CN116183910A - Immunofluorescence chromatography test paper for rapidly detecting human whole blood P-tau-181 protein and preparation method thereof - Google Patents
Immunofluorescence chromatography test paper for rapidly detecting human whole blood P-tau-181 protein and preparation method thereof Download PDFInfo
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Abstract
The application relates to the field of biological detection, and in particular discloses immunofluorescence chromatography test paper for rapidly detecting human whole blood P-tau-181 protein and a preparation method thereof. The immunofluorescence chromatography test paper comprises a sample pad, a combination pad, a coating film and a water absorption pad which are sequentially overlapped; the surface of the binding pad is sprayed with first fluorescent microspheres coupled with anti-P-tau-181 antibodies and second fluorescent microspheres coupled with monoclonal antibodies A; the surface of the coating film is provided with a detection area and a quality control area, the detection area is coated with a P-tau-181 antibody, and the quality control area is coated with a monoclonal antibody B which specifically recognizes the monoclonal antibody A. The immunofluorescence chromatography test paper integrates the characteristics of specificity of the antibody and high sensitivity of fluorescence, has the characteristics of accuracy, rapidness, high efficiency, specificity and sensitivity, is suitable for early diagnosis of Alzheimer's disease, and has important significance for timely and accurately guiding the use of medicines for patients.
Description
Technical Field
The application relates to the field of biological detection, in particular to immunofluorescence chromatography test paper for rapidly detecting human whole blood P-tau-181 protein and a preparation method thereof.
Background
Tau protein, also known as microtubule-associated protein (Microtubulue Association Protein, MAP), is present in the neuronal axons of normal brain tissue and has the effect of synthesizing and stabilizing the cellular skeleton of neurons. The cellular function of Tau protein in normal brain is to bind to tubulin and promote its polymerization to form microtubules; binds to the microtubules formed, maintains microtubule stability, reduces dissociation of tubulin molecules, and induces microtubule bundling. The Tau protein in normal brain contains 2-3 phosphate groups, while the Tau protein in brain of Alzheimer's Disease (AD) patients is abnormally hyperphosphorylated, and the Tau protein can contain 5-9 phosphate groups per molecule and lose normal biological functions.
In cells, tau protein can be modified, of which the most important is phosphorylation, whereas Tau protein function depends on its level of phosphorylation. There are data showing that soluble Tau protein is converted to insoluble after hyperphosphorylation, and that hyperphosphorylated Tau protein eventually forms neurofibrillary tangles (neuro fibrillary tangles, NFT).
Meanwhile, research shows that the main cause of the pathogenesis of AD is nerve fiber entanglement caused by Tau protein hyperphosphorylation in brain and beta amyloid (Abeta) in brain are deposited in a large amount to form senile plaques, thereby causing abnormal neuron function. It has been reported that the levels of Tau protein in cerebrospinal fluid are significantly increased in patients with mild cognitive impairment. Numerous studies have shown that Tau protein is closely related to cognitive function. Thus, as a nervous system specific protein, tau protein can be released from nerve cells to cerebrospinal fluid after brain injury, and further leak to peripheral blood through the disrupted blood brain barrier. Thus, the concentration of Tau protein in the cerebrospinal fluid and peripheral blood can reflect the degree of brain injury to some extent.
Studies have also shown that Tau protein hyperphosphorylation is an important initial step in AD brain neuron degradation, and an increase in the product P-Tau-181 protein of Tau protein hyperphosphorylation can initiate the generation of P-Tau-181 protein outside the blood brain barrier, and finally, the content of P-Tau-181 in blood is increased. Therefore, the detection of the content of the P-Tau-181 protein in whole blood has higher clinical value for diagnosing AD. However, currently, detection methods for P-tau-181 that are easy to operate, rapid, sensitive, accurate, and suitable for use in communities and primary hospitals are also lacking. In view of this, the present application is specifically proposed.
Disclosure of Invention
In order to solve the problems, the application provides the immunofluorescence chromatography test paper for rapidly detecting the human whole blood P-tau-181 protein and the preparation method thereof, and the immunofluorescence chromatography test paper integrates the characteristics of specificity of antibodies and high sensitivity of fluorescence, has the characteristics of accuracy, rapidness, high efficiency, specificity and sensitivity, is suitable for early diagnosis of Alzheimer's disease, and has important significance for timely and accurately guiding the use of medicines for patients.
The application adopts the following technical scheme:
in a first aspect, the present application provides an immunofluorescence chromatography test strip for rapid detection of human whole blood P-tau-181 protein, comprising a sample pad, a binding pad, a coating film and a water absorbing pad which are sequentially lapped;
the surface of the binding pad is sprayed with first fluorescent microspheres coupled with anti-P-tau-181 antibodies and second fluorescent microspheres coupled with monoclonal antibodies A;
the surface of the coating film is provided with a detection area and a quality control area, the detection area is coated with a P-tau-181 antibody, and the quality control area is coated with a monoclonal antibody B which specifically recognizes the monoclonal antibody A.
Further, the fluorescent microsphere is a polystyrene microsphere with streptavidin and rare earth elements loaded on the surface.
Further, the anti-P-tau-181 antibody is coupled to the first fluorescent microsphere through biotin; monoclonal antibody a was coupled to a second fluorescent microsphere via biotin.
Further, the monoclonal antibody A is sheep anti-chicken IgY or sheep anti-rabbit IgG; the monoclonal antibody B is chicken IgY or rabbit IgG.
In a second aspect, the present application provides a method for preparing the immunofluorescence chromatography test paper, which includes:
spraying the first fluorescent microsphere coupled with the anti-P-tau-181 antibody and the second fluorescent microsphere coupled with the monoclonal antibody A on a glass cellulose membrane to prepare a bonding pad;
coating the P-tau-181 antibody on a detection area of the nitrocellulose membrane, and coating the monoclonal antibody B on a quality control area of the nitrocellulose membrane to obtain a coating membrane;
preparing a sample pad, and sequentially overlapping and assembling the sample pad, the bonding pad, the coating film and the water absorption pad.
Further, the preparation method of the first fluorescent microsphere coupled with the anti-P-tau-181 antibody comprises the following steps:
mixing an anti-P-tau-181 antibody with biotin according to a mass ratio of 8-12:1, and carrying out shaking table reaction at room temperature for 0.5-1 h to obtain a biotin-anti-P-tau-181 antibody compound;
mixing the biotin-anti-P-tau-181 antibody complex with polystyrene microspheres with streptavidin and rare earth elements loaded on the surfaces, performing shaking table reaction for 0.5-1 h at room temperature, blocking with BSA, and purifying to obtain the SA-biotin-anti-P-tau-181 antibody complex.
Further, the preparation method of the bonding pad comprises the following steps:
mixing a solution containing the first fluorescent microspheres with a solution containing the second fluorescent microspheres, wherein the mass ratio of the anti-P-tau-181 antibody to the monoclonal antibody A in the obtained mixed solution is 8-12:1;
spraying the mixed solution onto the glass cellulose film with the spraying amount of 1-5 mu L/cm, and drying at 50-60 ℃ for 10-12 h.
Further, the preparation method of the coating film comprises the following steps:
marking a detection area and a quality control area on the nitrocellulose membrane, wherein the detection area is close to the binding pad;
diluting the P-tau-181 antibody to 0.5-1.0mg/mL, and then streaking and coating the detection area;
and diluting the monoclonal antibody B to 0.1-0.5mg/mL, and streaking and coating the monoclonal antibody B in a quality control area.
Further, in the scribing coating process, the scribing concentration is 1-2 mu L/cm, and the speed is 80-120 mm/s.
Further, the preparation method of the sample pad comprises the following steps:
smearing treatment liquid on the sample pad, and drying for 5-7 h at room temperature;
wherein the treatment liquid contains 1-5 wt% of phytohemagglutinin or erythrocyte antibody.
In summary, the application has the following beneficial effects:
1. the application adopts a double-antibody sandwich method, combines a solid-phase chromatography technology with a fluorescence immunoassay system to quantitatively detect the content of P-tau-181 in a whole blood sample, and adopts the detection principle that: during testing, a sample to be tested is dripped on a sample pad, chromatography is carried out from bottom to top under the action of capillary effect, P-tau-181 protein existing in the sample to be tested is combined with first fluorescent microspheres of coupling anti-P-tau-181 antibodies sprayed on a combining pad to form fluorescent microsphere-antibody-antigen combination, then the fluorescent microsphere-antibody-antigen combination is continuously migrated to a coating film under the action of chromatography, and the P-tau-181 antibodies in a detected area are intercepted to form a compound focused on the detected area. Meanwhile, due to the existence of the second fluorescent microsphere coupled with the monoclonal antibody A on the binding pad, the second fluorescent microsphere migrates to a quality control area under the chromatographic action and is intercepted by the monoclonal antibody B. Under the action of excitation light, fluorescent substances on the fluorescent microspheres emit light signals with a certain wavelength, are identified by a fluorescent immunochromatography reader and converted into a certain value, and then the concentration of the P-tau-181 protein in the sample is calculated according to the value according to a built-in calibration curve.
2. The application adopts the fluorescent immunoassay technology, greatly improves the signal intensity and sensitivity of the fluorescent immune reaction, ensures that the P-tau-181 protein with low content in the sample can generate a very strong fluorescent signal when being subjected to immune binding, and provides a more accurate, convenient, quick and simple method for detecting the P-tau-181 protein in human whole blood.
3. The scheme provided by the luminescence can overcome the HOOK effect (namely the HOOK effect) caused by the insufficient binding capacity of the traditional double-anti-sandwich antibody, namely the false negative phenomenon caused by the unsuitable proportion of the antigen and the antibody, wherein the excessive antibody is called a front band effect, and the excessive antigen is called a rear band effect, so that the detection range is greatly improved.
Drawings
FIG. 1 is a schematic structural diagram of an immunofluorescence chromatography test paper according to an embodiment of the present application;
FIG. 2 is a graph showing the sensitivity test results of the immunofluorescence chromatographic test paper in the experimental example of the present application;
FIG. 3 is a comparison of the consistency of serum and whole blood as measured by the immunofluorescence chromatography test paper in the experimental example of the present application.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the following examples, which are to be construed as merely illustrative and not limitative of the scope of the invention, but are not intended to limit the scope of the invention to the specific conditions set forth in the examples, either as conventional or manufacturer-suggested, nor are reagents or apparatus employed to identify manufacturers as conventional products available for commercial purchase.
The technical scheme of the application is as follows:
an immunofluorescence chromatography test paper for rapidly detecting human whole blood P-tau-181 protein is shown in figure 1, and comprises a support plate 1, wherein a sample pad 2, a binding pad 3, a coating film 4 and a water absorption pad 5 are sequentially arranged on the support plate 1, and in order to form a good solid phase chromatography effect, the ends of the sample pad 2, the binding pad 3, the coating film 4 and the water absorption pad 5 are sequentially overlapped. The coating film 4 is provided with a detection region (T region) and a quality control region (C region). Wherein, the detection area is close to the bonding pad 3, and the quality control area is close to the water absorption pad 5. The first fluorescent microsphere coupled with the anti-P-tau-181 antibody and the second fluorescent microsphere coupled with the monoclonal antibody A are loaded on the binding pad 3; the detection region (T region) of the coating film 4 is coated with P-tau-181 antibody, and the quality control region (C region) is coated with monoclonal antibody B which specifically recognizes the monoclonal antibody A.
Wherein, the monoclonal antibody A and the monoclonal antibody B used for quality control in the immunofluorescence chromatography test paper can be sheep anti-chicken IgY and chicken IgY; sheep anti-rabbit IgG and rabbit IgG are also contemplated. In the embodiment of the application, the sheep anti-chicken IgY and the chicken IgY are adopted as quality control antibodies, and the advantages are that the chicken IgY is more stable and the specificity of the antibody pair is high.
The preparation method of the immunofluorescence chromatography test paper for rapidly detecting the human whole blood P-tau-181 protein comprises the following steps:
(1) Preparing a sample pad:
the sample pad is smeared with a sample treatment solution and dried for 5 to 7 hours, preferably 5.5 to 6.5 hours at room temperature.
Wherein the sample treatment solution contains phytohemagglutinin or erythrocyte antibody with the mass concentration of 1-5 wt% (preferably 2-4 wt%). By smearing the treatment fluid on the sample pad, red blood cells in the sample to be detected are adsorbed on the sample pad during detection, and the red blood cells are prevented from being chromatographed to the combination pad and the detection area, so that the detection result is interfered, and the sensitivity and the accuracy of detection are improved.
(2) Preparing a bonding pad:
and spraying the first fluorescent microsphere coupled with the anti-P-tau-181 antibody and the second fluorescent microsphere coupled with the monoclonal antibody A on a glass cellulose membrane to prepare the bonding pad.
Further, the method for preparing the bonding pad comprises the following steps: mixing the solution containing the first fluorescent microspheres with the solution containing the second fluorescent microspheres, spraying the obtained mixed solution on a glass cellulose membrane with the spraying amount of 1-5 mu L/cm, and drying at 50-60 ℃ for 10-12 h.
Wherein the mass ratio of the anti-P-tau-181 antibody to the monoclonal antibody A in the obtained mixed solution is 8-12:1, preferably 9-11:1. When the ratio of the two antibodies is 8-12:1, the antigen-antibody reaction effect is high, the background value is low, the signal gradient of the test strip can be fully distinguished, and the performance is fully ensured.
In the spraying process, the spraying amount is 1-5 mu L/cm, preferably 2-4 mu L/cm, and the spraying is carried out in the spraying amount range, so that the optimal activity of the antibody is ensured by the spraying amount, and the occurrence of false negative phenomenon caused by excessive antibody can be avoided.
After spraying, the drying temperature of the bonding pad is 50-60 ℃, preferably 52-58 ℃; the drying time is 10 to 12 hours, preferably 11 hours. Drying was performed under such conditions.
Wherein, the fluorescent microsphere is a polystyrene microsphere with the surface loaded with Streptavidin (SA) and rare earth elements. And an anti-P-tau-181 antibody coupled to the first fluorescent microsphere by biotin; the monoclonal antibody A is coupled to the second fluorescent microsphere through biotin.
The fluorescent microsphere adopts rare earth elements as fluorescent markers, and can be: eu (Eu) 3+ 、Tb 3+ 、Sm 3+ 、Dy 3+ . The rare earth luminescent material has the following advantages: a. the luminous band is narrow, the color purity is high, and the color is bright; b. high light absorption and conversion efficiencyHigh; c. the emission wavelength distribution area is wide; d. fluorescence lifetime spans from nanoseconds to milliseconds up to 6 orders of magnitude; e. stable physical and chemical properties, high temperature resistance, and can bear the actions of high-power electron beams, high-energy radiation and strong ultraviolet light. The streptavidin loaded on the fluorescent microsphere does not contain glycosyl and has low isoelectric point, so that the nonspecific binding is low in the immunodetection, and the background dyeing is light. And because the streptavidin and the biotin have extremely strong affinity, high specificity and stability, the streptavidin can be coupled with 4 biotin groups, and finally an antibody-antigen-antibody-biotin-SA complex is formed, a large amount of fluorescent substances can be accumulated, so that the detection sensitivity is greatly improved.
The polystyrene microsphere with the particle size of 200nm-400nm by using streptavidin and rare earth elements can be prepared by adopting a conventional coupling reaction, and can also be directly prepared by adopting a commercial product. In the examples of the present application, commercial products were used, commercially available from Du Biotin Inc., under the product name Streptavidin (SA) modified time-resolved fluorescent microspheres.
Further, the preparation method of the first fluorescent microsphere coupled with the anti-P-tau-181 antibody comprises the following steps:
A. mixing an anti-P-tau-181 antibody with biotin according to a mass ratio of 8-12:1 (preferably 9-11:1), and carrying out shaking table reaction at room temperature for 0.5-1 h to obtain a biotin-anti-P-tau-181 antibody compound;
B. mixing the biotin-anti-P-tau-181 antibody complex with polystyrene microspheres with streptavidin and rare earth elements loaded on the surfaces, performing shaking table reaction at room temperature for 0.5-1 h, blocking with BSA, and purifying to obtain the SA-biotin-anti-P-tau-181 antibody complex.
Further, the monoclonal antibody A for quality control is goat anti-chicken IgY; the preparation method of the second fluorescent microsphere coupled with the monoclonal antibody A is similar.
(3) Preparing a coating film:
coating the P-tau-181 antibody on a detection area of the nitrocellulose membrane, and coating the monoclonal antibody B on a quality control area of the nitrocellulose membrane to obtain a coating membrane.
Further, the method for preparing the coating film comprises the following steps: marking a detection area and a quality control area on the nitrocellulose membrane, wherein the detection area is close to the binding pad; diluting the P-tau-181 antibody to 0.5-1.0mg/mL (preferably 0.6-0.9 mg/mL), and streaking and coating the detection zone; and diluting the monoclonal antibody B to 0.1-0.5mg/mL (preferably 0.2-0.4 mg/mL), and coating the streak on the quality control region.
Further, in the streaking coating process, the streaking concentration is 1 to 2. Mu.L/cm, preferably 1.2 to 1.8. Mu.L/cm; the speed is 80 to 120mm/s, preferably 90 to 110mm/s.
(4) And (3) assembling:
the prepared sample pad, the binding pad, the coating film and the water absorption pad are sequentially lapped and assembled to obtain the immunofluorescence chromatography test paper which can be used for rapidly detecting the human whole blood P-tau-181 protein.
The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
Examples
The embodiment provides an immunofluorescence chromatography test paper for rapidly detecting human whole blood P-tau-181 protein, and the preparation method comprises the following steps:
1. preparation of sample pad:
immersing the sample pad in the treatment liquid for 1 hour, taking out, and drying at room temperature for 6 hours; wherein the treatment fluid is a buffer solution containing 1% -5% of phytohemagglutinin or erythrocyte antibody, 0.5% of surfactant S9 (namely propylene oxide-ethylene oxide-vinyl diamine copolymer) and 0.2M pH7.4 PB.
2. Preparing a bonding pad:
A. preparation of SA-biotin-anti-P-tau-181 antibody complex:
(1) Dialysis against anti-P-tau-181 antibody: transferring the antibody solution into a dialysis bag by using a pipette, placing the dialysis bag into a beaker filled with 2M pH7.4PB buffer solution, changing the solution at intervals of 2 hours, repeating for 2 times, and standing for 16 hours;
(2) Buffer solution preparation: preparing 0.2M PH7.4+/-0.05 PBS buffer solution;
(3) Preparing a 10m mol biotin solution: 1mg of biotin was weighed out and added to 0.217ml of DMSO solution.
(4) Antibody coupling: adding 0.3mg of purified anti-P-tau-181 antibody into a centrifuge tube, wherein the mass ratio of biotin to antibody is 1:10, adding 10mol of biotin solution according to the proportion, uniformly mixing, and carrying out shaking table reaction at room temperature for 30-60min.
(5) Desalting and purifying: separating the biotin-antibody protein from the solution using a desalting column;
(6) SA-biotin-antibody coupling: 1ml of fluorescent microspheres (polystyrene microspheres with streptavidin and rare earth elements on the surface) were taken out from a refrigerator, frozen and centrifuged (19000 g, temperature 4 ℃ C., 30 min), and then the supernatant was aspirated by a pipette, and 2ml of preservation solution (preservation solution containing 1% BSA, 2-5%, tween 20.1%, 0.2M PH7.4 PB) was added, and the above steps were repeated twice, and vortexed and homogenized. Taking a proper container, diluting the solution by 100-300 times, adding the biotin-antibody solution, packaging with tinfoil paper, and performing shaking table reaction at room temperature for 30-60min.
(7) Closing: 1% BSA was added for blocking, and the mixture was stirred in a rotary incubator for 30-60min.
(8) Purifying: centrifuging the solution, freezing (11900 g, temperature 4 deg.C, 20 min), taking out the centrifuge tube, slowly sucking the supernatant with a pipette, and discarding; taking out the precipitate, sucking the same amount of preservation solution by using a pipetting gun, adding the solution into a centrifuge tube, completely scattering the precipitate by ultrasonic for 10 times, irradiating by using a purple flashlight after ultrasonic treatment, carefully observing the fluorescent microsphere solution, and repeating ultrasonic treatment for 2-3 times if the bright spots still exist in the solution to be aggregated, thereby obtaining the SA-biotin-anti-P-tau-181 antibody compound.
B. Preparation of SA-biotin-goat anti-chicken IgY complex:
(1) Antibody coupling: adding 0.3mg of purified goat anti-chicken IgY into a centrifuge tube, adding 10mol of biotin solution into the centrifuge tube according to the mass ratio of biotin to goat anti-chicken IgY of 1:10, uniformly mixing, performing shaking table reaction at room temperature for 30-60min, and purifying
(2) SA-biotin-goat anti-chicken IgY coupling: 1ml of fluorescent microspheres (polystyrene microspheres with streptavidin and rare earth elements on the surface) were taken out from a refrigerator, frozen and centrifuged (19000 g, temperature 4 ℃ C., 30 min), and then the supernatant was aspirated by a pipette, and 2ml of preservation solution (preservation solution containing 1% BSA, 2-5%, tween 20.1%, 0.2M PH7.4 PB) was added, and the above steps were repeated twice, and vortexed and homogenized. Taking a proper container, diluting the solution by 100-300 times, adding the biotin-sheep anti-chicken IgY solution, packaging with tinfoil paper, and performing shaking table reaction at room temperature for 30-60min.
(3) And (3) sealing and purifying: adding 1% BSA for blocking, uniformly mixing and reacting for 30-60min on a rotary incubator, and purifying to obtain the SA-biotin-goat anti-chicken IgY compound.
C. Mixing the prepared solution containing SA-biotin-anti-P-tau-181 antibody complex with SA-biotin-sheep anti-chicken IgY complex solution (the mass ratio of the P-tau-181 antibody to the sheep anti-chicken IgY in the obtained mixed solution is 100:10), spraying onto a glass cellulose membrane by a metal spraying film drawing instrument, spraying the solution with the spraying amount of 1-5 mu L/cm, placing into a 50-60 ℃ oven, and drying for 10-12 hours to obtain a bonding pad;
3. preparing a coating film:
(1) Marking one end of a nitrocellulose membrane (NC membrane) by a marker pen, wherein the distance of a C, T line is 0.5cm, and the distance of the lower edge of a T line NC membrane is 1cm;
(2) Detection zone (T line): the P-tau-181 antibody was diluted to 0.5-1.0mg/ml with 1% trehalose 0.2M PB pH7.4 for coating; the streaking concentration was 1.5. Mu.L/cm, speed 100mm/s;
(3) Quality control zone (line C): diluting chicken IgY antibody to 0.1-0.5mg/ml with 0.5% trehalose 0.2M PB pH7.4 for coating, and streaking at a concentration of 1.5 μL/cm and a speed of 100mm/s;
(4) And (3) placing the NC film coated with the C line and the T line into a 50-60 ℃ oven, and drying for 10-12 hours to obtain the coating film.
4. And (3) assembling:
the sample pad, the bonding pad, the coating film and the water absorbing pad are spliced and assembled in sequence. Wherein, the water absorption pad and the bonding pad are respectively overlapped at two ends of the coating film, and a detection area is formed on the surface of the coating film; the sample pad is overlapped on the bonding pad, and is cut into test strips with the width of 3.5-4mm after being assembled;
each test strip is arranged in the lower cover of the fluorescent card shell, wherein the water absorption pad end of the test strip is arranged at one end of the lower cover, the part of the middle coating film needs to be leveled, no bulge exists, and the sample adding hole is positioned in the middle of the sample pad; after the fluorescent card is assembled, the upper cover is covered and placed on the card pressing machine, so that the upper cover and the lower cover of the fluorescent card are tightly combined.
Performance test
1. Sensitivity test:
the analytical sensitivity refers to the lowest detectable concentration of the detection method. In the immunodetection, for working curves with positive correlation such as a sandwich method and an indirect method, the detection method with the lowest detection limit is the concentration value corresponding to the light average value +2SD value intensity of the 0 concentration reference signal on the working curve. For the working curve of negative correlation such as competition method, the intensity of 0 concentration reference signal intensity average value-2 SD value is on the working curve or the corresponding concentration value of the straight line of 0 point and LOB concentration (0 concentration blank sample contains dilution liquid of BSA or 0 concentration calibrator is used for replacement)
(1) The testing method comprises the following steps: the blank sample is a clinical sample with zero content of the object to be tested as a first choice, and is continuously tested for 20 times in one operation, and the luminescence value is recorded, as shown in table 1.
(2) Test results:
TABLE 1 sensitivity test results
After linear fitting, a correlation curve as shown in fig. 2 was obtained, y=0.0092x+0.0184 (R 2 =1), the blank detection results show that the minimum detection concentration (LOB) of the immunofluorescence chromatography test paper provided in the examples of the present application for P-tau-181 antibody is lower than 4pg/ml.
2. Serum and whole blood consistency comparison:
(1) The testing method comprises the following steps: the P-tau-181 in the homologous serum and whole blood samples were tested by the immunofluorescence chromatography test paper provided in the examples, and the results are shown in Table 2:
TABLE 2 results of serum and whole blood consistency comparisons
(2) Detection result:
the data in table 2 were linear fitted to give the correlation curves shown in fig. 3: y= 0.9415x-0.0497 (R 2 = 0.9868). Therefore, the immune fluorescent chromatography test paper provided by the embodiment is used for directly detecting the P-tau-181 in the human whole blood, and the detection result has high correlation with the detection serum, so that the immune fluorescent chromatography test paper can realize the simple, convenient, quick, sensitive and accurate detection of the P-tau-181.
3. Detecting an interfering substance:
the effect of the interferent on the experimental result is generally obtained by measuring the concentration of the interferent in a control sample or a basic sample, wherein the common endogenous interference (including triglyceride, bilirubin, hemoglobin, total protein and the like) is obtained by selecting two concentration levels of clinical samples, adding a solvent corresponding to the interferent into the control group and adding the interferent with corresponding concentrations into the experimental group, and the addition amount of the interferent is preferably not more than 5% of the total volume.
(1) Triglyceride (Gd)
170mg of triglyceride was weighed and dissolved in 1000ul of 0.9% NaCl aqueous solution to prepare 170mg/mL of triglyceride mother liquor. The clinical samples were added with triglyceride and tested by using the immunofluorescence chromatographic test paper provided in the examples, and the results are shown in table 3:
TABLE 3 influence of triglycerides on the detection results
(2) Bovine hemoglobin
40mg of bovine hemoglobin was dissolved in 1000uL of 0.9% NaCl aqueous solution to prepare 20mg/mL hemoglobin mother liquor. Bovine hemoglobin was added to the clinical samples and tested using the immunofluorescence chromatographic test paper provided in the examples, the results are shown in table 4:
TABLE 4 influence of bovine hemoglobin on the detection results
(3) Bilirubin
8mg of bilirubin is weighed and dissolved in 0.1M NaOH solution (2 mL) to prepare a mother liquor of 4 mg/mL. Bilirubin is added into clinical samples, and the results are shown in Table 5, wherein the results are detected by using the immunofluorescence chromatographic test paper provided in the examples:
TABLE 5 influence of bilirubin on detection results
As can be seen from tables 3 to 5, the immunofluorescence chromatography test paper provided by the embodiment of the application has good anti-interference effect, and the deviation between the immunofluorescence chromatography test paper and the test result of the control group is kept within +/-10% within the set two interference concentration ranges.
4. Specificity test:
high concentrations of AD7C-NTP and beta-amyloid 1-42 and recombinant antigens at different phosphorylation sites were tested and the assay results were evaluated for the presence of cross-reactivity.
The testing method comprises the following steps:
(1) Preparing 20000pg/ml AD7C-NTP and 2000pg/ml beta amyloid 1-42 by using a calibrator diluent, and testing concentration values of the calibration compound;
(2) The kit adopts Thr212 and Thr214 recognition site antibody to detect TUA-181, so that 100pg/ml of recombinant antigen which is not phosphorylated by Thr212 is prepared by using a calibrator diluent to detect the concentration value, and the detection result is smaller than LOB.
The test results are shown in table 6:
TABLE 6 specificity test results
As can be seen from Table 6, the immunofluorescence chromatography test paper provided by the embodiment of the application has good specificity, the detection results of the high-concentration AD7C-NTP and Abeta 1-42 are lower than the blank limit value, and simultaneously, the detection values of the antigen results of the phosphorylation of other multiple sites which are not phosphorylated by the Thr212 site after the detection are lower than LOB by adopting the Thr212 and Thr214 recognition site sandwich method, which indicates that the immunofluorescence chromatography test paper has good specificity.
The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.
Claims (10)
1. An immunofluorescence chromatography test paper for rapidly detecting human whole blood P-tau-181 protein is characterized by comprising a sample pad, a binding pad, a coating film and a water absorption pad which are sequentially overlapped;
the surface of the binding pad is sprayed with first fluorescent microspheres coupled with an anti-P-tau-181 antibody and second fluorescent microspheres coupled with a monoclonal antibody A;
the surface of the coating film is provided with a detection area and a quality control area, the detection area is coated with a P-tau-181 antibody, and the quality control area is coated with a monoclonal antibody B which specifically recognizes the monoclonal antibody A.
2. The immunofluorescence chromatography test paper according to claim 1, wherein said fluorescent microsphere is a polystyrene microsphere loaded with streptavidin and rare earth elements on the surface.
3. The immunofluorescence chromatography test strip of claim 2, wherein said anti-P-tau-181 antibody is coupled to said first fluorescent microsphere by biotin; the monoclonal antibody A is coupled to the second fluorescent microsphere through biotin.
4. The immunofluorescence chromatography test paper according to claim 1, wherein said monoclonal antibody a is goat anti-chicken IgY or goat anti-rabbit IgG; the monoclonal antibody B is chicken IgY or rabbit IgG.
5. A method of preparing an immunofluorescence chromatographic test strip according to any of claims 1-4 comprising:
spraying the first fluorescent microsphere coupled with the anti-P-tau-181 antibody and the second fluorescent microsphere coupled with the monoclonal antibody A on a glass cellulose membrane to prepare a bonding pad;
coating the P-tau-181 antibody on a detection area of the nitrocellulose membrane, and coating the monoclonal antibody B on a quality control area of the nitrocellulose membrane to obtain a coating membrane;
preparing a sample pad, and sequentially overlapping and assembling the sample pad, the bonding pad, the coating film and the water absorption pad.
6. The method for preparing immunofluorescence chromatography test paper according to claim 5, wherein the method for preparing the first fluorescent microsphere coupled with anti-P-tau-181 antibody comprises the following steps:
mixing the anti-P-tau-181 antibody with biotin according to a mass ratio of 8-12:1, and performing shaking table reaction at room temperature for 0.5-1 h to obtain a biotin-anti-P-tau-181 antibody compound;
mixing the biotin-anti-P-tau-181 antibody complex with polystyrene microspheres with streptavidin and rare earth elements loaded on the surfaces, performing shaking table reaction at room temperature for 0.5-1 h, blocking with BSA, and purifying to obtain the SA-biotin-anti-P-tau-181 antibody complex.
7. The method for preparing an immunofluorescence chromatographic test paper according to claim 5, wherein the method for preparing the binding pad comprises the following steps:
mixing a solution containing the first fluorescent microspheres with a solution containing the second fluorescent microspheres, wherein the mass ratio of the anti-P-tau-181 antibody to the monoclonal antibody A in the obtained mixed solution is 8-12:1;
and spraying the mixed solution onto the glass cellulose film, wherein the spraying amount is 1-5 mu L/cm, and drying at 50-60 ℃ for 10-12 h.
8. The method for preparing an immunofluorescence chromatography test paper according to claim 5, wherein the method for preparing the coating film comprises the following steps:
marking a detection area and a quality control area on the nitrocellulose membrane, wherein the detection area is close to the binding pad;
diluting the P-tau-181 antibody to 0.5-1.0mg/mL, and streaking and coating the detection area;
and diluting the monoclonal antibody B to 0.1-0.5mg/mL, and coating the streak on the quality control region.
9. The method for preparing the immunofluorescence chromatography test paper according to claim 8, wherein the streaking concentration is 1-2 mu L/cm and the streaking speed is 80-120 mm/s in the streaking coating process.
10. The method for preparing an immunofluorescence chromatographic test paper according to claim 5, wherein the method for preparing a sample pad comprises:
coating a treatment liquid on the sample pad, and drying for 5-7 hours at room temperature;
wherein the treatment liquid contains 1-5wt% of phytohemagglutinin or erythrocyte antibody.
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| CN117820472B (en) * | 2024-03-05 | 2024-05-10 | 南京诺唯赞医疗科技有限公司 | P-Tau 181 specific antibody and application thereof in Alzheimer disease auxiliary diagnosis kit |
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