CN114062683A - Test strip and kit for quantitatively detecting GDF-15 - Google Patents

Test strip and kit for quantitatively detecting GDF-15 Download PDF

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CN114062683A
CN114062683A CN202111345701.5A CN202111345701A CN114062683A CN 114062683 A CN114062683 A CN 114062683A CN 202111345701 A CN202111345701 A CN 202111345701A CN 114062683 A CN114062683 A CN 114062683A
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test strip
gdf
concentration
fluorescent microsphere
polyclonal antibody
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CN114062683B (en
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孙长贵
雷铮宇
王德健
曹志平
陈汝彬
陈珊珊
赵彦
孔佳能
沈林
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Hunan Yonghe Sunshine Biotechnology Co ltd
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    • G01N33/6854—Immunoglobulins
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Abstract

The invention provides a test strip for quantitatively detecting GDF-15, belonging to the technical field of immunobiology. The test strip for quantitatively detecting GDF-15 provided by the invention comprises a sample pad, a chromatographic membrane and absorbent paper; a fluorescent microsphere-labeled mouse anti-human GDF-15 monoclonal antibody and a fluorescent microsphere-labeled rabbit IgG polyclonal antibody are sprayed on the sample pad; the chromatographic membrane comprises a T detection line and a C quality control line, wherein the T detection line is coated with a mouse anti-human GDF-15 polyclonal antibody, and the C quality control line is coated with a goat anti-rabbit IgG polyclonal antibody. The test strip provided by the invention has the linear range of 50-2000pg/mL, the lowest detection limit of 50pg/mL and the precision of not more than 15%, the relative deviation of the test value and the theoretical value is within +/-15%, and the reagent can meet the performance parameters of the linear range, the lowest detection limit, the precision and the accuracy after being stored for 18 months at 4-30 ℃.

Description

Test strip and kit for quantitatively detecting GDF-15
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a test strip and a kit for quantitatively detecting GDF-15.
Background
Growth differentiation factor-15 (GDF-15) is a long-arm member of the transforming growth factor beta (TGF-beta) superfamily, and GDF-15 is a stress response protein, and GDF-15 is physiologically highly expressed in the prostate and placenta, and weakly expressed in most other tissues, including heart tissue. However, under pathological and environmental stress conditions, such as ischemia/reperfusion injury, high cardiac pressure load, heart failure, atherosclerosis and the like, GDF-15 is expressed in a large amount in myocardial cells and plays a role in regulating myocardial cell structures and apoptosis programs, and under special physiological pathologies, the GDF-15 expression level is also increased remarkably, such as myocardial, kidney, lung or liver injury caused by operation, ischemia, hypoxia and the like, and tumor focus parts such as prostate cancer, breast cancer, colon cancer and the like are also expressed highly. A large number of clinical and basic studies have demonstrated that GDF-15 is an important cardiovascular protective factor. As a new marker, GDF-15 levels are closely related to diagnosis, risk stratification and prognosis judgment of various cardiovascular diseases. GDF-15 levels are closely associated with a variety of tumors. Although the view of the altered levels of GDF-15 in various tumorigenesis is inconsistent, most studies indicate that GDF-15 expression is elevated in a variety of tumors. But from both gene and cell level, GDF-15 can be proved to be capable of inhibiting the growth, invasion and metastasis of tumor cells and promoting the apoptosis of the tumor cells.
GDF-15 promotes survival and differentiation of dopaminergic neurons in embryonic stage mice. Survival of damaged dopaminergic neurons can be promoted in vitro and in vivo, and is induced in neurons with damage to the cortex. In recent years, the relationship between GDF-15 levels and cerebrovascular disease has drawn increasing attention. Under the conditions of cerebrovascular diseases, brain injuries and the like, the GDF-15 level in serum is increased, the severity of the diseases can be predicted, but the protective effect of the GDF-15 is still controversial.
The time-resolved fluoroimmunoassay (TRFIA) is a non-isotopic immunoassay technology, and is characterized by that it uses lanthanide element to label antigen or antibody, and uses time-resolved technology to measure fluorescence according to the luminescent characteristics of lanthanide chelate, and at the same time it can detect two parameters of wavelength and time to make signal resolution, so that it can effectively eliminate interference of non-specific fluorescence and greatly raise analysis sensitivity. However, since the development of the GDF-15 detection in immunochromatography reagents has great difficulty, there is no report in the prior art on the use of time-resolved fluorescence immunoassay for measuring the content of GDF-15 in a sample.
Disclosure of Invention
In view of the above, the present invention provides a test strip without a binding pad, capable of quantitatively detecting GDF-15, which can accurately detect GDF-15 while simplifying the components of the test strip, and has the advantages of low detection limit, high detection precision and accuracy, and can meet the requirements of clinical diagnosis.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a test strip for quantitatively detecting GDF-15, which consists of a sample pad, a chromatographic membrane and absorbent paper;
the sample pad is coated with a fluorescent microsphere labeled mouse anti-human GDF-15 monoclonal antibody and a fluorescent microsphere labeled rabbit IgG polyclonal antibody;
the chromatographic membrane is provided with a T detection line and a C quality control line, the T detection line is coated with a mouse anti-human GDF-15 polyclonal antibody, and the C quality control line is coated with a goat anti-rabbit IgG polyclonal antibody.
Preferably, the sample pad is obtained after pretreatment with a sample pad treatment solution, the sample pad treatment solution comprises 0.02-0.04% by mass of an anti-erythrocyte antibody and 0.1-0.2M Tris-HCl buffer solution of 0.01-0.04% by mass of an HBR blocking agent, and the pH of the Tris-HCl buffer solution is 8.0.
Preferably, when the sample pad is coated with the fluorescent microsphere-labeled mouse anti-human GDF-15 monoclonal antibody, the used membrane spraying solution is 0.05-0.1M boric acid-borax buffer solution containing 0.5-1.5% of Tween20, 0.25-0.5% of casein, 2-5% of trehalose, 0.5-0.75% of PVP40000 and 0.9% of NaCl, and the pH value of the boric acid-borax buffer solution is 8.0.
Preferably, when the fluorescent microsphere labeled mouse anti-human GDF-15 monoclonal antibody and the fluorescent microsphere labeled rabbit IgG polyclonal antibody are prepared, the used confining liquid is 0.05-0.1M Tris-HCl solution containing 1% -2% BSA and 1% -2% Tween20, and the pH value of the Tris-HCl solution is 8.0.
Preferably, the concentration of the mouse anti-human GDF-15 monoclonal antibody marked by the fluorescent microspheres is 0.08-0.20 mg/mL.
Preferably, the concentration of the rabbit IgG polyclonal antibody marked by the fluorescent microspheres is 0.08-0.20 mg/mL.
Preferably, the spraying concentration of the mouse anti-human GDF-15 polyclonal antibody is 0.8-2.0 mg/mL.
Preferably, the spraying concentration of the goat anti-rabbit IgG polyclonal antibody is 0.8-2.0 mg/mL.
Preferably, the width of the T detection line is 0.5-1.5 mm, and the width of the C quality control line is 0.5-1.5 mm.
The invention also provides a kit for quantitatively detecting GDF-15, which comprises the test strip and diluent;
the diluent is a boric acid buffer solution containing 0.2-0.5% of BSA, 0.5-1.0% of PVP40000, 1.0-2.0% of sucrose and 0.5-1.0% of Tween20 in mass concentration, and the concentration of the boric acid buffer solution is 0.1-0.2M.
The invention has the beneficial effects that:
the invention provides the test strip capable of quantitatively detecting the GDF-15 for the first time, simplifies the components of the test strip and does not contain a combination pad. The invention carries out special pretreatment on the sample pad, and improves the performance of the test strip on the basis of simplifying the components of the test strip.
According to the invention, the coupling process formula, the microsphere film spraying liquid formula, the diluent formula, the marking concentration, the coating concentration and the like are improved, so that the performance of the test strip can meet the requirement of clinical detection of GDF-15. The test strip has the advantages of good linear range, low detection limit, high precision and accuracy and good stability, and the performance of the test strip can also meet the parameters of the linear range, the minimum detection limit, the precision and the accuracy after being stored for 18 months at 4-30 ℃.
Drawings
FIG. 1 is a structural diagram of the test strip for quantitatively detecting GDF-15 of the present invention.
Detailed Description
The invention provides a test strip for quantitatively detecting GDF-15, which consists of a sample pad, a chromatographic membrane and absorbent paper;
the sample pad is coated with a fluorescent microsphere labeled mouse anti-human GDF-15 monoclonal antibody and a fluorescent microsphere labeled rabbit IgG polyclonal antibody;
the chromatographic membrane is provided with a T detection line and a C quality control line, the T detection line is coated with a mouse anti-human GDF-15 polyclonal antibody, and the C quality control line is coated with a goat anti-rabbit IgG polyclonal antibody.
The test strip is not provided with a binding pad and consists of a sample pad, a chromatographic membrane and absorbent paper, a sample to be tested is dripped into the sample pad, a substance to be tested in the sample forms a compound with the antibody marked by the fluorescent microspheres on the sample pad under the action of chromatography, and the compound is continuously chromatographed to a T detection line of the chromatographic membrane and captured by the antibody. The more the substance to be detected in the sample, the more the fluorescent microspheres gathered in the T detection line, the stronger the fluorescent signal of the T detection line, and the positive correlation between the ratio of the fluorescent signals of the detection line and the C quality control line and the amount of the substance to be detected in the sample. The signal intensity of the fluorescent antibody can be detected and analyzed by a fluorescent immunity detector, so that the GDF-15 content in the sample can be accurately and quantitatively detected.
The specific materials of the sample pad, the chromatographic membrane and the absorbent paper are not particularly required, and the materials used for conventionally preparing the test strip in the field can be adopted. In the present invention, the sample pad is labeled with a fluorescent microsphere-labeled mouse anti-human GDF-15 monoclonal antibody purchased from Abcam and a fluorescent microsphere-labeled rabbit IgG polyclonal antibody purchased from yobo eugenol. In the invention, the fluorescent microsphere is obtained by copolymerizing metal europium element chelate and styrene monomer, and the manufacturer is Bangs Laboratories.
In the present invention, the sample pad is preferably obtained by pre-treating the sample pad with a sample pad treatment solution, the sample pad treatment solution preferably comprises 0.1-0.2M Tris-HCl buffer solution of anti-erythrocyte antibody with a mass concentration of 0.02-0.04% and HBR blocker with a mass concentration of 0.01-0.04%, the Tris-HCl buffer solution has a pH of 8.0, more preferably comprises 0.15M Tris-HCl buffer solution of anti-erythrocyte antibody with a mass concentration of 0.03% and HBR blocker with a mass concentration of 0.02-0.03%, the Tris-HCl buffer solution has a pH of 8.0, and the sample pad is pre-treated with the sample pad treatment solution of the present invention, such that the sample pad has functions of both a sample pad and a conjugate pad, and the test strip structure is simplified.
In the present invention, the preparation method of the fluorescent microsphere labeled mouse anti-human GDF-15 monoclonal antibody preferably comprises the following steps: mixing the fluorescent microsphere solution with boric acid-borax buffer solution, whirling, performing ultrasonic treatment, and centrifuging to obtain a first precipitate; activating and centrifuging the first precipitate by using an EDC solution to obtain a second precipitate; mixing the second precipitate with mouse anti-human GDF-15 monoclonal antibody solution and boric acid-borax buffer solution, reacting to obtain reactant, and centrifuging to obtain third precipitate; and (4) sealing the third precipitate by using a sealing solution overnight to obtain the mouse anti-human GDF-15 monoclonal antibody.
In the invention, the concentration of the fluorescent microsphere solution is preferably 5-10 mg/mL; the concentration of the boric acid-borax buffer solution is preferably 0.01-0.02mol/L, and the pH value is preferably 7.4-7.6. In the invention, the volume ratio of the fluorescent microsphere solution to the boric acid-borax buffer solution is preferably 1:9-1: 19.
The specific modes of the vortex and the ultrasound are not particularly limited in the invention, and the conventional vortex and ultrasound modes in the field can be adopted. In the invention, the vortex time is preferably 20-30s, the ultrasonic time is preferably 2-5 min, and the ultrasonic frequency is preferably 50-100 kHz. In the invention, the rotation speed of the centrifugation is preferably 14000-15000rpm, the temperature of the centrifugation is preferably 4-8 ℃, and the time of the centrifugation is preferably 15-20 min.
The source of the EDC solution is not particularly limited in the present invention, and any commercially available product that is conventional in the art may be used. In the invention, the concentration of the EDC solution is preferably 20-30g/L, and the activation time is preferably 15-30 min. In the present invention, the first precipitate obtained after treatment of 100. mu.l of the fluorescent microsphere solution is preferably activated with 20 to 30. mu.l of EDC solution.
In the invention, the concentration of the mouse anti-human GDF-15 monoclonal antibody solution is preferably 80-200 mug/mL. Each 100 mul of the fluorescent microsphere solution is treated to obtain a second precipitate which is preferably mixed with 100-. In the invention, the reaction time is preferably 2-4 h, and more preferably 3 h.
In the present invention, the blocking solution is preferably a 0.05-0.1M Tris-HCl solution containing 1% -2% BSA, 1% -2% Tween20, and the pH of the Tris-HCl solution is 8.0.
In the present invention, the preparation method of the fluorescent microsphere labeled rabbit IgG polyclonal antibody is the same as the preparation method of the fluorescent microsphere mouse anti-human GDF-15 monoclonal antibody, and the details are not repeated herein.
In the invention, the obtained fluorescent microsphere mouse anti-human GDF-15 monoclonal antibody and the fluorescent microsphere labeled rabbit IgG polyclonal antibody are respectively dissolved in a membrane spraying liquid, and then are respectively and uniformly sprayed on a sample pad, and the sample pad is obtained after drying for 3 hours at 37 ℃. The membrane spraying liquid is 0.05-0.1M boric acid-borax buffer solution containing 0.5-1.5% of Tween20, 0.25-0.5% of casein, 2-5% of trehalose, 0.5-0.75% of PVP40000 and 0.9% of NaCl, and the pH value of the boric acid-borax buffer solution is 8.0.
In the invention, the membrane spraying liquid contains fluorescent microspheres marked with mouse anti-human GDF-15 monoclonal antibodies, and the concentration of the mouse anti-human GDF-15 monoclonal antibody markers is preferably 0.08-0.20 mg/mL, more preferably 0.1-0.15 mg/mL; the membrane spraying liquid contains fluorescent microspheres marked with rabbit IgG polyclonal antibodies, and after the rabbit IgG polyclonal antibodies marked with the fluorescent microspheres are dissolved in the membrane spraying liquid, the concentration of the rabbit IgG polyclonal antibody markers is preferably 0.08-0.20 mg/mL, and more preferably 0.1-0.15 mg/mL.
In the invention, the T detection line is coated with a mouse anti-human GDF-15 polyclonal antibody, and the spraying concentration of the mouse anti-human GDF-15 polyclonal antibody is preferably 0.8-2.0 mg/mL, more preferably 1.5 mg/mL; the quality control line C is coated with a goat anti-rabbit IgG polyclonal antibody, and the spraying concentration of the goat anti-rabbit IgG polyclonal antibody is preferably 0.8-2.0 mg/mL, and more preferably 1 mg/mL. In the invention, the independent widths of the T detection line and the C quality control line are preferably 0.5-1.5 mm, and more preferably 1 mm. In the invention, the distance between the quality control line C and the absorbent paper is preferably 7.8-8.2 mm, and the distance between the T detection line and the quality control line C is preferably 14.8-15.2 mm.
Preferably, the T detection line and the C quality control line are scratched on the nitrocellulose membrane, and after the scratches disappear, the nitrocellulose membrane is dried for 24 hours at 37 ℃ in a dark place to obtain the chromatographic membrane.
The specific preparation method of the test strip is not particularly limited, and the preparation method of the conventional test strip in the field can be adopted, for example, the sample pad, the chromatographic membrane and the absorbent paper are sequentially lapped on a PVC plate and cut to obtain the test strip. In the specific embodiment of the invention, the cutting is carried out according to the specification of 4 +/-0.1 mm multiplied by 75 mm.
The invention also provides a kit for quantitatively detecting GDF-15, which comprises the test strip and diluent;
the diluent is a boric acid buffer solution containing 0.2-0.5% of BSA, 0.5-1.0% of PVP40000, 1.0-2.0% of sucrose and 0.5-1.0% of Tween20 in mass concentration, and the concentration of the boric acid buffer solution is 0.1-0.2M.
In the invention, the diluent is preferably a boric acid buffer solution containing 0.3-0.4% of BSA, 0.6-0.9% of PVP40000, 1.3-1.7% of sucrose and 0.6-0.8% of Tween20 by mass concentration, and the concentration of the boric acid buffer solution is preferably 0.15-0.18M.
The sources of BSA, PVP40000, sucrose, Tween and boric acid in the invention are not particularly limited, and any product which is conventional and commercially available in the field can be used. In the present invention, the kit for quantitatively detecting GDF-15 preferably further comprises an SD card for calibration and quality control, and the source of the SD card is not particularly limited in the present invention, and any commercially available product that is conventional in the art can be used.
When the kit is used for detecting the GDF-15 content in a sample to be detected, the sample to be detected is preferably serum or plasma. The use method of the kit of the invention preferably comprises the following steps: sucking 100 mu L of serum or plasma sample, adding into 300-500uL of diluent, uniformly mixing, sucking 100uL, and dripping into a sample pad for reaction for 10-15 min. The detection module of the dry fluorescence immunoassay analyzer emits special ultraviolet light through a light emitting diode, when the ultraviolet light irradiates a T detection line and a C quality control line on a chromatographic membrane, fluorescent microsphere double-antibody sandwich compound at the T detection line and the C quality control line presents a fluorescent strip under the irradiation of the ultraviolet light, a photodiode of the detection module of the dry fluorescence immunoassay analyzer converts the detected fluorescence into an electric signal to measure a T/C ratio, and the T/C ratio is converted into the concentration of GDF-15 through a software program of the dry fluorescence immunoassay analyzer.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation of sample pad:
sample pad pretreatment: the sample pad treatment solutions (Tris-HCl buffer solution (pH8.00) containing 0.02% by mass of anti-erythrocyte antibody and 0.01% by mass of HBR blocker at a concentration of 0.1M) were each sprayed uniformly onto glass fibers, and dried at 37 ℃ for 4 hours to obtain treated glass fibers.
Preparing a mouse anti-human GDF-15 monoclonal antibody marked by fluorescent microspheres: adding 100. mu.L of time-resolved fluorescent microspheres (10mg/mL) and 900. mu. LpH ═ 7.4 of 0.01mol/L boric acid-borax buffer solution into a 2mL EP tube, vortexing for 20s, and sonicating for 2 min; centrifuging the mixed solution at 15000rpm and 4 deg.C for 15min, and discarding the supernatant; the precipitate was reconstituted with 1ml of 0.01mol/L boric acid-borax buffer, pH 7.4, and then activated by addition of 20. mu.l of 30g/L EDC for 15 min. And centrifuging the activated mixed solution at 15000rpm and 4 ℃ for 15min, removing the supernatant, taking the precipitate, adding 100 mu g/mL of mouse anti-human GDF-15 monoclonal antibody and 1mL of 0.01mol/L boric acid-borax buffer solution with the pH value of 7.4 into the precipitate, reacting for 2h, directly adding 100 mu L of confining liquid (0.05M Tris-HCl solution containing 1% BSA and 1% Tween20 and the pH value of 8.0) into the suspension after the reaction is finished, confining overnight, and washing to obtain the fluorescent microsphere labeled mouse anti-human GDF-15 monoclonal antibody.
The preparation method of the fluorescent microsphere labeled rabbit IgG polyclonal antibody is the same as that of the fluorescent microsphere labeled mouse anti-human GDF-15 monoclonal antibody.
Respectively dissolving the fluorescent microsphere-marked mouse anti-human GDF-15 polyclonal antibody and the fluorescent microsphere-marked rabbit IgG polyclonal antibody in a membrane spraying solution containing 0.5 percent of Tween20, 0.25 percent of casein, 2 percent of trehalose, 0.5 percent of PVP40000 and 0.9 percent of NaCl in a 0.05M boric acid-borax buffer solution (the pH value is 8.0), enabling the concentration of the fluorescent microsphere-marked mouse anti-human GDF-15 monoclonal antibody to be 0.08mg/mL and the concentration of the fluorescent microsphere-marked rabbit IgG polyclonal antibody to be 0.08mg/mL, respectively and uniformly spraying the obtained solutions on the treated glass fibers, and drying at 37 ℃ for 3 hours to obtain the sample pad.
Preparation of chromatographic membrane:
pasting a chromatographic film: pasting a 25 x 300mm nitrocellulose membrane on a 78 x 300mm PVC base plate;
preparing a coating solution: respectively preparing coating solutions of a T line and a C line by using a phosphate buffer solution, wherein the concentration of a GDF-15 polyclonal antibody of the T line is 1.5mg/mL, and the concentration of a goat anti-rabbit polyclonal antibody of the C line is 1 mg/mL;
coating and drying the chromatographic membrane: the line C is marked at a position 8mm away from the end of the absorbent paper, the line T is marked at a position 15mm away from the end of the absorbent paper, and the widths of the line C and the line T are both 0.5 mm. Placing the mixture at room temperature until the scratch liquid on the NC membrane disappears, and then placing the mixture into an oven to dry for 24 hours at 37 ℃ in a dark place to obtain the chromatographic membrane.
The sample pad, the chromatographic membrane and the absorbent paper are sequentially lapped on a PVC plate and cut according to the specification of 4mm multiplied by 75mm to obtain the test paper strip, and the structure is shown in figure 1.
Example 2
The difference from example 1 is that the concentration of the mouse anti-human GDF-15 monoclonal antibody labeled by fluorescent microspheres sprayed on the treated glass fiber is 0.2mg/mL, the concentration of the rabbit IgG polyclonal antibody labeled by fluorescent microspheres is 0.2mg/mL, and the rest is the same as example 1.
Example 3
The difference from the example 1 is that the concentration of the GDF-15 polyclonal antibody on the T line on the chromatographic membrane is 0.8mg/mL, the concentration of the goat anti-rabbit polyclonal antibody on the C line is 0.8mg/mL, the widths of the C line and the T line are both 1.5mm, and the rest is the same as the example 1.
Example 4
The difference from the example 1 is that the concentration of the GDF-15 polyclonal antibody on the T line on the chromatographic membrane is 2.0mg/mL, the concentration of the goat anti-rabbit polyclonal antibody on the C line is 2.0mg/mL, the widths of the C line and the T line are both 1mm, and the rest is the same as the example 1.
Example 5
The test strip prepared in example 1 and a diluent are combined to form a kit capable of quantitatively detecting GDF-15, wherein the diluent is a boric acid buffer solution with the mass concentration of 0.2M containing BSA, 0.5% PVP40000, 1.0% sucrose and 1.0% Tween.
Example 6
Linear range of the kit obtained in example 5 was examined:
respectively using reference substances of 50pg/mL, 100pg/mL, 500pg/mL, 1000pg/mL and 2000pg/mL for detection, detecting each sample for three times, calculating an average value, fitting the measured average value with a theoretical concentration by a linear equation to obtain a linear regression equation, and calculating a linear correlation coefficient r, wherein the result is shown in Table 1. Wherein, the reference substance is GDF-15 antigen solution with corresponding concentration, and the solvent is 0.01M PBS containing 1% BSA and 10% mannitol by weight.
TABLE 1 Linear Range test results for the kit
Figure BDA0003353993140000091
As can be seen from Table 1, the linear range of the kit for detecting GDF-15 is 50 pg/mL-2000 pg/mL, and the linear correlation coefficient r is more than or equal to 0.990.
Example 7
Detection of minimum detection Limit of the kit obtained in example 5
The test was repeated 10 times using a 0.2M boric acid buffer solution containing 0.5% BSA, 0.5% PVP40000, 1.0% sucrose, and 1.0% tween by mass as a blank sample to obtain 10 test results, the average value (M) and Standard Deviation (SD) thereof were calculated, and the lowest detection limit was reported by adding two times the standard deviation (M +2SD) to the blank average value, and the results are shown in table 2.
TABLE 2 lowest detection Limit test results for the kit
Figure BDA0003353993140000092
Figure BDA0003353993140000101
As can be seen from Table 2, the lowest detection limit of the kit for detecting GDF-15 meets the technical standard requirement of less than or equal to 50 pg/mL.
Example 8
Precision of the kit obtained in example 5 was examined
The test was repeated 10 times using 200pg/mL and 1000pg/mL of the reference, respectively, and the mean M and standard deviation SD of the measurement values were calculated. According to the formula
Figure BDA0003353993140000102
And calculating the coefficient of variation CV. In the formula: CV is the coefficient of variation; SD is the standard deviation of 10 measurements; m is 10 measurementsThe results are shown in Table 3. Wherein, the reference substance is GDF-15 antigen solution with corresponding concentration, and the solvent is 0.01MPBS containing 1% BSA and 10% mannitol by weight.
TABLE 3 results of precision measurement of kit
Figure BDA0003353993140000103
Figure BDA0003353993140000111
As can be seen from Table 3, the CV values of the kit of the present invention satisfy the technical standard requirement of not more than 15%.
Example 9
Accuracy of detection of the kit obtained in example 5
The test was performed using 200pg/mL and 2000pg/mL reference samples as samples, and the measurement was repeated 3 times, and the average value was taken to calculate the relative deviation, and the results are shown in Table 4. Wherein, the reference substance is GDF-15 antigen solution with corresponding concentration, and the solvent is 0.01M PBS containing 1% BSA and 10% mannitol by weight.
TABLE 4 accuracy test results of the kit
Figure BDA0003353993140000112
As can be seen from Table 4, the relative deviation of the accuracy of the kit of the invention meets the technical standard requirement within + -15%.
Example 10
Stability of the kit obtained in example 5
After the kit obtained in example 5 is stored at 25 ℃ for 18 months, the linear range, the lowest detection limit, the precision and the accuracy of the kit are respectively detected, and the specific steps are respectively the same as those in examples 6-9. The results are shown in tables 5 to 8.
TABLE 5 Linear Range assay results for kits stored at ambient temperature
Figure BDA0003353993140000113
Figure BDA0003353993140000121
As can be seen from Table 5, the linear range of the kit for detecting GDF-15 after long-time storage at normal temperature is 50 pg/mL-2000 pg/mL, and the linear correlation coefficient r is more than or equal to 0.990.
TABLE 6 detection results of lowest detection limit of kit after storage at room temperature
Figure BDA0003353993140000122
As can be seen from Table 6, the lowest detection limit for detecting GDF-15 of the kit of the present invention can also meet the technical standard requirement of less than or equal to 50pg/mL after being stored at normal temperature for a long time.
TABLE 7 detection results of the precision of the kit after storage at room temperature
Figure BDA0003353993140000123
Figure BDA0003353993140000131
As can be seen from Table 7, the CV value of the kit of the invention still meets the technical standard requirement of not more than 15% after being stored for a long time at normal temperature.
TABLE 8 accuracy test results of the kit after storage at Normal temperature
Figure BDA0003353993140000132
As can be seen from Table 8, the relative deviation of the accuracy of the kit of the present invention after storage at normal temperature for a long time was still within the technical standards of. + -. 15%.
The kit obtained in example 5 was stored for 18 months at 4 ℃, 8 ℃, 12 ℃, 16 ℃, 20 ℃, 24 ℃, 28 ℃ and 30 ℃ respectively, and the above-mentioned linear range, minimum detection limit, precision and accuracy detection experiments were performed, and the results all met the linear range: 50-2000pg/mL, the lowest detection limit is less than or equal to 50pg/mL, the precision is not more than 15%, and the accuracy is as follows: the relative deviation of the test value from the theoretical value is within +/-15%.
After the kit is stored for 18 months at 4-30 ℃, the kit can meet the requirements of performance parameters such as linear range, minimum detection limit, precision, accuracy and the like, and has excellent stability.
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)

1. The test strip for quantitatively detecting GDF-15 is characterized by consisting of a sample pad, a chromatographic membrane and absorbent paper;
the sample pad is coated with a fluorescent microsphere labeled mouse anti-human GDF-15 monoclonal antibody and a fluorescent microsphere labeled rabbit IgG polyclonal antibody;
the chromatographic membrane is provided with a T detection line and a C quality control line, the T detection line is coated with a mouse anti-human GDF-15 polyclonal antibody, and the C quality control line is coated with a goat anti-rabbit IgG polyclonal antibody.
2. The test strip of claim 1, wherein the sample pad is pre-treated with a sample pad treatment solution comprising 0.02% to 0.04% by mass of an anti-erythrocyte antibody and 0.1% to 0.2M Tris-HCl buffer with 0.01% to 0.04% by mass of an HBR blocking agent, wherein the Tris-HCl buffer has a pH of 8.0.
3. The test strip of claim 1, wherein when the sample pad is coated with the fluorescent microsphere labeled mouse anti-human GDF-15 monoclonal antibody, the used membrane-spraying solution is 0.05-0.1M boric acid-borax buffer solution containing 0.5-1.5% Tween20, 0.25-0.5% casein, 2-5% trehalose, 0.5-0.75% PVP40000 and 0.9% NaCl, and the pH of the boric acid-borax buffer solution is 8.0.
4. The test strip of claim 1, wherein the blocking solution used for preparing the fluorescent microsphere labeled mouse anti-human GDF-15 monoclonal antibody and the fluorescent microsphere labeled rabbit IgG polyclonal antibody is 0.05-0.1M Tris-HCl solution containing 1-2% BSA and 1-2% Tween20, and the pH of the Tris-HCl solution is 8.0.
5. The test strip of claim 1, wherein the concentration of the fluorescent microsphere labeled mouse anti-human GDF-15 monoclonal antibody is 0.08-0.20 mg/mL.
6. The test strip of claim 1, wherein the concentration of the fluorescent microsphere labeled rabbit IgG polyclonal antibody is 0.08-0.20 mg/mL.
7. The test strip of claim 1, wherein the concentration of the polyclonal murine anti-human GDF-15 antibody sprayed is 0.8-2.0 mg/mL.
8. The test strip of claim 1, wherein the goat anti-rabbit IgG polyclonal antibody is sprayed at a concentration of 0.8-2.0 mg/mL.
9. The test strip of claim 1, wherein the width of the T detection line is 0.5-1.5 mm, and the width of the C quality control line is 0.5-1.5 mm.
10. A kit for quantitatively detecting GDF-15, comprising the test strip of any one of claims 1 to 9 and a diluent;
the diluent is a boric acid buffer solution containing 0.2-0.5% of BSA, 0.5-1.0% of PVP40000, 1.0-2.0% of sucrose and 0.5-1.0% of Tween20 in mass concentration, and the concentration of the boric acid buffer solution is 0.1-0.2M.
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