CN112698042A - Fluorescent immunochromatography test strip for detecting human growth differentiation factor-15 and preparation method and application thereof - Google Patents
Fluorescent immunochromatography test strip for detecting human growth differentiation factor-15 and preparation method and application thereof Download PDFInfo
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- 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
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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Abstract
The invention discloses a fluorescence immunochromatographic test strip for detecting human growth differentiation factor-15, a preparation method and an application thereof, wherein the test strip comprises a bottom plate, and a sample adding pad, a combination pad, a detection pad and a water absorption pad which are sequentially arranged on the bottom plate, immunofluorescence microspheres for marking mouse anti-human growth differentiation factor-15 monoclonal antibodies and immunofluorescence microspheres for marking rabbit IgG are coated on the combination pad, a detection line is coated with mouse anti-human growth differentiation factor-15 monoclonal capture antibodies, and a quality detection line is coated with goat anti-rabbit IgG polyclonal antibodies. The preparation method is improved, so that the sensitivity and the repeatability of the test strip in the detection of the human growth differentiation factor-15 by adopting a fluorescence quantitative method are improved, and a new technical means is provided for the detection of the human growth differentiation factor-15.
Description
Technical Field
The invention relates to a fluorescence immunochromatographic test strip and a preparation method and application thereof, in particular to a fluorescence immunochromatographic test strip for detecting human growth differentiation factor-15 and a preparation method and application thereof. The invention belongs to the technical field of medicines.
Background
Growth differentiation factor-15 (GDF 15) belongs to the family of Growth Differentiation Factors (GDFs), and is one of the members of the transforming growth factor-beta (TGF-B) superfamily. Studies have shown that the human GDF15 gene sequence is located at 19p 13.1-13.2, comprises 924 base pairs, consists of 2 exons and 1 intron, and is separated by its intron. The gene of GDF15 is first transcribed into a polypeptide of 308 amino acid residues, eventually forming a GDF15 mature protein comprising 224 amino acid residues.
GDF15 is generally highly expressed only in placenta and prostate, but is expressed in small amounts in heart, other organs and tissues, but is increased in various disease states such as tumor, pregnancy, neuronal damage and skeletal muscle dysplasia, and is also involved in the process of cardiovascular disease evolution. Researches show that the concentration of GDF15 in serum is closely related to various cardiovascular adverse events, has higher value for early prediction and prognosis risk suggestion of diseases such as heart failure, acute myocardial infarction, acute coronary syndrome and the like, and is a few cardiovascular disease biomarkers with great potential.
At present, the marker only stays in the laboratory research stage, and the research direction is mainly the disease evaluation of acute coronary syndrome and heart failure and the prognosis of diseases such as myocardial infarction, cerebral infarction and the like. The adopted enzyme-linked immunosorbent assay method has the defects of long antibody incubation time, multiple washing times and the like, and is not suitable for first-line clinical detection.
The invention patent application with the application number of 201610798041.9 discloses a test paper assembly for detecting human growth differentiation factors-15. The patent application uses magnetic particles to couple anti-human growth differentiation factor 15 monoclonal antibody to prepare a binding pad, anti-human growth differentiation factor 15 polyclonal antibody is coated on a detection line to capture the magnetic particles bound with GDF15 in a sample, and quantification is carried out through the magnetic strength of the detection line. The technology has the advantages of overcoming the defect of long time of enzyme-linked immunoassay, but has the defect that the number of magnetic immunoassay analyzers which are suitable for detecting cards and are based on magnetic field intensity detection in the market is less, thereby limiting the clinical popularization of the product.
Disclosure of Invention
The invention aims to provide a fluorescent immunochromatographic test strip for detecting growth differentiation factor-15 and a preparation method thereof, and the test strip can be used for fluorescent immunoassay.
In order to achieve the purpose, the invention adopts the following technical means:
the invention relates to a fluorescence immunochromatographic test strip for detecting human growth differentiation factor-15, which comprises a base plate, a sample adding pad, a combination pad, a detection pad and a water absorption pad, wherein the sample adding pad, the combination pad, the detection pad and the water absorption pad are sequentially arranged on the base plate, the sample adding pad and the combination pad are in lap joint, the water absorption pad and the detection pad are in lap joint, the detection pad is provided with a detection line and a quality detection line, the detection line is positioned at one end of the sample adding pad, the quality detection line is positioned at one end far away from the sample adding pad, the combination pad is coated with immunofluorescence microspheres for marking mouse anti-human growth differentiation factor-15 monoclonal antibody and immunofluorescence microspheres for marking rabbit IgG, the detection line is coated with mouse anti-human growth differentiation factor-15 monoclonal antibody, the quality detection line is coated with goat anti-rabbit IgG polyclonal capture antibody, and the mouse anti human growth differentiation factor-15 monoclonal antibody used for marking the immunofluorescence microspheres and the mouse anti human growth differentiation factor-15 monoclonal capture Monoclonal antibodies with different antigen recognition epitopes and with growth differentiation factor-15 are capable of forming a complex in the form of antibody-antigen-antibody.
Wherein, preferably, the mouse anti-human growth differentiation factor-15 monoclonal antibody used for marking the immunofluorescence microsphere is an anti-human growth differentiation factor-15 monoclonal antibody [23G10.F8], the cargo number ab105732, purchased from ABCAM company; the mouse anti-human growth differentiation factor-15 monoclonal capture antibody coated on the detection line is an anti-human growth differentiation factor-15 monoclonal antibody [7C12.B3.F2], cat ab106112, purchased from ABCAM.
Preferably, the immunofluorescence microsphere is a 200nm fluorescence microsphere with carboxyl modified on the surface.
Further, the invention also provides a method for preparing the fluorescence immunochromatographic test strip, which is characterized by comprising the following steps:
(1) bond pad pretreatment
Putting the polyester film into a clean tray with holes, treating the polyester film for 1 minute by running water of tap water, repeatedly cleaning the polyester film for 5 times by using purified water, rinsing the polyester film for 3 times by using pretreatment liquid, soaking the polyester film for 5 to 10 minutes, taking out the treated polyester film, and drying the polyester film in an oven at the temperature of between 35 and 40 ℃ for later use;
the pretreatment solution contains 1-10g/L of TRIS, 5-10g/L of sodium chloride, 0.5-6 g/L of surfactant and 8.0 +/-0.1 of pH value; preferably, the pretreatment solution contains TRIS 6.05g/L, sodium chloride 9g/L, surfactant 1.5g/L and pH value of 8.0 +/-0.1;
(2) preparation of fluorescent microspheres for labeling GDF-15 monoclonal antibody and rabbit IgG
Activating and washing the fluorescent microspheres, respectively adding a mouse anti-human growth differentiation factor 15 monoclonal antibody and rabbit IgG for coupling and sealing, washing to remove unbound antibodies, and adding a preservation buffer solution for preservation;
(3) bond pad preparation
Adding the fluorescent microspheres marked with the mouse anti-human GDF-15 antibody and the fluorescent microspheres marked with the rabbit IgG into a binding buffer solution, spraying the binding buffer solution on a polyester film to prepare a binding pad, drying the binding pad in an oven, and storing the binding pad for later use; the combination buffer comprises 0.1-0g/dL of TRIS, 2-8g/dL of BSA, 10-20g/dL of sucrose and 1-10g/dL of D-trehalose, and has a pH value of 8.0 +/-0.1, preferably, the combination buffer comprises 0.605g/dL of TRIS, 5g/dL of BSA, 15g/dL of sucrose and 5g/dL of D-trehalose, and has a pH value of 8.0 +/-0.1;
(4) preparation of human growth differentiation factor 15 antibody coating and detection pad
Preparing a detection line working solution from a mouse anti-human growth differentiation factor-15 monoclonal capture antibody, wherein the concentration of a coating antibody is 0.5-1.0 mg/ml, preparing a quality inspection line working solution from a goat anti-rabbit IgG, wherein the concentration of the coating antibody is 0.8-1.2 mg/ml, respectively pointing the detection line working solution and the quality inspection line working solution on a nitrocellulose membrane by a membrane scribing instrument to obtain a detection line and a quality inspection line, and drying and refrigerating for later use;
(5) assembly
Attaching the detection pad, the combination pad, the sample adding pad and the water absorption pad to a bottom plate according to a fixed sequence, assembling into a large test paper plate, and storing for later use under a dry condition;
(5) cutting of
The assembled large test strip plate is cut into test strips with the width of 4mm (+ -0.05 mm) by a slitter and stored under a dry condition for later use.
Wherein, preferably, the surfactant is monododecyl nonaethylene glycol ether, polyvinylpyrrolidone K30, polyoxyethylene distyryl phenyl ether, lauryl alcohol polyoxyethylene ether, 3- [3- (choleamidopropyl) dimethylamino ] propane sulfonic acid inner salt, 3- [ (3-cholesterylaminopropyl) dimethylamino ] -2-hydroxy-1-propane sulfonic acid, octyl phenol polyoxyethylene ether, dodecyl-beta-D-maltoside, N-nonanoyl-N-methylglucamine or polyethylene glycol octyl phenyl ether, and preferably, the surfactant is monododecyl nonaethylene glycol ether.
Preferably, the method further comprises the steps of embedding the cut test paper strips into a single-hole test card box, rolling tightly to manufacture test cards, putting each test card into an aluminum foil bag, putting one desiccant bag, and sealing by using a sealing machine.
Preferably, the labeling buffer used for labeling the GDF-15 monoclonal antibody and the rabbit IgG in the step (2) contains 0.1-1g/dL of TRIS, 1-10g/dL of BSA, 10-20g/dL of sucrose, 1-10g/dL of D-trehalose, 0.5-6 g/L of a surfactant and the pH value is 8.0 +/-0.1, and preferably contains 0.605g/dL of TRIS, 5g/dL of BSA5g/dL, 15g/dL of sucrose, 5g/dL of D-trehalose and 1.5g/L of a surfactant.
Wherein, preferably, the surfactant is monododecyl nonaethylene glycol ether, polyvinylpyrrolidone K30, polyoxyethylene distyryl phenyl ether, lauryl alcohol polyoxyethylene ether, 3- [3- (choleamidopropyl) dimethylamino ] propane sulfonic acid inner salt, 3- [ (3-cholesterylaminopropyl) dimethylamino ] -2-hydroxy-1-propane sulfonic acid, octyl phenol polyoxyethylene ether, dodecyl-beta-D-maltoside, N-nonanoyl-N-methylglucamine or polyethylene glycol octyl phenyl ether, and preferably, the surfactant is monododecyl nonaethylene glycol ether.
Preferably, the distance between the detection line and the quality detection line is 5 mm.
Furthermore, the invention also provides application of the fluorescence immunochromatographic test strip in preparation of a reagent for detecting the human growth differentiation factor-15.
The detection principle of the test strip is as follows: after the sample is dripped into the sample adding hole of the detection card, the human growth differentiation factor 15 in the sample is combined with the fluorescent substance labeled anti-human growth differentiation factor 15 monoclonal antibody in the combination pad to form a reaction compound, and the reaction compound moves forwards along the Nitrocellulose (NC) membrane along with the chromatography action and is captured by the corresponding monoclonal antibody on the detection line of the Nitrocellulose (NC) membrane to form the compound. The capture amount of the human growth differentiation factor 15 in the sample is positively correlated with the signal intensity of the detection area, under the action of an excitation light source, a fluorescent substance emits a fluorescent signal with a specific wavelength, a fluorescence immunoassay instrument captures the fluorescent signal, the fluorescent signal is automatically converted into a quantitative value through signal conversion and a set calibration curve, the concentration of the human growth differentiation factor 15 in the sample is calculated, and the quantitative detection of the human growth differentiation factor 15 in a serum, plasma or whole blood sample is realized.
Compared with the prior art, the invention has the beneficial effects that:
1. the fluorescent detection test strip produced according to the conventional preparation method and the principle can be used for detecting the growth differentiation factor 15, but the repeatability and the sensitivity of the prepared product are poor. Therefore, the invention adopts special binding solution for preparing the binding pad, and the repeatability is improved to a certain extent. The use of a special conjugate pad pretreatment procedure for washing and desensitizing the conjugate pad greatly improves reproducibility and sensitivity.
2. The detection card is manufactured based on an immunofluorescence quantitative technology, carboxyl fluorescent microspheres (exciting light 365nm and emitting light 610nm) with good detection sensitivity are selected, and the detection test paper strip with good sensitivity and high detection speed is obtained by screening the particle size of the fluorescent microspheres and a marking buffer solution used for manufacturing the combined pad.
3. The preparation method is improved, so that the sensitivity and the repeatability of the test strip in the detection of the human growth differentiation factor-15 by adopting a fluorescence quantitative method are improved, and a new technical means is provided for the detection of the human growth differentiation factor-15.
Drawings
FIG. 1 is a schematic view of a test strip assembly;
FIG. 2 is a schematic view of the assembly of the test card.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Example 1 preparation of a fluorescent immunochromatographic test strip for detecting growth differentiation factor-15
1. Experimental materials: mouse anti-human GDF-15 monoclonal antibodies [23G10.F8], [7C12.B3.F2] (available from ABCAM, cat # ab105732, ab106112), recombinant human GDF-15 antigen ((available from ABCAM, cat # ab50077)), nitrocellulose membrane, blotter, 6cm × 30cm PVC plate, rabbit IgG (SIGMA, cat # I5006), goat anti-rabbit IgG (Thermo Scientific, cat # A32731), 200nm fluorescent microspheres (1.02g/100g, available from Thermo Scientific, cat # 93470520010150), 2- (N-morpholine) ethanesulfonic acid monohydrate (MES, available from SIGMA), N-hydroxysuccinimide (NHS, available from SIGMA), Tween-20 (available from BBI), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (GMA, available from SIGMA).
2. Coupling of human growth differentiation factor 15 labeled antibody and fluorescent microsphere and preparation of binding pad
The preparation of the immunofluorescence microsphere suspension for detecting GDF-15 by using EDC/NHS comprises the steps of activation, coupling, sealing and storage, and the process flow is as follows:
(1) preparation of fluorescent microsphere for marking GDF-15 monoclonal antibody
Initial washing of fluorescent microspheres:
100 μ l of 0.2 μm fluorescent microspheres (Thermo) with 1% solids content was added to the centrifuge tube, 900 μ l of activation buffer (MES 1.95g/dL) was added to the centrifuge tube, and mixed by vortexing with a vortex mixer for 30 s. Centrifuge at 13000rpm for 10min at 4 ℃ and aspirate the supernatant.
Activation of fluorescent microspheres:
1ml of activation buffer (MES 1.95g/dL) was added, after resuspension by sonication, 50. mu.l (20mg/ml) of EDC and 50. mu.l (20mg/ml) of NHS were added, the mixture was stirred and mixed by a vortex mixer, then the reaction was carried out at room temperature for 30min by rotation, the microspheres after activation were centrifuged at 13000rpm for 10min at 4 ℃ and the supernatant was aspirated.
Mouse anti-human growth differentiation factor monoclonal antibody labeling:
1ml of a labeling buffer (0.62 g/dL of boric acid, 0.955g/L of borax decahydrate, pH 8.0) was added, the microspheres after activation were ultrasonically resuspended, centrifuged at 13000rpm at 4 ℃ for 10min, and the supernatant was aspirated. After 1ml of labeling buffer was added and resuspended by sonication, 80ug of GDF15 monoclonal antibody [23G10.F8] was added and the reaction was allowed to rotate at room temperature for 2 hours.
Sealing the fluorescent microspheres:
mu.l of blocking solution (BSA0.5g/dL, ethanolamine 0.2ml/dL, Proclin-3000.2ml/dL) was added thereto, and the mixture was subjected to a rotary reaction at room temperature for 30 minutes. Centrifuging at 13000rpm for 10min at 4 ℃ and removing the supernatant.
Final washing of the fluorescent microspheres:
1ml of cleaning solution (BSA0.5g/dL, Tween 20100ul/dL, Proclin-300100ul/dL) is added for carrying out ultrasonic resuspension on the microspheres after being sealed, the microspheres are centrifuged at 13000rpm at 4 ℃ for 10min, and the supernatant is sucked off. The above procedure was repeated (twice in total). Adding 100ul of preservation buffer (NaH2PO4 & 2H2O 0.0593g/dL, Na2HPO4 & 12H2O 0.58g/dL, NaCl 0.85g/dL, sucrose 1g/dL, Bovine Serum Albumin (BSA)0.5g/dL, Tween 20(Tween-20)50 ul/dL, Proclin-300100ul/dL, pH 7.3) into the precipitate, carrying out ultrasonic resuspension, and refrigerating at 2-8 ℃ for later use.
(2) Preparation of fluorescent microspheres for labeling rabbit IgG
The steps of washing and activating the fluorescent microspheres are the same as those in (1).
Labeling rabbit IgG:
1ml of a labeling buffer (0.62 g/dL of boric acid, 0.955g/L of borax decahydrate, pH 8.0) was added, the microspheres after activation were ultrasonically resuspended, centrifuged at 13000rpm at 4 ℃ for 10min, and the supernatant was aspirated. Then adding 1ml of marking buffer solution, adding 80ug of rabbit IgG antibody after the microspheres are subjected to ultrasonic resuspension activation, and carrying out rotary reaction for 2 hours at room temperature.
The fluorescent microspheres are sealed and finally washed as in (1).
(3) Fabrication of bond pads
Mu.l of the fluorescent microspheres labeled with mouse anti-human GDF-15 antibody and 45. mu.l of the fluorescent microspheres labeled with rabbit IgG were added to 4912.5. mu.l of binding buffer (TRIS 0.605g/dL, BSA5g/dL, sucrose 15g/dL, D trehalose 5g/dL, pH 8.0. + -. 0.1) and mixed by vortexing with a vortex mixer. Spraying the mixture on a polyester film by using a gold marking machine in a proper amount (the parameter of the gold marking machine is the spraying amount is 5.0 mu l/cm, the length is 300mm), drying the mixture for at least 2 hours in an environment with the humidity of not more than 40% and the temperature of 18-37 ℃, and sealing and storing the mixture for later use under the drying condition.
3. Preparation of human growth differentiation factor 15 antibody coating and detection pad
(1) Preparation of working solution for detection line
Mouse anti-human GDF-15 monoclonal antibody [7C12.B3.F2] was diluted to 0.6mg/mL with coating buffer (sodium dihydrogen phosphate 0.0296g/dL, disodium hydrogen phosphate 0.29g/dL, D trehalose 1g/dL, pH 7.3) and refrigerated for further use.
(2) Preparation of quality control line working solution
Goat anti-rabbit IgG was diluted to 1mg/mL with coating buffer (sodium dihydrogen phosphate 0.0296g/dL, disodium hydrogen phosphate 0.29g/dL, D-trehalose 1g/dLpH 7.3) and refrigerated for future use.
(3) Preparation of detection pad
And sticking the nitrocellulose membrane on a PVC bottom plate to form a nitrocellulose membrane adhesive plate with good adhesion. And respectively pointing the detection line working solution and the quality control line working solution at the center of the same membrane by using a membrane scribing instrument, and calibrating by using a standard plate, wherein the distance between the quality control line and the upper end of the bottom plate is 35mm, and the distance between the detection line and the upper end of the bottom plate is 40 mm. (the wider side of the PVC plate is the upper end), the spotting amount is 1.0. mu.l/cm. The distance between the two lines is 5mm, and the positions of the detection line and the quality control line are marked on the spot and bad lines are marked; drying for at least 2 hours in the environment with the humidity not more than 40% and the temperature of 18-37 ℃, and then sealing and storing for later use under the drying condition.
4. Assembling and preparing the test strip
(1) The sample pad was untreated glass fiber. Taking a sample pad with the specification of 200 x 300mm, and cutting the sample pad into strips with the specification of 20mm x 300mm
(2) A water-absorbing pad with a specification of 200X 300mm is taken and cut into long strips with a specification of 28mm X300 mm.
(3) The bonding pad was cut into a long piece of 9mm × 305 mm.
(4) Taking the coated detection diaphragm plate (the wider side of the PVC plate blank is the upper side, the narrower side of the PVC plate blank is the lower side), removing the anti-sticking paper above, taking a 28mm × 300mm strip water absorption pad to be stuck above the detection diaphragm plate, wherein the upper end of the water absorption pad is flush with the upper edge of the bottom plate, and the lower end of the water absorption pad is pressed on the reaction membrane and is overlapped for 2 mm. The release paper below the middle base plate is removed, the combination pad with the specification of 9mm multiplied by 305mm is pasted below the bottom plate, and the combination pad is pressed on the detection pad and overlapped by 1 mm. A sample pad with the specification of 20mm multiplied by 300mm is taken and pasted below the bottom plate, the lower end of the sample pad is flush with the lower edge of the bottom plate, and the upper end of the sample pad is pressed at the lower end of the combination pad and is overlapped by 1 mm. The assembly is schematically shown in fig. 1.
(5) Cutting and preparing single test paper strip
The assembled large test strip plate is cut into test strips with the width of 4mm (+ -0.05 mm) by a slitter and stored under a dry condition for later use.
(6) Card installation and preparation detection card
The cut test strip is embedded into a single-hole detection card box, and a sample adding pad (area A) is placed at one end of a shell of the detection card, which is provided with a sample adding hole. And (5) rolling tightly to prepare the detection card. Each detection card is put into an aluminum foil bag, and meanwhile, a drying agent bag is put into the detection card and is sealed by a sealing machine. The schematic diagram of the detection card is shown in fig. 2.
After the sample is dripped into the small hole of the sample adding area of the detection card, the human growth differentiation factor 15 in the sample flows to the area B under the action of chromatography, the mouse anti-human growth differentiation factor 15 monoclonal antibody coated on the surface of the fluorescent microsphere in the area B and the binding pad is combined to form a reaction compound, the reaction compound enters the area C along with the chromatography, moves forwards along a Nitrocellulose (NC) membrane of the area C and is captured by the corresponding monoclonal antibody on a Nitrocellulose (NC) membrane detection line to form the compound. The capture amount of the human growth differentiation factor 15 in the sample is positively correlated with the signal intensity of the detection area, the fluorescent microspheres emit fluorescent signals with specific wavelengths under the action of an excitation light source, the fluorescent signal is captured by a fluorescence immunoassay instrument and automatically converted into quantitative values through signal conversion and a set calibration curve, the concentration of the human growth differentiation factor 15 in the sample is calculated, and the quantitative detection of the human growth differentiation factor 15 in a serum, plasma or whole blood sample is realized.
Example 2 preparation of a fluorescence immunochromatographic test strip for detecting growth differentiation factor-15
The following formulation was used for the labeling buffer: TRIS0.605g/dL, BSA5g/dL, sucrose 15g/dL, D-trehalose 5g/dL, and monolauryl nonaethylene glycol ether 1.5g/L, with a pH of 8.0. + -. 0.1, the procedure was otherwise as in example 1.
Wherein the monododecyl nonaethylene glycol ether can be replaced by polyvinylpyrrolidone K30, polyoxyethylene distyrenated phenyl ether, polyoxyethylene lauryl ether, 3- [3- (cholestyramidopropyl) dimethylamino ] propanesulfonic acid inner salt, 3- [ (3-cholesteryl aminopropyl) dimethylamino ] -2-hydroxy-1-propanesulfonic acid, octylphenol polyoxyethylene ether, dodecyl-beta-D-maltoside, N-nonanoyl-N-methylglucamine or polyethylene glycol octylphenyl ether.
Example 3 preparation of fluorescent immunochromatographic test strip for detecting growth differentiation factor-15
A bond pad pretreatment
The polyester film is put into a clean tray with holes, treated by running water for 1 minute, repeatedly washed by purified water for 5 times, rinsed by pretreatment liquid for 3 times, and soaked for 5-10 minutes. And taking out the treated polyester film, and drying the polyester film in an oven at 35-40 ℃ for later use.
The formula of the pretreatment solution is0.605g/dL of TRIS, 9g/L of sodium chloride, 1.5g/L of monododecyl nonaethylene glycol ether and the pH value is 8.0 +/-0.1.
Wherein the monododecyl nonaethylene glycol ether can be replaced by polyvinylpyrrolidone K30, polyoxyethylene distyrenated phenyl ether, polyoxyethylene lauryl ether, 3- [3- (cholestyramidopropyl) dimethylamino ] propanesulfonic acid inner salt, 3- [ (3-cholesteryl aminopropyl) dimethylamino ] -2-hydroxy-1-propanesulfonic acid, octylphenol polyoxyethylene ether, dodecyl-beta-D-maltoside, N-nonanoyl-N-methylglucamine or polyethylene glycol octylphenyl ether.
B labeling buffer formulation example 2.
C the remaining preparation steps were as in example 1.
Example 4, and application and comparison of the fluorescence immunochromatographic test strip obtained in examples 1 to 3 in detecting growth differentiation factor-15
The product obtained in example 1 is GDF15-1, the product obtained in example 2 is GDF15-2, and the product obtained in example 3 is GDF 15-3.
1. Detection conditions and detection parameters
A detection instrument: dry fluoroimmunoassay analyzer (AFS-1000) Guangzhou blue Bob Biotech Co., Ltd
Detecting parameters:
a pre-read time (120); a test time (900); sample addition amount (100); number of peaks (3); a buffer amount (100); the amount of the mixed liquid (100); peak number (X1); peak algorithm (9. fluorescence peak area)
Standard curve: and (3) diluting the GDF-15 standard substance by PBS (phosphate radical 20mmol/L, NaCl150mmol/L) to obtain standard substance solutions with final concentrations of 10, 25, 50, 100, 250 and 500ng/L respectively, loading 100 mu L of the standard substance solution to a loading hole of the detection card respectively, and detecting after the standard substance solution is placed for 15 minutes. And detecting the fluorescent signals on the detection line and the quality detection line by using an instrument. And (3) taking the fluorescence signal and the GDF-15 concentration as parameters, making a quantitative detection standard curve, and detecting the concentration of the sample.
2. Detecting linearity
Diluting the high-value sample with the concentration of 5000pg/mL by PBS (phosphate 20mmol/L and NaCl150mmol/L), mixing the high-value sample with the high-value sample into 5 samples (xi) with different gradients of dilution ratios of 1/50, 1/10, 1/5, 1/2 and 1, and testing the detection cards respectively. The test was repeated for 3 test cards for each diluted concentration sample, and the mean value (yi) of the measurement results was obtained. The dilution concentration (xi) is used as an independent variable, and the measurement result mean value (yi) is used as a dependent variable to calculate a linear regression equation and a linear regression coefficient. The results are shown in tables 1-3 below:
TABLE 1 test results for GDF15-1
| 1 | 2 | 3 | 4 | 5 | |
| Test 1 | 110.31 | 487.79 | 839.41 | 2691.02 | 4278.6 |
| Test 2 | 131.33 | 467.96 | 1110.99 | 2358.51 | 4528.73 |
| Test 3 | 89.9 | 511.11 | 941.35 | 2842.5 | 6012.8 |
| Mean value | 110.51 | 488.95 | 963.92 | 2630.68 | 4940.04 |
| Concentration of dilution | 0.02 | 0.1 | 0.2 | 0.5 | 1 |
| r | 0.9993 | Slope of | 4976 | Intercept of a beam | -0.003 |
TABLE 2 test results for GDF15-2
| 1 | 2 | 3 | 4 | 5 | |
| Test 1 | 111.16 | 532.65 | 891.49 | 2586.62 | 5067.91 |
| Test 2 | 119.72 | 518.04 | 1040.55 | 2575.85 | 4452.39 |
| Test 3 | 99.72 | 499.81 | 1035.96 | 2689.44 | 5083.59 |
| Mean value | 110.20 | 516.83 | 989.33 | 2617.30 | 4867.96 |
| Concentration of dilution | 0.02 | 0.1 | 0.2 | 0.5 | 1 |
| r | 0.9992 | Slope of | 4884 | Intercept of a beam | -0.008 |
TABLE 3 test results for GDF15-3
It can be seen that the linearity of all three preparation methods can meet the requirements.
3. Minimum limit of detection
PBS (phosphate 20mmol/L, NaCl150mmol/L) was used as a blank sample, and the test card prepared according to the present invention was used for 20 measurements, and regression was performed by taking the mean value of the test signal plus two times the standard deviation into the fitted curve. As a result, the minimum detection limit of GDF15-1 was 27.48pg/mL, the minimum detection limit of GDF15-2 was 20.93pg/mL, and the minimum detection limit of GDF15-3 was 11.76 pg/mL. The preparation method of example 3 was found to be the most sensitive (table 4).
TABLE 4 lowest detection Limit
4. Repeatability of
The product prepared by the invention continuously measures 2 levels of samples 10 times respectively, and calculates the mean value, standard deviation and Coefficient of Variation (CV).
TABLE 5 results of repeated experiments
The results are shown in Table 5, which shows that example 3 has the best reproducibility and can meet clinical requirements. The product prepared by the general method in the embodiment 1 has poor repeatability, and can only achieve the semi-quantitative purpose.
5. Human serum reference range
120 normal human serum samples and 120 normal plasma samples are respectively detected, the 95 percent site is 900pg/mL, and GDF15-1, GDF15-2 and GDF15-3 determine that the reference ranges of healthy people are all less than 900 pg/mL.
Claims (10)
1. The fluorescence immunochromatographic test strip for detecting the human growth differentiation factor-15 is characterized by comprising a base plate, a sample adding pad, a combination pad, a detection pad and a water absorption pad which are sequentially arranged on the base plate, wherein the sample adding pad is in lap joint with the combination pad, the detection pad is in lap joint with the combination pad, the water absorption pad is in lap joint with the detection pad, the detection pad is provided with a detection line and a quality detection line, the detection line is positioned at one end of the sample adding pad, the quality detection line is positioned at one end far away from the sample adding pad, the combination pad is wrapped with immunofluorescence microspheres for marking the mouse anti-human growth differentiation factor-15 monoclonal antibody and immunofluorescence microspheres for marking the rabbit IgG, the detection line is wrapped with the mouse anti-human growth differentiation factor-15 monoclonal antibody, the quality detection line is wrapped with the goat anti-rabbit IgG polyclonal antibody, the mouse anti-human growth differentiation factor-15 monoclonal antibody used for marking the immunofluorescence microspheres and the anti-human growth differentiation factor-15 monoclonal antibody wrapped on the detection 15 monoclonal capture antibodies are monoclonal antibodies with different antigen recognition epitopes and are capable of forming a complex with growth differentiation factor-15 in the form of antibody-antigen-antibody.
2. The fluorescence immunochromatographic test strip of claim 1, wherein the mouse anti-human growth differentiation factor-15 monoclonal antibody used for labeling the immunofluorescent microsphere is anti-human growth differentiation factor-15 monoclonal antibody [23G10.F8], cat ab105732, purchased from ABCAM company; the mouse anti-human growth differentiation factor-15 monoclonal capture antibody coated on the detection line is an anti-human growth differentiation factor-15 monoclonal antibody [7C12.B3.F2], cat ab106112, purchased from ABCAM.
3. The fluorescence immunochromatographic test strip of claim 1, wherein the immunofluorescence microsphere is a 200nm fluorescence microsphere with a carboxyl group modified on the surface.
4. A method for preparing the fluorescent immunochromatographic test strip of any one of claims 1 to 3, comprising the steps of:
(1) bond pad pretreatment
Putting the polyester film into a clean tray with holes, treating the polyester film for 1 minute by running water of tap water, repeatedly cleaning the polyester film for 5 times by using purified water, rinsing the polyester film for 3 times by using pretreatment liquid, soaking the polyester film for 5 to 10 minutes, taking out the treated polyester film, and drying the polyester film in an oven at the temperature of between 35 and 40 ℃ for later use;
the pretreatment solution contains 1-10g/L of TRIS, 5-10g/L of sodium chloride, 0.5-6 g/L of surfactant and 8.0 +/-0.1 of pH value; preferably, the pretreatment solution contains TRIS 6.05g/L, sodium chloride 9g/L, surfactant 1.5g/L and pH value of 8.0 +/-0.1;
(2) preparation of fluorescent microspheres for labeling GDF-15 monoclonal antibody and rabbit IgG
Activating and washing the fluorescent microspheres, respectively adding a mouse anti-human growth differentiation factor 15 monoclonal antibody and rabbit IgG for coupling and sealing, washing to remove unbound antibodies, and adding a preservation buffer solution for preservation;
(3) bond pad preparation
Adding the fluorescent microspheres marked with the mouse anti-human GDF-15 antibody and the fluorescent microspheres marked with the rabbit IgG into a binding buffer solution, spraying the binding buffer solution on a polyester film to prepare a binding pad, drying the binding pad in an oven, and storing the binding pad for later use; the combination buffer comprises 0.1-0g/dL of TRIS, 2-8g/dL of BSA, 10-20g/dL of sucrose and 1-10g/dL of D-trehalose, and has a pH value of 8.0 +/-0.1, preferably, the combination buffer comprises 0.605g/dL of TRIS, 5g/dL of BSA, 15g/dL of sucrose and 5g/dL of D-trehalose, and has a pH value of 8.0 +/-0.1;
(4) preparation of human growth differentiation factor 15 antibody coating and detection pad
Preparing a detection line working solution from a mouse anti-human growth differentiation factor-15 monoclonal capture antibody, wherein the concentration of a coating antibody is 0.5-1.0 mg/ml, preparing a quality inspection line working solution from a goat anti-rabbit IgG, wherein the concentration of the coating antibody is 0.8-1.2 mg/ml, respectively pointing the detection line working solution and the quality inspection line working solution on a nitrocellulose membrane by a membrane scribing instrument to obtain a detection line and a quality inspection line, and drying and refrigerating for later use;
(5) assembly
Attaching the detection pad, the combination pad, the sample adding pad and the water absorption pad to a bottom plate according to a fixed sequence, assembling into a large test paper plate, and storing for later use under a dry condition;
(5) cutting of
The assembled large test strip plate is cut into test strips with the width of 4mm (+ -0.05 mm) by a slitter and stored under a dry condition for later use.
5. The method of claim 4, wherein the surfactant is monododecyl nonaethylene glycol ether, polyvinylpyrrolidone K30, polyoxyethylene distyrylphenyl ether, polyoxyethylene lauryl ether, 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt, 3- [ (3-cholesterylaminopropyl) dimethylamino ] -2-hydroxy-1-propanesulfonic acid, octylphenol polyoxyethylene ether, dodecyl-beta-D-maltoside, N-nonanoyl-N-methylglucamine or polyethylene glycol octylphenyl ether, preferably the surfactant is monododecyl nonaethylene glycol ether.
6. The method of claim 4, further comprising inserting the cut test strips into a single-hole test cartridge, rolling the test cartridge to form test cards, placing each test card in an aluminum foil pouch, placing a desiccant pouch in the pouch, and sealing the pouch with a sealing machine.
7. The method of claim 4, wherein the GDF-15 monoclonal antibody and rabbit IgG are labeled in step (2) using a labeling buffer comprising TRIS 0.1-1g/dL, BSA 1-10g/dL, sucrose 10-20g/dL, D-trehalose 1-10g/dL, and a surfactant 0.5-6 g/L at a pH of 8.0 ± 0.1, preferably TRIS0.605g/dL, BSA5g/dL, sucrose 15g/dL, D-trehalose 5g/dL, and a surfactant 1.5 g/L.
8. The method of claim 7, wherein the surfactant is monododecyl nonaethylene glycol ether, polyvinylpyrrolidone K30, polyoxyethylene distyrylphenyl ether, polyoxyethylene lauryl ether, 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt, 3- [ (3-cholesterylaminopropyl) dimethylamino ] -2-hydroxy-1-propanesulfonic acid, octylphenol polyoxyethylene ether, dodecyl-beta-D-maltoside, N-nonanoyl-N-methylglucamine or polyethylene glycol octylphenyl ether, preferably the surfactant is monododecyl nonaethylene glycol ether.
9. The method of claim 7, wherein the detection line is 5mm from the quality detection line.
10. Use of the fluorescent immunochromatographic test strip of any one of claims 1 to 3 in the preparation of a reagent for detecting human growth differentiation factor-15.
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