: 3500r/min, time: 10min), and extracting serum/plasma for detection.
2. A calibrator and a sample are sequentially added into a reaction vessel (hereinafter referred to as a "well"), 20 mul of calibrator/sample +380 mul of sample diluent are taken, mixed evenly, and 10 mul/well is added for detection.
3. mu.L of magnetic particle suspension was added to each well.
4. 100 μ L of enzyme conjugate was added to each well.
5. After mixing, the mixture was incubated at 37 ℃ for 15 minutes.
6. Washing with the cleaning solution was performed 5 times.
7. Adding 100 mu L of chemiluminescence substrate into each hole, and detecting the luminescence value after uniformly mixing.
8. And (4) calculating a result: the kit recommends adopting a four-parameter fitting mode, taking the concentration value of the calibrator as an x axis, and taking the log value of the luminous value of the calibrator as a y axis to establish a calibration curve. And (4) calculating a corresponding concentration value according to the luminous value of the sample to be detected. The automatic operating system of the instrument can automatically calculate the test result of the sample through the stored calibration curve and the luminous value obtained by the test of the sample.
Example 3 Performance testing of the kits of the invention
1. Assay sensitivity detection
And (3) carrying out analysis sensitivity assessment according to an EP17A protocol for determining detection limit and quantitative limit, preparing 5 clinical samples with a value close to 0, repeating each sample for 3 times for 4 days in total to obtain 60 non-negative data results, if the number of the samples is less than 60, carrying out experiments according to requirements and complementing 60, and calculating the analysis sensitivity.
TABLE 1 detection of sensitivity of the kit of the invention
As can be seen from the results in Table 1, the kit of the invention has the analysis sensitivity of 0.02 mug/ml, which is much lower than that of the existing product of 0.1 mug/ml, and has higher analysis sensitivity.
2. Detection of precision
The precision evaluation was carried out according to "precision evaluation of EP05A 2-quantitative determination method", wherein two batches of reagents were used together with high/medium/low value quality control materials, and the variation of the measured concentration was calculated 20 times each, and the measurement results are shown in Table 2.
TABLE 2 results of the detection of the precision of the kit of the present invention
Table 2 the data results show that: the kit has the variation coefficient of less than 5 percent and good precision.
3. Accuracy detection
Accuracy was assessed by measuring standard substances according to CLSI Standard EP 15-A2. The NIBSC standard 93/560 was diluted in a gradient to 10. mu.g/ml, 5. mu.g/ml, 2. mu.g/ml, 1. mu.g/ml/0.5. mu.g/ml, and the two batches were subjected to duplicate well assay with calculated deviations, the assay results are shown in Table 3.
TABLE 3 accuracy test results of the kit of the present invention
The results in Table 3 show that the deviation between the detection result and the standard substance of the kit is less than 5 percent, and the accuracy is high.
4. Correlation analysis
At present, the kit for full-automatic detection of insulin-like growth factor binding protein 3 at home and abroad only contains Western seeds, the market acceptance is high, a comparison kit 1 is set, a sample diluent is physiological saline containing 0.1% of ProClin300, and other components in the kit are the same as those in the kit of the embodiment 1.
The kit, the control kit 1 and the western sub-kit are adopted to simultaneously detect 50 samples of different age groups, the age is 8 months to 20 years, and 25 samples of men and women are respectively detected, the kit and the control kit 1 are respectively subjected to correlation analysis with the detection results of the western sub-kit, and the results are shown in tables 4 to 5 and fig. 1 to 2.
TABLE 4 correlation analysis results of the kit of the present invention and the Western sub-kit
TABLE 5 correlation analysis results of control kit 1 and Western sub-kits
As can be seen from tables 4-5 and FIGS. 1-2, the clinical compliance rate and clinical relevance of the kit are consistent with the height of the west seed, the compliance rate is 100%, the compliance rate of the control kit 1 and the west seed adopting other sample diluents is 88, and the results show that the clinical relevance of the kit is remarkably improved by adopting the specific sample diluent, and the clinical compliance rate is improved from 88% to 100%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
- The quantitative detection kit for insulin-like growth factor binding protein 3 is characterized by comprising magnetic particles coated with an IGFBP-3 monoclonal antibody, an enzyme-labeled IGFBP-3 monoclonal antibody and a sample diluent;the sample diluent is of hydrochloric acid, citric acid, malic acid, phosphoric acid and sulfosalicylic acid.
- 2. The quantitative determination kit according to claim 1, wherein the magnetic fine particles have a particle diameter of 1.00 to 3 μm and a magnetic content of 40%.
- 3. The quantitative detection kit according to claim 1, wherein the enzyme-labeled IGFBP-3 monoclonal antibody is a horseradish peroxidase-labeled IGFBP-3 monoclonal antibody.
- 4. The quantitative detection kit according to claim 3, wherein the horseradish peroxidase-labeled IGFBP-3 monoclonal antibody is prepared from horseradish peroxidase, an IGFBP-3 monoclonal antibody and an enzyme diluent, the enzyme diluent is composed of a buffer solution, a protective protein and a preservative, bovine serum albumin, and the pH of the enzyme diluent is 6.0-8.0.
- 5. The kit of claim 4, wherein the buffer is any of Bis-Tris, Tris-NaCl and PBS.
- 6. The kit according to claim 4, wherein the protective protein is or two of bovine serum albumin, bovine serum and casein.
- 7. The kit according to claim 4, wherein the preservative is any or more of ProClin300, BRONIDOX (5-bromo-5 nitro-1, 3-dioxane), MIT (2-methyl-4-isothiazolin-3-one hydrochloride), NaN3 (sodium azide).
- 8. The quantitative detection kit according to claim 1, further comprising a chemiluminescent substrate, wherein the chemiluminescent substrate is luminol or isoluminol.
- 9. The quantitative detection kit according to claim 1, further comprising an IGFBP-3 antigen calibrator, wherein the IGFBP-3 antigen calibrator is prepared from an IGFBP-3 antigen and a calibrator diluent, and the calibrator diluent is a Tris-NaCl buffer containing 2% bovine serum albumin.
- 10. The quantitative detection kit according to claim 9, wherein the concentration of the IGFBP-3 antigen calibrator is 0-16 μ g/mL.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111398598A (en) * | 2020-03-16 | 2020-07-10 | 迪瑞医疗科技股份有限公司 | Chemiluminescence detection kit for insulin-like growth factor binding protein-3 and preparation method thereof |
| CN114544978A (en) * | 2022-03-04 | 2022-05-27 | 美康生物科技股份有限公司 | IGF-1 detection kit, preparation method and detection method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104697829A (en) * | 2015-02-10 | 2015-06-10 | 深圳市新产业生物医学工程股份有限公司 | Acid treatment agent for IGF-I chemiluminesent immunoassay, sample pretreatment method, kit and detection method |
| CN107991492A (en) * | 2017-11-17 | 2018-05-04 | 广州赛莱拉干细胞科技股份有限公司 | A kind of detection method of the immue quantitative detection reagent box of insulin-like growth factor binding protein-3 and its non-diagnostic purpose |
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- 2019-11-27 CN CN201911182975.XA patent/CN110736843A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104697829A (en) * | 2015-02-10 | 2015-06-10 | 深圳市新产业生物医学工程股份有限公司 | Acid treatment agent for IGF-I chemiluminesent immunoassay, sample pretreatment method, kit and detection method |
| CN107991492A (en) * | 2017-11-17 | 2018-05-04 | 广州赛莱拉干细胞科技股份有限公司 | A kind of detection method of the immue quantitative detection reagent box of insulin-like growth factor binding protein-3 and its non-diagnostic purpose |
Non-Patent Citations (1)
| Title |
|---|
| MYBIOSOURCE: "Human IGFBP-3 (Insulin Like Growth Factor Binding Protein 3) CLIA Kit", 《MYBIOSOURCE》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111398598A (en) * | 2020-03-16 | 2020-07-10 | 迪瑞医疗科技股份有限公司 | Chemiluminescence detection kit for insulin-like growth factor binding protein-3 and preparation method thereof |
| CN114544978A (en) * | 2022-03-04 | 2022-05-27 | 美康生物科技股份有限公司 | IGF-1 detection kit, preparation method and detection method thereof |
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Application publication date: 20200131 |