CN114252599A - Test strip for detecting oligomeric cartilage matrix protein and detection method - Google Patents
Test strip for detecting oligomeric cartilage matrix protein and detection method Download PDFInfo
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- CN114252599A CN114252599A CN202111589049.1A CN202111589049A CN114252599A CN 114252599 A CN114252599 A CN 114252599A CN 202111589049 A CN202111589049 A CN 202111589049A CN 114252599 A CN114252599 A CN 114252599A
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/105—Osteoarthritis, e.g. cartilage alteration, hypertrophy of bone
Abstract
The invention discloses a test strip for detecting oligomeric cartilage matrix protein and a detection method, and relates to the technical field of quantitative detection by an immunochromatography method. Compared with the prior art, the detection test strip provided by the invention has the following advantages: the kit has the advantages of convenient manufacture, small volume, portability and lower cost, overcomes the defects of complicated manufacture, large volume, inconvenience in carrying and the like of an enzyme-linked immunosorbent assay (ELISA), and can realize single-person detection. The test strip has high detection sensitivity and can meet the requirement of quantitative detection. The result is interpreted by the immunoassay analyzer, so that automation can be realized, the influence of subjective factors is reduced, and a convenient, rapid, reliable and repeatable diagnosis result is provided. The test strip is suitable for detection requirements of various medical sites.
Description
Technical Field
The invention relates to the technical field of quantitative detection by immunochromatography, in particular to a test strip for detecting oligomeric cartilage matrix protein and a detection method.
Background
Cartilage Oligomeric Matrix Protein (COMP), also known as cartilage oligomeric matrix protein, cartilage oligomeric matrix protein or cartilage oligomeric matrix protein. The oligomeric cartilage matrix protein belongs to a bone metabolism marker, is a pentameric protein formed by connecting five independent secondary units through disulfide bonds, is originally found in cartilage, and is rarely shown in tendon and synovial membrane in recent years, but has not been reported in connective tissues such as skin and lung. Cartilage, due to disease, releases protein molecules into the synovial fluid upon disruption, and these proteins, such as oligomeric cartilage matrix proteins, are also found in blood.
The detection of the oligomeric cartilage matrix protein has important clinical significance for early diagnosis, prognosis and curative effect observation of osteoarthritis, rheumatoid arthritis cartilage pathological changes and other diseases. At present, enzyme-linked immunosorbent assay (ELISA) is a common method for clinically detecting the oligomeric cartilage matrix protein at home and abroad. The method has the problems of long time consumption, low sensitivity, high background value and complicated operation, and is easy to generate false positive and false negative results, and the ELISA detection needs professional personnel to operate, so that the method is not suitable for the clinical requirement on the rapid and accurate quantitative detection of the oligomeric cartilage matrix protein. At present, the domestic detection of the oligomeric cartilage matrix protein products is less, and the establishment of a product or a method for quickly, conveniently and accurately detecting the oligomeric cartilage matrix protein is urgently needed clinically.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a test strip for detecting oligomeric cartilage matrix protein and a detection method so as to solve the technical problems.
The invention is realized by the following steps:
the invention provides a test strip for detecting oligomeric cartilage matrix protein, which comprises a substrate, wherein a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the substrate, a tracer labeled anti-oligomeric cartilage matrix protein antibody 1 and a tracer labeled quality control line ligand 1 are fixed on the combination pad, a detection line and a quality control line are arranged on the nitrocellulose membrane, an anti-oligomeric cartilage matrix protein antibody 2 is fixed on the detection line, and a ligand 2 capable of being specifically combined with the quality control line ligand 1 is fixed on the quality control line.
The above antibody 1 against the oligomeric cartilage matrix protein and the antibody 2 against the oligomeric cartilage matrix protein are antibodies against two different epitopes of the oligomeric cartilage matrix protein. Commercially available antibodies having high affinity for the oligomeric cartilage matrix protein may be selected as desired.
The invention can realize the rapid and accurate detection of the oligomeric cartilage matrix protein in the sample to be detected by utilizing the characteristic that the specific antibody for resisting the oligomeric cartilage matrix protein in the test strip is combined with the oligomeric cartilage matrix protein (antigen) in the sample to be detected.
Compared with the prior art, the detection test strip provided by the invention has the following advantages: the kit has the advantages of convenient manufacture, small volume, portability and lower cost, overcomes the defects of complicated manufacture, large volume, inconvenience in carrying and the like of an enzyme-linked immunosorbent assay (ELISA), and can realize single-person detection.
The test strip has high detection sensitivity and can meet the requirement of quantitative detection. The result is interpreted by the immunoassay analyzer, so that automation can be realized, the influence of subjective factors is reduced, and a convenient, rapid, reliable and repeatable diagnosis result is provided. The test strip is suitable for detection requirements of various medical sites.
In a preferred embodiment of the present invention, the tracer is a substance that emits light and binds to anti-AGP antibody 1 and CDL 1.
In an alternative embodiment, the tracer includes, without limitation, colloidal gold, quantum dots, fluorescent microspheres, up-converting luminescence, time-resolved fluorescence, or gold magnetic nanoparticles. The tracer is a substance that emits light and binds to the anti-oligopolychondral matrix protein antibody 1 and the quality control line ligand 1. The intensity of the tracer luminescence can be quantitatively interpreted or detected using a specific instrument. And the formed coupled compound is traced by coupling the tracer with the anti-oligomeric cartilage matrix protein antibody 1 and the quality control line ligand 1, under the action of chromatography, the anti-oligomeric cartilage matrix protein antibody 1 in the coupled compound is firstly combined with oligomeric cartilage matrix protein in a sample and then is combined with the anti-oligomeric cartilage matrix protein antibody 2 on a detection line together, the quality control line ligand 1 in the coupled compound is directly combined with the ligand 2 on a nitrocellulose membrane, finally, the tracer content at the detection line position is in positive correlation with the concentration of the oligomeric cartilage matrix protein in the sample to be detected, and the tracer content on the quality control line is basically unchanged in the same test strip. The quantitative detection of the oligomeric cartilage matrix protein is realized by comparing and analyzing a standard curve established by detecting the oligomeric cartilage matrix protein with different known concentrations on a test strip.
In a preferred embodiment of the present invention, the above-mentioned quality control line ligand 1 and ligand 2 are a pair of biomaterials capable of specific binding.
In a preferred embodiment of the present invention, the quality control line ligand 1 is selected from any one of the following substances: rabbit anti-bovine serum albumin, dinitrophenol conjugate and X IgY, wherein X is rabbit, sheep, cattle, horse, pig, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose; the ligand 2 is selected from any one of the following substances: bovine serum albumin and dinitrophenol conjugate, and Z anti-X IgY; z is rabbit, sheep, cattle, horse, pig, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose, and X is different from Z; in a preferred embodiment of the present invention, Z is sheep and X is chicken.
In a preferred embodiment of the present invention, the concentration of the coating of antibody 2 against oligomeric cartilage matrix protein on the detection line is 1-1.2 mg/mL;
the coating concentration of the ligand 2 on the quality control line is 1-1.2 mg/mL.
The coating concentration can meet the requirement of fast and high-sensitivity detection of the oligomeric cartilage matrix protein.
In a preferred embodiment of the present invention, the test strip further includes a card housing, the card housing has a cavity, the test strip is fixedly disposed in the cavity, the card housing is further provided with a sample adding hole and an observation window, the sample adding hole is disposed corresponding to the sample pad, and the observation window is disposed corresponding to the detection line and the quality control line of the nitrocellulose.
In a preferred embodiment of the present invention, the sample pad is further provided with a erythrocyte retention membrane; in an alternative embodiment, the erythrocyte retention membrane is a blood filtration membrane. The sample pad is used to filter red blood cells or other impurities in the sample.
When the sample contains red blood cells, the red blood cells are blocked by the blood filtering membrane, so that the influence of the red blood cells on the detection result can be reduced, and the detection accuracy is improved. Therefore, the test strip provided by the invention can meet the detection requirement of a whole blood sample.
The invention also provides a method for detecting the oligomeric cartilage matrix protein by using the test strip for detecting the oligomeric cartilage matrix protein, which is used for the purpose of non-disease diagnosis and comprises the following steps:
and (3) loading the sample on a sample pad, detecting a detection line of the test strip by using an instrument capable of quantitatively detecting the content of the tracer, and obtaining a quantitative result of the oligomeric cartilage matrix protein according to a standard curve (such as a standard curve obtained by five-point sampling) formulated by detecting the oligomeric cartilage matrix protein with known concentration on the test strip.
In a preferred embodiment of the present invention, the content of the above-mentioned oligomeric cartilage matrix protein is calculated according to the ratio of the content of the detection line tracer to the content of the quality control line tracer.
The invention has the following beneficial effects:
the invention can realize the rapid and accurate detection of the oligomeric cartilage matrix protein in the sample to be detected by utilizing the characteristic that the specific antibody for resisting the oligomeric cartilage matrix protein in the test strip is combined with the oligomeric cartilage matrix protein (antigen) in the sample to be detected.
Compared with the prior art, the detection test strip provided by the invention has the following advantages: the kit has the advantages of convenient manufacture, small volume, portability and lower cost, overcomes the defects of complicated manufacture, large volume, inconvenience in carrying and the like of an enzyme-linked immunosorbent assay (ELISA), and can realize single-person detection.
The test strip has high detection sensitivity and can meet the requirement of quantitative detection. The result is interpreted by the immunoassay analyzer, so that automation can be realized, the influence of subjective factors is reduced, and a convenient, rapid, reliable and repeatable diagnosis result is provided. The test strip is suitable for detection requirements of various medical sites.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic view of a partial cross-sectional structure of a test strip of the present invention;
FIG. 2 is a schematic diagram of the overall cross-sectional structure of the test strip of the present invention;
FIG. 3 is a standard curve chart plotted in Experimental example 1.
Reference numerals: 1-PVC base plate; 2-sample pad; 3-a conjugate pad; 4-nitrocellulose membrane; 5-water absorption pad; 6-clamping shell.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a test strip for quantitatively detecting oligomeric cartilage matrix protein and a detection method. Referring to fig. 1, the test strip includes a PVC base plate 1 (i.e., a substrate), and a sample pad 2, a conjugate pad 3, a nitrocellulose membrane 4, and a water absorbent pad 5 are sequentially disposed on the PVC base plate 1. Specifically, the sample pad 2 is lapped on the conjugate pad 3, and the conjugate pad 3 and the absorbent pad 5 are respectively lapped on both ends of the nitrocellulose membrane 4 in the direction of chromatography.
In this example, quantum dot-labeled anti-chondroprotein matrix antibody 1 and quantum dot-labeled chicken IgY are immobilized on the conjugate pad 3, and the coating amount of the chicken IgY is 3.0. mu.l/cm. The amount of coating of the anti-oligomeric cartilage matrix protein antibody 1 was 3.0. mu.l/cm. The quantum dot marking method comprises the following steps: firstly, activating carboxyl on the surface of the quantum dot by using EDC-hcl, then uniformly mixing the activated quantum dot with a labeled antibody, reacting for 2 hours, then using a sealant to seal, removing supernatant liquid by a centrifugal mode after each step, and finally storing in a storage solution.
The nitrocellulose membrane 4 is provided with a detection line and a quality control line, the detection line is fixed with an antibody 2 for resisting another epitope of the oligomeric cartilage matrix protein, the antibody 2 is purchased from Hytest, the quality control line is fixed with goat anti-chicken IgY, and the raw materials are from Beijing Baiqiao Ruizian biotechnology limited.
The coating concentration of the antibody 2 for resisting the oligomeric cartilage matrix protein on the detection line is 1 mg/mL;
the coating concentration of the ligand 2 on the quality control line is 1 mg/mL.
Preparing a coating buffer solution before NC membrane coating, and respectively preparing an antibody 2 of another epitope of the anti-oligomeric cartilage matrix protein into an antibody 2 coating working solution for subsequent T-line membrane scribing; the goat anti-chicken IgY is prepared into coating working solution of the antibody on the quality control line (C line).
Coating the coating working solution onto the corresponding position of NC (line C is 1.6cm away from the lower edge of NC, line spacing of C, T is 0.6cm) by a film-scribing instrument at a coating amount of 1uL/cm and a coating speed of 50mm/s, baking in an oven at 37 ℃ for 12-24h, and sealing in an aluminum foil bag filled with a desiccant for later use.
The conjugate pad was prepared as follows:
1) preparing a probe: preparing a probe preparation reagent.
(1) 100. mu.l of the quantum dot suspension was dispersed in 200. mu.l of a reaction buffer (25mM PB buffer (pH 6.0. + -. 0.2)) and mixed well.
(2) 12 μ l of coupling agent EDC (10mg/ml) was added and incubated for 0.5h with constant temperature (30 ℃ C.) mixing. Centrifuging at 15000rpm for 20min, removing supernatant, dispersing quantum dots in 100 μ l reaction buffer, and ultrasonically dispersing uniformly.
(3) Dispersing 100 mu g of anti-cartilage oligomeric matrix protein antibody 1 in 200 mu l of reaction buffer solution, quickly adding the activated quantum dots into the antibody, and uniformly mixing; mixing and incubating for 1h at constant temperature (30 ℃).
(4) Centrifugation was carried out at 12000rpm for 15min to remove free antibody.
(5) Adding 200 μ l of confining liquid, dispersing with ultrasound (power 10-20%), mixing at constant temperature (30 deg.C), and incubating for 2 h.
(6) Centrifuging at 12000rpm for 15min, removing supernatant, adding 200 μ l of preservation solution, and performing ultrasonic treatment (power 10-20%) to obtain quantum dot labeled anti-cartilage oligomeric matrix protein antibody 1 probe solution. The chicken IgY probe solution marked by the quantum dots is prepared by the same method.
2) Preparing a probe diluent: respectively diluting the quantum dot labeled anti-oligomeric cartilage matrix protein antibody 1 probe solution and the quantum dot labeled chicken IgY probe solution by using a probe diluent (0.01M PB buffer solution) to prepare a probe working solution (wherein the dilution of the anti-oligomeric cartilage matrix protein antibody 1 probe solution is 1:80, and the dilution of the chicken IgY quantum dot probe solution is 1:200), then uniformly spraying the probe working solution onto the treated binding pad by using a gold spraying instrument at the coating amount of 3ul/cm and the coating speed of 50mm/s, repeatedly spraying the probe working solution once, baking the probe working solution in a 45 ℃ baking oven for 12-24h, and then filling the probe working solution into an aluminum foil bag filled with a drying agent for sealing for later use.
Referring to fig. 2, the test strip further comprises a card shell 6, the card shell 6 has a cavity, a sample adding hole and an observation window are arranged on the cavity, and the PVC base plate 1, the sample pad 2, the combination pad 3, the nitrocellulose membrane 4 and the absorbent pad 5 are all fixed in the cavity.
The method for detecting the oligomeric cartilage matrix protein by using the test strip comprises the following steps:
and adding a sample to be detected from the sample adding hole, wherein the oligomeric cartilage matrix protein in the sample passes through the sample pad 2, is combined with the anti-oligomeric cartilage matrix protein antibody marked by the quantum dots in the combination pad 3 and flows laterally along the nitrocellulose membrane 4 to the direction of the absorbent paper 5, and is combined by the antibody of the other epitope of the anti-oligomeric cartilage matrix protein and stays on the detection line when flowing through the detection line. Under the drive of the liquid in the sample, the chicken IgY marked by the quantum dots in the combined pad flows laterally along the nitrocellulose membrane 4 to the direction of the absorbent paper 5, and is combined by the chicken IgY on the quality control line and stays on the quality control line when flowing through the quality control line.
And detecting the test strip from the observation window by adopting a quantum dot fluorescence immunoassay analyzer capable of quantitatively detecting the content of the quantum dots. The fluorescence intensity (T) of the quantum dots at the detection line position is in positive correlation with the concentration of the oligomeric cartilage matrix protein in the sample to be detected, and the fluorescence intensity (C) of the quantum dots of the chicken IgY marked by the quantum dots formed on the quality control line is a constant value which is basically consistent on the same batch of test strips. The method comprises the steps of detecting the oligomeric cartilage matrix protein with different known concentrations on a test strip, calculating corresponding T/C values and formulating a calibration curve, and realizing quantitative detection of the oligomeric cartilage matrix protein by using the T/C values of a sample on the test strip and combining the calibration curve during detection.
Example 2
In this example, colloidal gold is used as the tracer. The coating amount of the antibody 1 of the colloidal gold-labeled anti-oligomeric cartilage matrix protein and the chicken IgY labeled with the colloidal gold on the combined pad 3 is 3.0 mul/cm. The method of colloidal gold labeling is well established and will not be described in detail herein. The labeled amount of the colloidal gold-labeled anti-chondroprotein matrix protein antibody 1 was the same as in example 1.
Compared with the example 1, the detection instrument is a colloidal gold immunoassay analyzer. The other detection methods and the preparation method of the test strip are the same as those in example 1.
Example 3
In this example, fluorescent microspheres are used as the tracer. The coating amount of the anti-cartilage oligomeric matrix protein antibody 1 marked by the fluorescent microspheres and the chicken IgY marked by the fluorescent microspheres on the binding pad 3 is 3.0 mu l/cm. The method for labeling the fluorescent microspheres comprises the following steps: dissolving the fluorescent microspheres with dimethyl sulfoxide, desalting and replacing a buffer system with the antibody to be labeled, detecting the protein concentration, mixing the fluorescent microspheres and the antibody to be labeled in proportion, shaking and mixing uniformly for 2 hours in a dark place, adding sodium hydroxide solution to stop the reaction, desalting with a dialysis membrane, and calculating the labeling ratio for later use. The labeled amount of the fluorescent microsphere-labeled anti-chondroprotein antibody 1 was the same as that in example 1.
In contrast to example 1, the detection instrument was a fluorescence immunoassay analyzer. The other detection methods and the preparation method of the test strip are the same as those in example 1.
Example 4
In this embodiment, the tracer is gold magnetic nanoparticles. The coating amount of the gold magnetic nano-labeled anti-cartilage matrix protein antibody 1 and the gold magnetic nano-labeled chicken IgY on the combination pad 3 is 3.0 mu l/cm. The labeling amount of the gold magnetic nano-labeled anti-chondroprotein matrix protein antibody 1 was the same as that of example 1.
Compared with the embodiment 1, the detection instrument is a gold magnetic nano immunoassay analyzer. The other detection methods and the preparation method of the test strip are the same as those in example 1.
Example 5
In this example, the tracer employs up-conversion luminescence. The conjugate pad 3 has the anti-chondroglobin matrix protein antibody 1 labeled by up-conversion luminescence and chicken IgY labeled by up-conversion luminescence, and the coating amount is 3.0. mu.l/cm. The labeled amount of the up-converting luminescent-labeled anti-chondroprotein matrix protein antibody 1 was the same as that in example 1.
The detection instrument was an up-converting luminescence immunoassay instrument, compared to example 1. The other detection methods and the preparation method of the test strip are the same as those in example 1.
Example 6
In this example, the tracer employs time-resolved fluorescence. The coating amount of the anti-oligomeric cartilage matrix protein antibody 1 with time-resolved fluorescence labeling and the chicken IgY with time-resolved fluorescence labeling on the binding pad 3 is 3.0 mu l/cm. The amount of the time-resolved fluorescence-labeled anti-chondroprotein matrix protein antibody 1 was the same as in example 1.
In contrast to example 1, the detection instrument was a time-resolved fluorescence analyzer. The other detection methods and the preparation method of the test strip are the same as those in example 1.
Example 7
In this embodiment, the tracer is a quantum dot. The coating amount of the anti-cartilage oligomeric matrix protein antibody 1 marked by the quantum dots and the chicken IgY marked by the quantum dots on the binding pad 3 is 3.0 mu l/cm. The labeling amount of the quantum dot-labeled anti-chondroprotein antibody 1 was the same as that in example 1.
The rest of this example is the same as example 1.
Example 8
Compared with the embodiment 1, the difference is only that the blood filtering membrane is adopted or added in the sample pad, when the sample contains red blood cells, the red blood cells are blocked by the blood filtering membrane, and the influence of the red blood cells on the detection result can be reduced. The test strip provided by the invention is suitable for detecting a whole blood sample.
Experimental example 1
The method comprises the steps of detecting the oligomeric cartilage matrix protein with different known concentrations on a test strip, calculating corresponding T/C values and formulating a calibration curve, and realizing quantitative detection of the oligomeric cartilage matrix protein by using the T/C values of a sample on the test strip and combining the calibration curve during detection. And during actual calibration, selecting 5-11 different concentrations of the oligomeric cartilage matrix protein calibration stock in a detection linear range, detecting by using a test strip, calculating the T/C value of each concentration calibration stock, performing binary polynomial fitting on a curve by using the T/C value as an abscissa and the concentration as an ordinate, and calculating Y-aX2The values of a, b, c of + bX + c. And when the sample is detected, substituting the T/C value obtained by detecting the sample into a standard curve formula to obtain the concentration of the oligomeric cartilage matrix protein in the sample.
And (3) carrying out repeated detection for 3 times by using working calibrators Q0-Q7, recording corresponding T/C values, calculating an average value, and carrying out curve fitting by taking the concentration of the working calibrators as a vertical coordinate and the average value of the T/C values corresponding to the working calibrators as a horizontal coordinate.
Quantitative measurement of aggrecan is shown in the following table, and a standard curve prepared therefrom is shown in FIG. 3.
The rest of the experimental example is the same as example 1, and the characteristics not explained in this example are explained by example 1.
From the above test results, the analytical sensitivity was 0.2 ng/mL. Linear range: in the concentration range of 0.2-50ng/mL, R2The detection linear correlation coefficient of the kit can meet the requirement that r is more than or equal to 0.990.
Experimental example 2
And detecting the C value of the optical intensity of the tracer on the quality control line of each batch of test strips, calculating the average value and SD, setting a range for the C value during detection, and judging that the test strips have problems in the detection process when the C value is higher or lower than the range, and giving an alarm or not reporting the result by an instrument. Can play a role in quality control.
The rest of this embodiment is the same as embodiment 1, and the features not explained in this embodiment are explained by embodiment 1.
Experimental example 3
This example was subjected to stability testing.
Through the real-time stability (2-8 ℃, 8-30 ℃) experiments of 201001, 201101 and 201102 batches of test strips for detecting aggrecan prepared in example 1, all performance indexes of the products meet the requirements after 20 months.
The test results of the unsealing stability tests of 201001, 201101 and 201102 batches (the test strip for detecting aggrecan prepared in example 1) show that the test results are not affected within 30 minutes after the aluminum foil bags are torn open.
The results of accelerated stability tests (37 ℃) on 201001, 201101 and 201102 batches (test strips for detecting aggrecan prepared in example 1) show that the stability of the aggrecan determination kit (quantum dot fluorescence immunochromatography) produced by the invention can ensure that the validity period is 18 months.
The results of transportation stability tests (simulating high and low temperature transportation) on 201001, 201101 and 201102 batches (test strips for detecting aggrecan prepared in example 1) of products show that the test strips for detecting aggrecan (quantum dot fluorescence immunochromatography) can withstand extreme weather of over-high temperature and over-low temperature in the transportation process.
The stability test of the serum, plasma and whole blood samples at the storage conditions of 2-8 ℃ and-20 ℃ shows that the serum, plasma and whole blood samples can be stored for 5 days at the temperature of 2-8 ℃ and can be stored for 3 months at the temperature of-20 ℃.
Experimental example 4
The test strip provided in example 1 was subjected to an analytical reproducibility test and an accuracy test.
Repeatability of
The detection reagent of the same batch performs repeated detection 10 times on a sample with a certain concentration in the detection range, calculates the average value (M) and Standard Deviation (SD) of 10 measurement results, and obtains the coefficient of variation according to the formula CV as SD/M multiplied by 100%.
Inter-batch precision
The three batches of the detection reagent are repeatedly tested 10 times for each sample with a certain concentration in the detection range, the average value (M) and the Standard Deviation (SD) of 30 measurement results are calculated, and the variation coefficient is obtained according to the formula CV as SD/M multiplied by 100%.
Accuracy of
The detection of the same batch of detection reagent is repeated for 3 times for a sample with a certain concentration in the detection range. The relative deviation B is calculated according to equation (1).
B＝(M-T)/T×100％ (1)
In the formula: b-relative deviation;
m-mean value of measured concentration;
t-calibration concentration.
The result shows that the coefficient of variation CV value of the repeatability test is 9.6 percent; the batch-to-batch precision CV value was 12.1%. The accuracy test relative deviation was 7.5%.
In addition, the experimental example also examined the hook effect: it was found that the test strip provided in example 1 did not produce a hook effect at a concentration range of 3000 ng/mL.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The test strip for detecting the oligomeric cartilage matrix protein is characterized by comprising a substrate, wherein a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the substrate, a tracer-labeled anti-oligomeric cartilage matrix protein antibody 1 and a tracer-labeled quality control line ligand 1 are fixed on the combination pad, a detection line and a quality control line are arranged on the nitrocellulose membrane, an anti-oligomeric cartilage matrix protein antibody 2 is fixed on the detection line, and a ligand 2 capable of being specifically combined with the quality control line ligand 1 is fixed on the quality control line.
2. The strip for detecting an oligomeric cartilage matrix protein according to claim 1, wherein said tracer is a substance capable of emitting light and binding to said anti-oligomeric cartilage matrix protein antibody 1 and said quality control line ligand 1;
preferably, the tracer is colloidal gold, quantum dots, fluorescent microspheres, up-conversion luminescence, time-resolved fluorescence or gold magnetic nanoparticles.
3. The strip of claim 2, wherein the quality control line ligand 1 and the ligand 2 are a pair of biomaterials capable of specific binding.
4. The strip of claim 3, wherein the quality control line ligand 1 is selected from any one of the following substances: rabbit anti-bovine serum albumin and dinitrophenol conjugate, XIgY, wherein X is rabbit, sheep, cattle, horse, pig, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose; the ligand 2 is selected from any one of the following substances: bovine serum albumin and dinitrophenol conjugate, and Z anti-X IgY; z is rabbit, sheep, cattle, horse, pig, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose, and X is different from Z;
preferably, Z is sheep and X is chicken.
5. The strip for detecting oligomeric cartilage matrix protein according to claim 3, wherein the coating concentration of antibody 2 against oligomeric cartilage matrix protein on the detection line is 1-1.2 mg/mL; the coating concentration of the ligand 2 on the quality control line is 1-1.2 mg/mL.
6. The test strip for detecting oligomeric cartilage matrix protein according to claim 1, further comprising a card housing, wherein the card housing has a cavity, the test strip is fixedly disposed in the cavity, the card housing is further provided with a sample adding hole and an observation window, the sample adding hole is disposed corresponding to the sample pad, and the observation window is disposed corresponding to the detection line and quality control line of the nitrocellulose.
7. The strip of claim 1, wherein the sample pad is further provided with a erythrocyte-cutting membrane.
8. The strip of claim 7, wherein the erythrocyte-cutting membrane is a hemofiltration membrane.
9. A method for detecting an oligomeric cartilage matrix protein using the test strip for detecting an oligomeric cartilage matrix protein according to any one of claims 1 to 8, comprising:
and (3) loading the sample onto a sample pad, detecting the detection line and the quality control line of the test strip by adopting an instrument capable of quantitatively detecting the content of the tracer, and obtaining the quantitative result of the oligomeric cartilage matrix protein according to a standard curve formulated by signals of the detection line and the quality control line of the oligomeric cartilage matrix protein with known concentration on the test strip.
10. The method of claim 9, wherein the amount of oligomeric cartilage matrix protein is calculated from the ratio of detection line tracer amount to quality control line tracer amount.
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| CN113466448A (en) * | 2021-06-30 | 2021-10-01 | 广东牧玛生命科技有限公司 | Placental protein 13 test strip and quantitative detection method |
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