CN109444406A - Test strips of quantitative detection marker cyfra21-1 and preparation method thereof and detection method - Google Patents
Test strips of quantitative detection marker cyfra21-1 and preparation method thereof and detection method Download PDFInfo
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- CN109444406A CN109444406A CN201811329449.7A CN201811329449A CN109444406A CN 109444406 A CN109444406 A CN 109444406A CN 201811329449 A CN201811329449 A CN 201811329449A CN 109444406 A CN109444406 A CN 109444406A
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Abstract
本发明涉及一种定量检测标志物cyfra21‑1的试纸条及其制备方法和检测方法，该试纸条包括样本垫，喷涂有处理液；结合垫，喷涂有近红外荧光纳米微球标记的抗cyfra21‑1的第一抗体；硝酸纤维素膜，设置有由抗cyfra21‑1的第二抗体形成的检测带和由羊抗鼠多克隆抗体形成的质控带；以及吸水垫。本发明的定量检测标志物cyfra21‑1的试纸条及其制备方法和检测方法是针对尿液中cyfra21‑1进行定量检测，灵敏度高，特异性高，可用于膀胱癌的早期检测和复发监测，直接将尿液滴至试纸条上便可以快速检测，而且该试纸条制备过程简单，可批量化生产，成本低，试纸检测手段快速，省时省力，适合临床广泛应用。
The invention relates to a test strip for quantitative detection of marker cyfra21-1 and a preparation method and detection method thereof. The test strip comprises a sample pad, which is sprayed with a treatment liquid; a binding pad, which is sprayed with a near-infrared fluorescent nano-microsphere marker an anti-cyfra21-1 primary antibody; a nitrocellulose membrane, provided with a detection zone formed by an anti-cyfra21-1 secondary antibody and a quality control zone formed by a goat-anti-mouse polyclonal antibody; and an absorbent pad. The test strip for quantitatively detecting the marker cyfra21-1 and its preparation method and detection method of the present invention are for quantitatively detecting cyfra21-1 in urine, have high sensitivity and high specificity, and can be used for early detection and recurrence monitoring of bladder cancer The test strip can be quickly detected by directly dripping urine onto the test strip, and the test strip has a simple preparation process, can be mass-produced, and has low cost.
Description
Technical Field
The invention relates to the technical field of detection and analysis, in particular to a test strip for quantitatively detecting a marker cyfra21-1, a preparation method and a detection method thereof.
Background
Bladder cancer is a common malignant tumor of the urinary system, and the incidence rate of male bladder cancer in China tends to rise year by year and to be younger. Despite the great advances in the diagnostic and therapeutic techniques for bladder cancer in recent years, the mortality rate of bladder cancer remains high. Bladder cancer currently lacks effective therapeutic targets and postoperative assessment indices, with approximately 5% to 50% of patients experiencing postoperative recurrence or metastasis.
At present, the diagnosis of bladder cancer, especially the monitoring of postoperative recurrence, mainly takes the cytology examination of urine abscission and the urethral cystoscopy examination. However, cystoscopy is invasive, inconvenient to operate, painful to the patient, and has risks of infection, bleeding, and the like, and the detection cost is expensive. Urine exfoliative cytology has good specificity but low sensitivity, and is easily interfered by factors such as urinary tract infection. Therefore, both of these examination methods have certain limitations in clinical applications.
In recent years, scholars at home and abroad are dedicated to searching sensitive, specific, reliable and effective bladder cancer biomarkers from urine and establishing a noninvasive detection method to improve the early diagnosis rate of bladder cancer.
cyfra21-1 is a soluble fragment of cytokeratin 19 with a molecular weight of about 30000 Da. In malignant lung cancer tissues, cyfra21-1 is abundant, and is highly expressed in lung squamous carcinoma. At present, the kit is mainly used as a tumor marker, has great significance for the diagnosis of lung cancer, and is helpful for doctors to predict the treatment effect and prognosis. Has certain early diagnosis value for oropharyngeal squamous cell carcinoma, esophageal cancer and colorectal cancer. cyfra21-1 was also valuable for the diagnosis of bladder cancer, with sensitivity of 69.6% and 91.9% for low-grade and high-grade bladder cancer, respectively. Another study showed that the optimal cutoff concentration of urine for detection of primary bladder tumors by cyfra21-1 was 4.9. mu.g/L, sensitivity was 79.3%, and specificity was 88.6%; the optimal detection threshold for detecting recurrent bladder tumors was 4.04. mu.g/L, sensitivity was 76.2%, and specificity was 84.2%. One META analysis in 2014 showed a collective sensitivity of cyfra21-1 for bladder cancer diagnosis of 71% with a collective specificity of 75%.
Conventionally, for the detection of cyfra21-1 in urine, methods such as ELISA and colloidal gold immunochromatography have been generally used. The ELISA method needs to pretreat urine to extract protein, is not suitable for rapid detection, is rapid in colloidal gold immunochromatography, but has a large false negative proportion, can be qualitative or semi-quantitative, and has to be improved in specificity and sensitivity. Therefore, there is a need to develop a new means for early detection of bladder cancer urine by combining new methods, new technologies and new materials, and to improve the specificity and sensitivity for bladder cancer diagnosis.
Disclosure of Invention
The invention aims to provide a test strip for quantitatively detecting a marker cyfra21-1 and a preparation method and a detection method thereof, and solves the problem that the detection method for cyfra21-1 in urine in the prior art cannot achieve rapid, high-specificity and high-sensitivity detection at the same time.
The technical scheme adopted by the invention for solving the technical problem is as follows: a test strip for quantitatively detecting a marker cyfra21-1, comprising:
a sample pad sprayed with a treatment fluid;
the combination pad is sprayed with a first anti-cyfra 21-1 antibody marked by near-infrared fluorescent nano microspheres;
a nitrocellulose membrane provided with a detection band formed by a second antibody against cyfra21-1 and a quality control band formed by a goat anti-mouse polyclonal antibody;
and a water absorbent pad.
In the test strip of the invention, the first antibody of the near-infrared fluorescent nano-microsphere labeled anti-cyfra 21-1 is labeled by the following method:
s1, initial washing: absorbing 550 mu L of near-infrared fluorescent nano microspheres, adding initial washing liquid, ultrasonically mixing, absorbing and removing supernatant after centrifugation, resuspending precipitate by using the initial washing liquid, ultrasonically mixing, absorbing and removing supernatant after centrifugation, repeatedly washing at least once, resuspending by using the initial washing liquid after the last washing, ultrasonically mixing, and ensuring that the near-infrared fluorescent nano microspheres are in a monodisperse state;
s2, activation: adding 80-120 mu L of 20mg/mL EDC and 350 mu L of 20mg/mL NHS 250-350 mu L which are prepared in situ, ultrasonically mixing, starting timing and activating for at least 15min from adding EDC and NHS, centrifuging at the temperature of 2-8 ℃, sucking and removing supernatant, and re-suspending with coupling solution;
s3, coupling: ultrasonically mixing the solution in a microsphere solution in which the coupling solution is suspended again, centrifuging the solution, discarding the supernatant, ultrasonically mixing the solution, centrifuging the solution, discarding the supernatant, and repeatedly washing the solution at least once; adding coupling solution for resuspension, adding a first antibody, wherein the adding amount of the first antibody is 30 mug of the first antibody per 100 mul of microspheres, ultrasonically mixing uniformly, and oscillating by using a shaking table, wherein the ultrasonic treatment is performed once every 20min-40 min;
s4, sealing: adding 2-5 mu L of ethanolamine after coupling, adding a sealing solution for heavy suspension, oscillating by using a shaking table, centrifuging at the temperature of 2-8 ℃ after sealing, and removing a supernatant;
s5, final washing: resuspending with final washing solution, ultrasonically mixing, centrifuging at 2-8 deg.C, removing supernatant, and repeatedly washing at least once; the precipitate was dissolved in 400-600. mu.L of final wash.
In the test strip of the present invention, step S1 specifically includes: absorbing 500 mu L of fluorescent microspheres, adding 1mL of primary washing liquid, fully mixing by ultrasound, centrifuging at a high speed, removing the supernatant by suction, resuspending the precipitate by using 1mL of primary washing liquid, fully mixing by ultrasound for 2min, centrifuging at a high speed, removing the supernatant by suction, repeatedly washing at least once, resuspending by using 1mL of primary washing liquid after the last washing, fully mixing by ultrasound, and ensuring that the microspheres are in a monodisperse state;
step S2 specifically includes: adding 100 mu L of 20mg/mL EDC and 300 mu L of 20mg/mL NHS which are prepared in situ, ultrasonically mixing the mixture, starting to add EDC and NHS, timing and activating the mixture for 15min, centrifuging the mixture at a high speed at 4 ℃, sucking and removing supernatant, and resuspending the mixture by using 1mL of coupling solution;
step S3 specifically includes: ultrasonically mixing the solution in 1mL of coupling solution resuspended microsphere solution, centrifuging at a high speed, discarding the supernatant, ultrasonically mixing the solution, centrifuging at a high speed, discarding the supernatant, and repeatedly washing at least once; adding 1000 μ L coupling solution for resuspension, adding 150 μ g first antibody, mixing, shaking for 3 hr, and performing ultrasonic treatment for 2min every 30 min;
step S4 specifically includes: adding 3 mu L of ethanolamine stock solution after coupling is finished, adding 800 mu L of confining liquid for heavy suspension, oscillating for 2.5h by a shaking table, centrifuging at high speed at 4 ℃ after confining is finished, and removing supernatant;
step S5 specifically includes: resuspending with 1mL of final washing solution, ultrasonically mixing, centrifuging at high speed at 4 ℃, removing supernatant, and repeatedly washing at least once; the precipitate was dissolved in 500. mu.L of the final wash.
In the test strip, the processing solution sprayed on the sample pad is prepared from Tris, BSA and Tween-20, the concentration of Tris is 0.01mol/L, the concentration of BSA is 0.5g/100mL, and the concentration of Tween-20 is 0.5g/100 mL;
in step S1, the initial washing solution is a buffer solution with pH6.0 prepared by MES, and the concentration of MES is 0.1 mol/L;
in steps S2 and S3, the coupling solution is a buffer solution with pH6.5 prepared by boric acid and sodium tetraborate, the concentration of the boric acid is 0.2mol/L, and the concentration of the sodium tetraborate is 0.01 mol/L;
in step S4, the blocking solution is a buffer solution of pH7.2 prepared from PBS, BSA and Tween-20, and the concentration of PBS is 0.01mol/L, the concentration of BSA is 0.5g/100mL, and the concentration of Tween-20 is 0.4g/100 mL;
in step S5, the final wash solution is a buffer solution of pH7.2 prepared from PBS, BSA, and Tween-20, and the concentration of PBS is 0.01mol/L, the concentration of BSA is 0.5g/100mL, and the concentration of Tween-20 is 0.4g/100 mL.
In the test strip, the near-infrared fluorescent nano-microspheres are europium chelate PS-COOH microspheres, and the average particle size distribution of the near-infrared fluorescent nano-microspheres is 0.18-0.22 mu m; the sample pad and the conjugate pad are each a glass cellulose membrane; the water absorption pad is water absorption paper or water absorption cloth.
In the test strip of the present invention, the sample pad, the conjugate pad, the nitrocellulose membrane, and the absorbent pad are fixed by an adhesive base plate, and the sample pad, the conjugate pad, the nitrocellulose membrane, and the absorbent pad are bonded to the adhesive base plate.
The invention also provides a preparation method of the test strip, which comprises the following steps:
A. marking a first antibody of anti-cyfra 21-1 by using near-infrared fluorescent nano microspheres;
B. spraying a first anti-cyfra 21-1 antibody marked by near-infrared fluorescent nano microspheres on the bonding pad, and drying for later use;
C. spraying a treatment liquid on the sample pad, and drying for later use;
D. selecting a nitrocellulose membrane, preparing a second antibody of anti-cyfra 21-1 and a goat anti-mouse polyclonal antibody into a detection band and a quality control band on the nitrocellulose membrane by using a scribing machine, and drying for later use.
In the preparation method of the present invention, step D is followed by step E: and sequentially bonding the nitrocellulose membrane, the combination pad, the sample pad and the water absorption pad on the viscous bottom plate, then cutting the test paper into test paper strips by a cutting machine, putting the test paper strips into the plastic clamping grooves, and assembling to obtain a finished product.
The invention also provides a cyfra21-1 detection method of the test strip, which comprises the following steps:
s10, diluting a cyfra21-1 standard substance in multiple times to obtain a plurality of standard samples with different concentrations, respectively sucking the plurality of standard samples by a microsyringe, respectively dripping the standard samples on the test strips corresponding to each standard sample, standing at room temperature, putting each test strip in a near-infrared fluorescence scanner for reading, recording a Dr value, measuring each standard sample with each concentration at least twice, and making a standard curve graph of the sample concentration by using the Dr value after averaging; wherein, Dr value is the ratio of the fluorescence value of the detection line divided by the fluorescence value of the quality control line;
s20, sucking the urine sample to be detected to the test strip, standing at room temperature, and sending the test strip to a near infrared fluorescence scanner for reading to obtain the concentration of cyfra 21-1.
In the detection method, in step S10, the absorption amount of each standard sample is 80-120 mul, and the standard sample is dripped on a test strip and then stands for 12-20 min at room temperature;
in step S20, dropwise adding the urine sample to be tested on the test strip, and standing at room temperature for 12-20 min; wherein, the urine sample to be detected is collected from the patient and stored at the temperature of 2-8 ℃, the suction volume of the urine sample to be detected is 80-120 mul, and the detection is required to be completed within 4 h.
The test strip for quantitatively detecting the marker cyfra21-1, the preparation method and the detection method thereof have the following beneficial effects: the test strip for quantitatively detecting the marker cyfra21-1, the preparation method and the detection method thereof carry out quantitative detection on cyfra21-1 in urine, have high sensitivity and high specificity, can be used for early detection and recurrence monitoring of bladder cancer, can carry out rapid detection by directly dripping urine on the test strip, has simple preparation process, can be produced in batches, has low cost, rapid test strip detection means, saves time and labor, and is suitable for wide clinical application.
Drawings
FIG. 1 is a schematic structural diagram of a test strip for quantitatively detecting the marker cyfra 21-1;
FIG. 2 is a standard graph of cyfra21-1 testing.
Detailed Description
The test strip for quantitatively detecting the marker cyfra21-1, the preparation method and the detection method thereof will be further described with reference to the accompanying drawings and examples:
as shown in FIG. 1, the test strip for quantitatively detecting the marker cyfra21-1 of the present invention comprises a sample pad 2, a binding pad 3, a nitrocellulose membrane 4 and a water absorbent pad 7, wherein a detection band 5 and a quality control band 6 are fixed on the nitrocellulose membrane 4.
The test strip for quantitatively detecting the marker cyfra21-1 utilizes a fluorescence immunochromatography technology. The fluorescence immunochromatography technology is a novel membrane detection technology based on antigen-antibody specific immunoreaction. The technology takes strip-shaped fiber chromatographic materials fixed with a detection band 5 (coating antibody) and a quality control band 6 (antibody) as a stationary phase, a test solution as a mobile phase, a fluorescence labeling antibody fixed on a binding pad 3 and an analyte moving on the chromatographic strip through capillary action. Usually, a "sandwich" type double-antibody sandwich immunochromatography method is adopted, i.e., an analyte is firstly combined with a fluorescence labeled antibody under the action of a mobile phase, and then combined with a coating antibody to form a "sandwich" type double-antibody sandwich when reaching the detection zone 5.
The test strip for quantitatively detecting the marker cyfra21-1, the preparation method and the detection method thereof utilize a fluorescent quantitative immunochromatography technology, have the advantages of simple and convenient operation, quick detection and strong portability, and realize the accurate quantification of the detection result by a fluorescent tracing enhancement technology. Compared with the traditional rapid detection technology, the technology has the following advantages: (1) the sensitivity is higher: the fluorescence quantitative immunochromatography product directly detects an excited fluorescence signal by a functionalized nano microsphere carrier technology and a fluorescence marker probe. The detection signal has higher signal-to-noise ratio and higher signal detection amount and detection sensitivity, and meanwhile, carboxyl with proper density is modified on the surface of the nano fluorescent microsphere and is used for covalent coupling with an antibody, so that the stability of the marker is improved; (2) the detection range is wider: different from the traditional photometric percentage analysis and optical density scanning analysis technologies, the fluorescence immunochromatography technology adopts a fluorescence direct excitation luminescence detection means, the fluorescence signal intensity is linearly related to the number of fluorescent microspheres, the problems of catalytic efficiency, substrate amount limitation and the like existing in an enzyme catalysis luminescence technology are solved, the quantitative range is directly related to the specific protein amount participating in the reaction in a reaction system, the technical sensitivity of detecting cyfra21-1 by the test strip can reach more than 1.2ng/ml, and the performance index is far higher than that of other rapid detection technologies; (3) the price is low: compared with the traditional quantitative detection technology, the technology has the advantages of high speed, low price and the like.
As shown in FIG. 1, the test strip for quantitatively detecting the marker cyfra21-1 of the present invention comprises a sample pad 2, a binding pad 3, a nitrocellulose membrane 4, and a water absorption pad 7. Wherein, the sample pad 2 is sprayed with the treatment liquid; a first antibody which is labeled by near-infrared fluorescent nano microspheres and is used for resisting cyfra21-1 is sprayed on the binding pad 3; on the nitrocellulose membrane 4, a detection band 5 formed of a second antibody against cyfra21-1 and a quality control band 6 formed of a goat anti-mouse polyclonal antibody were provided.
Wherein, the processing liquid sprayed on the sample pad 2 is prepared by Tris, BSA and Tween-20, the concentration of Tris is 0.01mol/L, the concentration of BSA is 0.5g/100mL, and the concentration of Tween-20 is 0.5g/100 mL.
The near-infrared fluorescent nano-microsphere is a europium chelate PS-COOH microsphere, the manufacturer of the europium chelate PS-COOH microsphere is Bangs Laboratories, Inc, the average particle size distribution of the near-infrared fluorescent nano-microsphere is 0.18-0.22 mu m, and the average particle size of the near-infrared fluorescent nano-microsphere is preferably 0.196 mu m. The primary antibody against cyfra21-1 was Fitzgerald brand cat # 10-2689 at a concentration of 4 mg/mL. The second antibody against cyfra21-1 was Fitzgerald brand cat # 10-2732 at a concentration of 4 mg/mL. The goat anti-mouse polyclonal antibody is of Arista Biologicals inc brand cat # ABGAM-0500.
The sample pad 2 and the conjugate pad 3 are each a glass cellulose membrane; the absorbent pad 7 is absorbent paper or absorbent cloth.
The sample pad 2, the combination pad 3, the nitrocellulose membrane 4 and the absorbent pad 7 are fixed by the adhesive base plate 1, and the sample pad 2, the combination pad 3, the nitrocellulose membrane 4 and the absorbent pad 7 are adhered to the adhesive base plate 1.
The preparation method of the test strip for quantitatively detecting the marker cyfra21-1 comprises the following steps:
A. marking a first antibody of anti-cyfra 21-1 by using near-infrared fluorescent nano microspheres;
B. spraying a first anti-cyfra 21-1 antibody marked by near-infrared fluorescent nano microspheres on the bonding pad 3, and drying for later use; the drying temperature is 35-40 ℃, and the drying time is 8-15 h; preferably drying in a strong convection air oven at 37 ℃ for 10 hours;
C. spraying a treatment liquid on the sample pad 2, and drying for later use; the drying temperature is 35-40 ℃, and the drying time is 8-15 h; preferably drying in a strong convection air oven at 37 ℃ for 10 hours; the treatment solution is prepared from Tris, BSA and Tween-20, the concentration of Tris is 0.01mol/L, the concentration of BSA is 0.5g/100mL, and the concentration of Tween-20 is 0.5g/100 mL;
D. selecting a nitrocellulose membrane 4, preparing a second antibody of anti-cyfra 21-1 and a goat anti-mouse polyclonal antibody on the nitrocellulose membrane 4 by using a scribing machine to prepare a detection band 5 and a quality control band 6, and drying for later use; the drying temperature is 35-40 ℃, and the drying time is 8-15 h; preferably in a strong convection air oven at 37 c for 10 hours.
E. And sequentially bonding the nitrocellulose membrane 4, the combination pad 3, the sample pad 2 and the water absorption pad 7 on the viscous bottom plate 1, then cutting the test paper into test paper strips by a cutting machine, putting the test paper strips into the plastic clamping grooves, and assembling to obtain a finished product.
In step A, the first antibody against cyfra21-1 labeled by the near-infrared fluorescent nanospheres is labeled by the following method:
s1, initial washing: absorbing 550 mu L of near-infrared fluorescent nano microspheres, adding initial washing liquid, ultrasonically mixing, absorbing and removing supernatant after centrifugation, resuspending precipitate by using the initial washing liquid, ultrasonically mixing, absorbing and removing supernatant after centrifugation, repeatedly washing at least once, resuspending by using the initial washing liquid after the last washing, ultrasonically mixing, and ensuring that the near-infrared fluorescent nano microspheres are in a monodisperse state; wherein the primary washing solution is a buffer solution with pH6.0 prepared by MES, the concentration of the MES is 0.1mol/L, and the MES refers to 2- (N-morpholine) ethanesulfonic acid; the dosage of the initial washing liquid is 0.8-1.5ml preferably each time;
s2, activation: adding 80-120 mu L of 20mg/mL EDC and 350 mu L of 20mg/mL NHS 250-chitosan, ultrasonically mixing, starting timing and activating for at least 15min from the addition of EDC and NHS, centrifuging at the temperature of 2-8 ℃, sucking and removing supernatant, and re-suspending with coupling solution; wherein the coupling solution is a buffer solution with pH of 6.5 prepared from boric acid and sodium tetraborate, the concentration of the boric acid is 0.2mol/L, and the concentration of the sodium tetraborate is 0.01 mol/L; EDC is a pharmaceutical chemical reagent for 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; NHS is a pharmaceutical chemical reagent for N-hydroxysuccinimide; the dosage of the coupling solution is preferably 0.8-1.5ml each time;
s3, coupling: ultrasonically mixing the solution in a microsphere solution in which the coupling solution is suspended again, centrifuging the solution, discarding the supernatant, ultrasonically mixing the solution, centrifuging the solution, discarding the supernatant, and repeatedly washing the solution at least once; adding coupling solution for resuspension, adding a first antibody, wherein the adding amount of the first antibody is 30 mug of the first antibody per 100 ul of microspheres, ultrasonically mixing uniformly, and oscillating by using a shaker, wherein the shaking by using a shaker with 100-200 turns is preferred, the oscillating time is preferably 2-4h, and the ultrasound is performed once every 20-40 min; it should be noted that, the adding amount of the first antibody is 30 μ g of the first antibody per 100 μ l of the microspheres, which means that 30 μ g of the first antibody is added to each 100 μ l of the microspheres which absorb the near-infrared fluorescent nanoparticles during initial washing;
s4, sealing: after the coupling is finished, adding 2-5 mu L of ethanolamine, wherein the ethanolamine is 100% stock solution, adding a sealing solution for heavy suspension, the sealing solution is preferably 600-1000 mu L, oscillating by using a shaker, the shaking by using a shaker with 100-200 revolutions is preferred, the oscillating time is preferably 2-3.5h, and after the sealing is finished, centrifuging at the temperature of 2-8 ℃ and removing supernatant; wherein the blocking solution is a buffer solution with pH7.2 prepared from PBS, BSA and Tween-20, the concentration of the PBS is 0.01mol/L, the concentration of the BSA is 0.5g/100mL, and the concentration of the Tween-20 is 0.4g/100 mL;
s5, final washing: resuspending with final washing solution, preferably 0.8-1.5ml, ultrasonically mixing, centrifuging at 2-8 deg.C, removing supernatant, and repeatedly washing at least once; the precipitate was dissolved in 400-600. mu.L of final wash; wherein the final washing solution is a buffer solution with pH7.2 prepared by PBS, BSA and Tween-20, the concentration of the PBS is 0.01mol/L, the concentration of the BSA is 0.5g/100mL, and the concentration of the Tween-20 is 0.4g/100 mL.
Preferably, step S1 specifically includes: absorbing 500 mu L of fluorescent microspheres, adding 1mL of primary washing liquid, fully mixing by ultrasound, centrifuging at a high speed, removing the supernatant by suction, resuspending the precipitate by using 1mL of primary washing liquid, fully mixing by ultrasound for 2min, centrifuging at a high speed, removing the supernatant by suction, repeatedly washing at least once, resuspending by using 1mL of primary washing liquid after the last washing, fully mixing by ultrasound, and ensuring that the microspheres are in a monodisperse state;
step S2 specifically includes: adding 100 mu L of 20mg/mL EDC and 300 mu L of 20mg/mL NHS which are prepared in situ, ultrasonically mixing the mixture, starting to add EDC and NHS, timing and activating the mixture for 15min, centrifuging the mixture at a high speed at 4 ℃, sucking and removing supernatant, and resuspending the mixture by using 1mL of coupling solution;
step S3 specifically includes: ultrasonically mixing the solution in 1mL of coupling solution resuspended microsphere solution, centrifuging at a high speed, discarding the supernatant, ultrasonically mixing the solution, centrifuging at a high speed, discarding the supernatant, and repeatedly washing at least once; adding 1mL of coupling solution for resuspension, adding 150 μ g of the first antibody, ultrasonically mixing, and oscillating on a shaker for 3h, wherein the ultrasonic treatment is carried out for 2min every 30 min;
step S4 specifically includes: adding 3 mu L of ethanolamine stock solution after coupling is finished, adding 800 mu L of confining liquid for heavy suspension, oscillating for 2.5h by a shaking table, centrifuging at high speed at 4 ℃ after confining is finished, and removing supernatant;
step S5 specifically includes: resuspending with 1mL of final washing solution, ultrasonically mixing, centrifuging at high speed at 4 ℃, removing supernatant, and repeatedly washing at least once; the precipitate was dissolved in 500. mu.L of the final wash.
The rotation speed of each centrifugation is preferably 10000-15000rpm, and the centrifugation time is preferably 10-20 min.
The method for detecting cyfra21-1 by the test strip comprises the following steps:
s10, diluting a cyfra21-1 standard substance in multiple times to obtain a plurality of standard samples with different concentrations, respectively sucking the plurality of standard samples by using a microsyringe, respectively dripping the standard samples on the test strips corresponding to each standard sample, standing at room temperature for 12-20 min, putting each test strip into a near-infrared fluorescence scanner for reading, recording a Dr value, measuring each standard sample with each concentration at least twice, averaging, and making a standard curve graph of the Dr value to the sample concentration as shown in figure 2; wherein, Dr value is the ratio of the fluorescence value of the detection line divided by the fluorescence value of the quality control line; the amount of each standard sample to be sucked is 80-120. mu.l;
s20, sucking the urine sample to be tested to the test strip, standing at room temperature for 12-20 min, and collecting the urine sample
The test strip is sent to a near infrared fluorescence scanner for reading, and the concentration of cyfra21-1 is obtained. Wherein,
the urine sample to be detected is collected from the patient and stored at the temperature of 2-8 ℃, the suction amount of the urine sample to be detected is 80-120 mu l, and the detection is required to be completed within 4 h.
The test strip prepared by the invention is suitable for detecting the content of cyfra21-1 in urine. The midmorning urine of the patient with bladder cancer is collected and immediately transferred to a laboratory, and is stored at low temperature for later use, and the detection is completed within 4 hours.
The following is a detailed description of specific examples.
Example 1
Selecting a glass cellulose membrane as a solid phase material of the binding pad 3, spraying a first anti-cyfra 21-1 antibody marked by near-infrared fluorescent nano microspheres on the binding pad 3, and drying in a strong convection air oven at 37 ℃ for 10 hours for later use. Selecting a glass cellulose membrane as a sample pad 2, spraying a treatment solution on the sample pad 2, and drying in a strong convection air oven at 37 ℃ for 10 hours for later use. Selecting a nitrocellulose membrane 4, preparing a second antibody of anti-cyfra 21-1 and a goat anti-mouse polyclonal antibody into a detection band 5 and a quality control band 6 on the nitrocellulose membrane 4 by using a scribing machine, and drying for 10 hours in a strong convection air oven at 37 ℃ for later use. And sequentially bonding the nitrocellulose membrane 4, the combination pad 3, the sample pad 2 and the water absorption pad 7 on the viscous bottom plate 1, then cutting the test paper into test paper strips by a cutting machine, putting the test paper strips into the plastic clamping grooves, and assembling to obtain a finished product.
The method for labeling the anti-cyfra 21-1 first antibody by using the near-infrared fluorescent nano microspheres comprises the following steps: initial washing: absorbing 500 mu L of near-infrared fluorescent nano microspheres, adding 1mL of initial washing solution, fully mixing the mixture by using an ultrasonic cleaner, centrifuging the mixture at 13000rpm for 15min, absorbing and discarding the supernatant, resuspending the precipitate by using 1mL of initial washing solution, fully mixing the precipitate by using the ultrasonic cleaner for 2min, centrifuging the mixture at 13000rpm for 15min, absorbing and discarding the supernatant, repeatedly washing the precipitate for 3 times, resuspending the precipitate by using 1mL of initial washing solution after the last washing, fully mixing the precipitate by using the ultrasonic cleaner, and ensuring that the near-infrared fluorescent nano microspheres are in a monodisperse state; and (3) activation: adding 100 μ L of 20mg/mL EDC and 300 μ L of 20mg/mL NHS (used as prepared) and ultrasonically mixing, activating for 15min (starting timing by adding an activating agent), centrifuging for 15min at 13000rpm at 4 ℃, removing supernatant by suction, and resuspending by using 1mL coupling solution; coupling: ultrasonically mixing the solution in 1mL of coupling solution resuspended microsphere solution, centrifuging the solution at 13000rpm for 15min, discarding the supernatant, ultrasonically mixing the solution, centrifuging the solution at 13000rpm for 15min, and repeatedly washing the solution for 3 times; adding 1ml of coupling solution for resuspension, adding 150 mu g of first antibody, ultrasonically mixing uniformly, and carrying out ultrasonic treatment for 2min every 30min at 140-rotation table for 3 h; and (3) sealing: adding 3 mu L of ethanolamine after coupling, adding 800 mu L of confining liquid for heavy suspension, sealing by a 140-rotation table for 2.5h, centrifuging at 4 ℃, 13000rpm for 15min after sealing, and removing supernatant; final washing: resuspending with 1mL of final washing solution, ultrasonically mixing, centrifuging at 4 deg.C and 13000rpm for 15min, removing supernatant, and repeatedly washing for 3 times; the precipitate was dissolved in 500. mu.L of the final wash.
Diluting cyfra21-1 standard substance in multiple times, sucking 100 μ L of standard substance with a microsyringe, dripping into a sample port of a test strip, standing at room temperature for 15 minutes, reading the test strip in a near infrared fluorescence scanner, recording the measured value (the fluorescence value of a detection line is divided by the fluorescence value of a quality control line), measuring the concentration of each sample three times, averaging, and plotting the measured value on the concentration of the sample. The midmorning urine of the patient with bladder cancer is collected and immediately transferred to a laboratory, and is stored at low temperature for later use, and the detection is completed within 4 hours. And (3) sucking 100 mu L of a urine sample to be detected to a sample port of the test strip, standing for 15 minutes at room temperature, sending the test strip into a near infrared fluorescence scanner for reading, and directly giving out the concentration of cyfra21-1 by the instrument.
The test paper strip in the embodiment is used for carrying out a detection experiment, the results are shown in table 1, and the sensitivity of detecting the initial onset and recurrence of the bladder cancer is calculated to be 100%, the specificity is 100%, the false positive rate is 0% and the false negative rate is 0% by taking 40ng/ml as a cutoff value.
Table 1:
example 2
Selecting a glass cellulose membrane as a solid phase material of the combination pad 3, spraying a first anti-cyfra 21-1 antibody marked by near-infrared fluorescent nano microspheres on the combination pad 3, and drying in a strong convection air oven at 40 ℃ for 8 hours for later use. Selecting a glass cellulose membrane as a sample pad 2, spraying a treatment solution on the sample pad 2, and drying in a strong convection air oven at 40 ℃ for 8 hours for later use. Selecting a nitrocellulose membrane 4, preparing a second antibody of anti-cyfra 21-1 and a goat anti-mouse polyclonal antibody into a detection band 5 and a quality control band 6 on the nitrocellulose membrane 4 by using a scribing machine, and drying for 8 hours in a strong convection air oven at 40 ℃ for later use. And sequentially bonding the nitrocellulose membrane 4, the combination pad 3, the sample pad 2 and the water absorption pad 7 on the viscous bottom plate 1, then cutting the test paper into test paper strips by a cutting machine, putting the test paper strips into the plastic clamping grooves, and assembling to obtain a finished product.
The method for labeling the anti-cyfra 21-1 first antibody by using the near-infrared fluorescent nano microspheres comprises the following steps: initial washing: absorbing 550 mu L of near-infrared fluorescent nano microspheres, adding 1.5mL of initial washing solution, fully mixing the mixture by using an ultrasonic cleaning instrument, centrifuging the mixture at 15000rpm for 10min, sucking and discarding the supernatant, resuspending the precipitate by using 1.5mL of initial washing solution, fully mixing the precipitate by using the ultrasonic cleaning instrument for 3min, centrifuging the mixture at 15000rpm for 10min, sucking and discarding the supernatant, repeatedly washing the precipitate for 2 times, resuspending the precipitate by using 1.5mL of initial washing solution after the last washing, fully mixing the precipitate by using the ultrasonic cleaning instrument, and ensuring that the near-infrared fluorescent nano microspheres are in a monodisperse state; and (3) activation: adding 120 μ L of 20mg/mL EDC and 350 μ L of 20mg/mL LNHS (for use now), ultrasonically mixing, activating for 20min (adding activating agent for timing), centrifuging at 2 deg.C and 15000rpm for 20min, removing supernatant, and resuspending with 1.5mL coupling solution; coupling: ultrasonically mixing the solution in 1.5mL of coupling solution resuspended microsphere solution, centrifuging the solution at 15000rpm for 10min, discarding supernatant, ultrasonically mixing the solution, centrifuging the solution at 15000rpm for 10min, and repeatedly washing the solution for 2 times; adding 1.5ml of coupling solution for resuspension, adding 165 mu g of first antibody, ultrasonically mixing uniformly, and carrying out ultrasonic treatment for 2min every 20min at 200-rotation shaking table; and (3) sealing: after the coupling is finished, adding 5 mu L of ethanolamine, then adding 1000 mu L of confining liquid for heavy suspension, sealing for 2h by a 200-rotation table, centrifuging for 10min at the temperature of 2 ℃ and the rotational speed of 15000rpm, and removing supernatant; final washing: resuspending with 1.5mL of final washing solution, ultrasonically mixing, centrifuging at 2 deg.C and 15000rpm for 10min, removing supernatant, and repeatedly washing for 2 times; the pellet was dissolved in 600. mu.L of the final wash.
Diluting cyfra21-1 standard substance in multiple times, sucking 120 μ L of standard substance with a microsyringe, dripping into a sample port of a test strip, standing at room temperature for 20 minutes, reading the test strip in a near infrared fluorescence scanner, recording the measured value (the fluorescence value of a detection line is divided by the fluorescence value of a quality control line), measuring the concentration of each sample three times, and after averaging, plotting the measured value on the concentration of the sample. The midmorning urine of the patient with bladder cancer is collected and immediately transferred to a laboratory, and is stored at low temperature for later use, and the detection is completed within 4 hours. And (3) sucking 120 mu L of a urine sample to be detected to a sample port of the test strip, standing at room temperature for 20 minutes, sending the test strip into a near infrared fluorescence scanner for reading, and directly giving out the concentration of cyfra21-1 by the instrument.
The test strip in this example was used to perform a test, the results are shown in table 2, and 40ng/ml was used as the cutoff value, and the sensitivity of detecting the initial onset and recurrence of bladder cancer was calculated to be 91.67, the specificity was 100%, the false positive rate was 0%, and the false negative rate was 8.33%.
Table 2:
example 3
Selecting a glass cellulose membrane as a solid phase material of the combination pad 3, spraying a first anti-cyfra 21-1 antibody marked by near-infrared fluorescent nano microspheres on the combination pad 3, and drying in a strong convection air oven at 35 ℃ for 15 hours for later use. Selecting a glass cellulose membrane as a sample pad 2, spraying a treatment solution on the sample pad 2, and drying in a strong convection air oven at 35 ℃ for 15 hours for later use. Selecting a nitrocellulose membrane 4, preparing a second antibody of anti-cyfra 21-1 and a goat anti-mouse polyclonal antibody into a detection band 5 and a quality control band 6 on the nitrocellulose membrane 4 by using a scribing machine, and drying for 15 hours in a strong convection air oven at 35 ℃ for later use. And sequentially bonding the nitrocellulose membrane 4, the combination pad 3, the sample pad 2 and the water absorption pad 7 on the viscous bottom plate 1, then cutting the test paper into test paper strips by a cutting machine, putting the test paper strips into the plastic clamping grooves, and assembling to obtain a finished product.
The method for labeling the anti-cyfra 21-1 first antibody by using the near-infrared fluorescent nano microspheres comprises the following steps: initial washing: absorbing 450 mu L of near-infrared fluorescent nano microspheres, adding 0.8mL of initial washing liquid, fully and uniformly mixing by using an ultrasonic cleaner, centrifuging for 20min at 10000rpm, sucking and discarding the supernatant, resuspending the precipitate by using 0.8mL of initial washing liquid, fully and uniformly mixing for 1.5min by using the ultrasonic cleaner, centrifuging for 20min at 10000rpm, sucking and discarding the supernatant, repeatedly washing for 1 time, resuspending by using 0.8mL of initial washing liquid after the last washing, fully and uniformly mixing by using the ultrasonic cleaner, and ensuring that the near-infrared fluorescent nano microspheres are in a monodisperse state; and (3) activation: adding 80 μ L of 20mg/mL EDC and 250 μ L of 20mg/mL LNHS (for use in preparation), ultrasonically mixing, activating for 15min (adding an activating agent for timing), centrifuging at 8 ℃, 10000rpm for 20min, removing supernatant, and resuspending with 0.8mL of coupling solution; coupling: ultrasonically mixing the solution in 0.8mL of coupling solution-resuspended microsphere solution, centrifuging the solution at 10000rpm for 20min, discarding the supernatant, ultrasonically mixing the solution, centrifuging the solution at 10000rpm for 20min, and repeatedly washing the solution for 1 time; adding 0.8ml of coupling solution for resuspension, adding 135 mu g of first antibody, ultrasonically mixing uniformly, and rotating a shaker for 4h at 100 rpm for 2min every 40 min; and (3) sealing: adding 2 μ L ethanolamine after coupling, adding 600 μ L confining liquid for resuspension, sealing with 100 rotary table for 3.5h, centrifuging at 8 deg.C and 10000rpm for 20min, and removing supernatant; final washing: resuspending with 0.8mL final wash solution, mixing with ultrasound, centrifuging at 8 deg.C and 10000rpm for 20min, removing supernatant, and washing repeatedly for 1 time; the precipitate was dissolved in 400. mu.L of the final wash.
Diluting cyfra21-1 standard substance in multiple times, sucking 80 μ L of standard substance with a microsyringe, dripping into a sample port of a test strip, standing at room temperature for 12 minutes, reading the test strip in a near infrared fluorescence scanner, recording the measured value (the fluorescence value of a detection line is divided by the fluorescence value of a quality control line), measuring the concentration of each sample twice, averaging, and plotting the measured value on the concentration of the sample. The midmorning urine of the patient with bladder cancer is collected and immediately transferred to a laboratory, and is stored at low temperature for later use, and the detection is completed within 4 hours. And (3) sucking 80 mu L of a urine sample to be detected to a sample port of the test strip, standing at room temperature for 12 minutes, sending the test strip into a near infrared fluorescence scanner for reading, and directly giving out the concentration of cyfra21-1 by the instrument.
The test paper strip in the embodiment is used for carrying out a detection experiment, the results are shown in table 3, and the sensitivity of detecting the initial onset and recurrence of the bladder cancer is 91.67, the specificity is 100%, the false positive rate is 0% and the false negative rate is 8.33% by calculation by taking 40ng/ml as a cutoff value.
Table 3:
it will be understood that modifications and variations can be made by persons skilled in the art in light of the above teachings, and all such modifications and variations are intended to be included within the scope of the invention as defined in the appended claims.
Claims (10)
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| PCT/CN2019/116380 WO2020094103A1 (en) | 2018-11-09 | 2019-11-07 | Test strip for quantificationally detecting marker cyfra21-1 and preparation method therefor and detection method thereof |
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| WO2020094103A1 (en) * | 2018-11-09 | 2020-05-14 | 深圳市众循精准医学研究院 | Test strip for quantificationally detecting marker cyfra21-1 and preparation method therefor and detection method thereof |
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| CN118465271A (en) * | 2024-07-09 | 2024-08-09 | 宁波奥丞生物科技有限公司 | Immunochromatography test paper box for semi-quantitative detection of bladder cancer |
| CN120908461A (en) * | 2025-10-13 | 2025-11-07 | 四川省医学科学院·四川省人民医院 | Preparation method and application of HPV16/18 type E6/E7 protein multi-target detection test strip |
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| CN118599001B (en) * | 2024-06-24 | 2025-05-30 | 湖南华瑞精检生物科技有限公司 | Cancer early screening antigen, detection kit and application thereof |
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