CN115327108A - Human AD7c-NTP colloidal gold detection test strip as well as preparation method and application thereof - Google Patents
Human AD7c-NTP colloidal gold detection test strip as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a human AD7C-NTP colloidal gold detection test strip and a preparation method and application thereof, the test strip comprises a PVC base plate and an adsorption layer, the adsorption layer is formed by sequentially overlapping a sample pad, a glass fiber film, a nitrocellulose film and a water absorption pad, the glass fiber film contains a colloidal gold-labeled AD7C-NTP protein monoclonal antibody A, the nitrocellulose film is provided with a quality control line C and a detection line T, the quality control line C is coated with an goat anti-mouse IgG antibody, and the detection line T is coated with an AD7C-NTP protein monoclonal antibody B. According to the human AD7c-NTP colloidal gold detection test strip, the AD7c-NTP protein monoclonal antibodies A and B and goat anti-mouse IgG are selected, so that the human AD7c-NTP protein can be quickly and simply detected, the sensitivity and the specificity are high, and the test strip has the advantages of convenience, rapidness, simplicity in operation, accurate result and the like, and is suitable for clinical rapid detection.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnosis, in particular to a human AD7c-NTP colloidal gold detection test strip as well as a preparation method and application thereof.
Background
Alzheimer-related neurofilament protein (AD 7 c-NTP) is a transmembrane phosphoprotein expressed in neuronal cells. In 1996, researchers separated AD7c-NTP cDNA from the brains of AD patients for the first time, detected clinical specimens found that the concentration of cerebrospinal fluid AD7c-NTP of early AD patients is significantly higher than that of a control group and a non-AD nervous system disease control group, and the research result arouses wide attention on AD7c-NTP as a potential AD biomarker. At present, a great deal of research shows that AD7c-NTP is an AD biomarker with the expression quantity specifically increased in brain tissues, cerebrospinal fluid and urine of AD patients, the urine and the cerebrospinal fluid AD7c-NTP detection result have similar specificity and sensitivity, and the level of the AD7c-NTP in the urine and the cerebrospinal fluid is in positive correlation with the severity of dementia.
AD7c-NTP is transmembrane protein and can easily permeate cell barriers; the molecular weight is only 41KDa, and the urine can be entered through the kidney; the isoelectric point is 9.89, the protein is alkaline protein, and is tightly combined with most acidic plasma protein in plasma, after reaching the kidney, because a layer of negatively charged adhesive glycoprotein is arranged on a glomerular basement membrane, the positively charged AD7c-NTP is concentrated in the raw urine through ultrafiltration. Under the double balance system of charge and concentration, the content of AD7c-NTP protein in urine is stable, and the detection is relatively accurate and reliable.
At present, the detection method of the AD7c-NTP protein in the human urine mainly adopts an ELISA detection method, but the ELISA method has long operation time, needs devices such as an incubation concussion instrument, an enzyme-labeling instrument and the like, and is not suitable for on-site rapid detection. Therefore, it is necessary to establish a rapid and simple detection method.
Disclosure of Invention
Aiming at the technical problems in the related art, the invention provides a human AD7c-NTP colloidal gold detection test strip as well as a preparation method and application thereof, which can overcome the defects in the prior art.
In order to achieve the technical purpose, the technical scheme of the invention is realized as follows:
the utility model provides a human AD7C-NTP colloidal gold test paper strip, includes PVC bottom plate and adsorbed layer, the adsorbed layer comprises sample pad, glass fiber membrane, nitrocellulose membrane and the pad that absorbs water overlap joint in proper order, the last AD7C-NTP albumen monoclonal antibody A that contains the colloidal gold mark of glass fiber membrane, be provided with quality control line C and detection line T on the nitrocellulose membrane, the parcel has goat anti mouse IgG's antibody on quality control line C, the parcel has AD7C-NTP albumen monoclonal antibody B on the detection line T.
Preferably, people AD7C-NTP colloidal gold test paper strip still includes the shell of packing, the shell of packing includes epitheca and inferior valve, the epitheca is fixed with inferior valve joint, be provided with fixed the knot in the inferior valve, PVC bottom plate and adsorbed layer pass through fixed the knot with the inferior valve is connected, the epitheca is equipped with the observation window of quality control line C and detection line T corresponding to the position of nitrocellulose membrane, the epitheca is equipped with the application of sample hole corresponding to the position of sample pad.
Preferably, the outer casing is made of a polyvinyl chloride material.
Preferably, the absorbent pad is made of a paper material.
Preferably, the side of the nitrocellulose membrane is coated with an adhesive.
According to another aspect of the present invention, there is provided a method for preparing a human AD7c-NTP colloidal gold test strip, comprising the steps of:
s1, preparing a colloidal gold solution by adopting a sodium citrate reduction method: adding 1mL of chloroauric acid into 100mL of deionized water, heating to boil, quickly adding 10mL of trisodium citrate, continuously boiling for 15min, cooling, and supplementing with deionized water to the original volume to obtain a colloidal gold solution;
s2, carrying out colloidal gold labeling on the AD7c-NTP protein monoclonal antibody A under the conditions that the pH value is 8.0 and the concentration of the AD7c-NTP protein monoclonal antibody A is 20 mu g/mL: adding lmL and 0.lM K into 10mL of colloidal gold solution 2 CO 3 Stirring the solution for 30min, adding 200 μ g of AD7c-NTP protein monoclonal antibody A, stirring for 30min, adding lmL 10% BSA, stirring for 30min, centrifuging at 4 ℃ for 30min at 10000g, removing the supernatant, and suspending the precipitate with 0.01M sodium phosphate buffer solution to obtain the colloidal gold particle-labeled AD7c-NTP protein monoclonal antibody A;
s3, soaking, spraying and drying the glass cellulose membrane to obtain a marker-combined glass fiber membrane;
s4, respectively spraying the AD7C-NTP protein monoclonal antibody B and goat anti-mouse IgG on a nitrocellulose membrane, and drying to obtain the nitrocellulose membrane coated with a detection line T and a quality control line C;
s5, a sample pad, a glass fiber membrane, a nitrocellulose membrane and a water absorption pad are stuck on the PVC base plate, and the sample pad, the glass fiber membrane, the nitrocellulose membrane and the water absorption pad are sequentially overlapped.
The invention also provides application of the human AD7c-NTP colloidal gold test strip in non-diagnostic purpose urine AD7c-NTP protein detection.
The invention has the beneficial effects that: according to the human AD7c-NTP colloidal gold detection test strip, the AD7c-NTP protein monoclonal antibodies A and B and goat anti-mouse IgG are selected, so that the human AD7c-NTP protein can be quickly and simply detected, the sensitivity and the specificity are high, the detection linear range is 10 ng/ml-2000 ng/ml, and the test strip has the advantages of convenience, rapidness, simplicity in operation, accurate result and the like, and is suitable for clinical quick detection.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
Fig. 1 is a schematic structural diagram of a test strip according to an embodiment of the present invention, wherein: 1. the detection device comprises a sample pad, 2, a glass fiber membrane, 3, a nitrocellulose membrane, 4, a water absorption pad, 5, a PVC bottom plate, 6, detection lines T,7 and a quality control line C;
FIG. 2 is a schematic diagram of a theoretical detection result of the test strip according to the embodiment of the present invention;
FIG. 3 shows the results of a semi-quantitative experiment on AD7c-NTP antigen standard according to an embodiment of the present invention;
FIG. 4 shows the results of a semi-quantitative test on clinical urine specimens according to an embodiment of the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.
Example 1
As shown in fig. 1, the human AD7C-NTP colloidal gold test strip according to the embodiment of the present invention includes a PVC base plate 5 and an adsorption layer, the adsorption layer is formed by sequentially overlapping a sample pad 1, a glass fiber membrane 2, a nitrocellulose membrane 3, and a water absorption pad 4, the glass fiber membrane 2 contains a colloidal gold-labeled AD7C-NTP protein monoclonal antibody a, the nitrocellulose membrane 3 is provided with a quality control line C7 and a detection line T6, the quality control line C7 is coated with an antibody against goat IgG, and the detection line T6 is coated with an AD7C-NTP protein monoclonal antibody B. The AD7c-NTP protein monoclonal antibodies A and B recognize different epitopes of the AD7c-NTP protein.
The absorbent pad 4 is made of paper materials, and the side face of the nitrocellulose membrane 3 is coated with adhesive.
In some other embodiments of the present invention, the human AD7C-NTP colloidal gold test strip further includes an outer packaging shell, the outer packaging shell includes an upper shell and a lower shell, the upper shell and the lower shell are fixed in a clamping manner, a fixing buckle is disposed in the lower shell, the PVC base plate and the adsorption layer are connected to the lower shell through the fixing buckle, an observation window of a quality control line C and a detection line T is disposed at a position of the upper shell corresponding to the nitrocellulose membrane, a sample adding hole is disposed at a position of the upper shell corresponding to the sample pad, and the outer packaging shell is made of polyvinyl chloride.
The human AD7c-NTP colloidal gold test strip detects urine AD7c-NTP protein by a double-antibody sandwich method. When in detection, the AD7c-NTP protein antigen in the sample is firstly combined with the AD7c-NTP protein monoclonal antibody A marked by colloidal gold particles on the glass fiber membrane, and the complex moves on the nitrocellulose membrane under the action of absorbent paper. When moving to the detection line T, the immobilized DNA fragment binds to the AD7c-NTP protein monoclonal antibody B, thereby being immobilized on the detection line T of the nitrocellulose membrane. The more AD7c-NTP protein in the sample, the more complexes on the detection line T, and the more significant the color development on the strip. Next, the goat anti-mouse IgG is combined with the control line C to develop color, which is used as the basis for controlling whether the sample is sufficient and the test strip is working normally, and the theoretical detection result is shown in fig. 2.
Example 2
The preparation method of the human AD7c-NTP colloidal gold test strip in the embodiment 1 comprises the following steps:
s1, preparing a colloidal gold solution:
the colloidal gold is prepared by a sodium citrate reduction method. Adding 1mL of chloroauric acid into 100mL of deionized water, heating to boil, quickly adding 10mL of trisodium citrate, continuously boiling for 15min, cooling, supplementing with deionized water to the original volume (100 mL), and recovering the volume evaporated during boiling.
Colloidal gold labeling of the S2 monoclonal antibody a:
determining the optimal condition for binding the S21 monoclonal antibody A and the colloidal gold:
the classical antibody labeling method of colloidal gold mainly comprises electrostatic adsorption, wherein the whole labeled protein is negatively charged by adjusting the pH value of a solution, and the electrostatic adsorption mode is easily interfered by factors such as ionic strength, pH value, labeled protein molecular weight, protein isoelectric point and the like of the labeled solution. Therefore, it is necessary to search for optimal labeling conditions for different labeled proteins.
Screening the optimal concentration of the S211 monoclonal antibody A combined with the colloidal gold:
adding the antibody solution to be labeled into a 40kD ultrafiltration tube, centrifuging at 10000g for 10min, resuspending by using a colloidal gold solution until the concentration of the antibody solution is 5-50 mu g/mL (shown in table 1), uniformly mixing by vortex, and standing at room temperature for 10min. Add 100. Mu.L of 10% NaCl per tube, vortex, mix well and let stand at room temperature for 10min. The minimum antibody concentration with red color is the optimal concentration, and finally the optimal concentration of the AD7c-NTP protein monoclonal antibody A is determined to be 20 mu g/mL.
TABLE 1 screening of optimal concentration of monoclonal antibody A binding to colloidal gold
S212 monoclonal antibody A and colloidal gold are combined for optimal pH screening:
using K 2 CO 3 The pH of the colloidal gold solution is adjusted to 3-10 (shown in table 2), 50 mu L of 20 mu g/mLAD7c-NTP protein monoclonal antibody A is added into each colloidal gold solution, and the colloidal gold solution is vortexed, mixed uniformly and then stood for 10min at room temperature. Add 100. Mu.L of 10% NaCl per tube, vortex, mix well and let stand at room temperature for 10min. The lowest pH at which the color was red was the optimum pH, which was finally determined to be 8.
TABLE 2 monoclonal antibody A and colloidal gold binding optimum pH screening
| pH | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Monoclonal antibody A (ug) | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Colloidal gold labeling of S213 monoclonal antibody a:
taking 10mL of colloidal gold solution, adding lmL and 0.lM K 2 CO 3 The solution was stirred for 30min. Adding 200 μ g of AD7c-NTP protein monoclonal antibody A, and stirring for 30min. BSA (lmL 10%) was added and stirred for 30min. Centrifuging at 4 ℃ 10000g for 30min, removing supernatant, and suspending the precipitate with 0.01M sodium phosphate buffer solution to obtain the AD7c-NTP protein monoclonal antibody A marked by colloidal gold particles.
S3, preparing a glass fiber membrane:
the glass fiber membrane was soaked for 1 hour in 0.01M PBS buffer solution having pH 8.0 containing 3% BSA, 0.1% Tween20, and then dried at room temperature. And (3) spraying the AD7c-NTP protein monoclonal antibody A marked by the colloidal gold particles on the glass fiber membrane by using a gold spraying instrument, and drying the glass fiber membrane in an oven at 37 ℃ overnight to obtain the glass fiber membrane combined with the marker.
S4, preparation of a nitrocellulose membrane:
the nitrocellulose membrane was soaked for 1 hour with 0.01M PBS buffer having pH 8.0 containing 3% BSA, 0.1% Tween20, and then dried at room temperature. The AD7c-NTP protein monoclonal antibody B was formulated to be 1mg/ml and goat anti-mouse IgG was formulated to be 1.5mg/ml using 0.01M PBS at pH 8.0. And (3) marking the AD7C-NTP protein monoclonal antibody B on a nitrocellulose membrane by using a membrane spraying instrument according to the parameter of 20 mu L/cm, coating a detection line T, marking the goat anti-mouse IgG according to the parameter of 1.0 mu L/cm, and coating a quality control line C. And drying in a 24 ℃ oven to obtain the nitrocellulose membrane coated with the detection line T and the quality control line C.
Assembly of S5 test strip
The test strip is formed by overlapping a sample pad, a glass fiber membrane, a nitrocellulose membrane and a water absorption pad in sequence, wherein a piece of PVC base plate is taken, the nitrocellulose membrane is adhered to the center, and the water absorption pad and the glass fiber membrane are respectively adhered to the upper part and the lower part of the nitrocellulose membrane, and are overlapped by 2mm; a sample pad was attached under the fiberglass membrane. The test paper was cut into 8cm x 0.5cm strips.
Wherein, the goat anti-mouse IgG antibody is purchased from Zhengzhou Dengning biotechnology limited, and the product number is DK239; the AD7c-NTP protein monoclonal antibody A is purchased from Beijing Boaosen, and the product number is V3003; the AD7c-NTP protein monoclonal antibody B is purchased from Beijing Boaosen, and the product number is V3005.
Example 3
The method for detecting the urine AD7c-NTP protein by adopting the human AD7c-NTP colloidal gold detection test strip disclosed by the embodiment 1 of the invention is compared with other methods for technical indexes, and the technical indexes are shown in a table 3.
TABLE 3 comparison of technical indices of different methods
Urine samples were collected from 30 confirmed alzheimer patients and 30 healthy controls. And (3) sucking a urine sample by using a disposable suction tube, vertically dropping 2-3 drops on the sample pad of the test strip in the embodiment 1, reacting for 10min, and judging the detection result. Meanwhile, the clinical samples are detected by using a kit which is a kit obtained from national medical instrument registration certificate, namely an Alzheimer related neurofilament protein (AD 7 c-NTP) detection kit (enzyme linked immunosorbent assay), and the detection results are analyzed. Shows that the two methods have good correlation of the test results and the correlation coefficient R 2 ＝0.99。
The result can judge that the human AD7c-NTP colloidal gold test strip has good performance, and has the advantages of simple and convenient operation, quick reaction, accurate and reliable result, suitability for field detection and the like.
Example 4
The test strip of embodiment 1 of the invention can judge the qualitative test result by naked eyes, and can realize semi-quantitative detection if a scanner and analysis software are further combined to count the gray value.
The test strip provided by the embodiment 1 of the invention is used for detecting AD7c-NTP antigen standard products with different concentrations, and the experimental results are shown in a table 4 and a figure 3.
TABLE 4 semi-quantitative test results of AD7c-NTP antigen standard
30 urine samples of patients with Mild Cognitive Impairment (MCI), AD dementia and Health Control (HC) are collected, a disposable suction tube is used for sucking the urine samples, 2-3 drops of the urine samples are vertically dripped on the sample pad of the test paper strip in the embodiment 1, the reaction is carried out for 10min, and the detection result is judged. The grey scale values were counted using a scanner in combination with computer analysis software and the experimental results are shown in table 5.
TABLE 5 semi-quantitative test results of clinical urine specimens
Meanwhile, the clinical samples are detected by adopting an Alzheimer's related neurofilament protein (AD 7 c-NTP) detection kit (enzyme-linked immunosorbent assay) which is a kit for obtaining a national medical instrument registration certificate, the detection results are analyzed, and the negative and positive diagnosis coincidence rates are compared. The test strip of the embodiment 1 of the invention is calculated to obtain the positive diagnosis coincidence rate (AD 86.67%; MCI 56.67%) and the negative diagnosis coincidence rate (90%). The ROC curve was plotted, and the AUC value of AD patients quantified using the test strip of example 1 of the present invention was 0.8811, and the AUC value of patients in the early stage of MCI was 0.6450 (see fig. 4).
According to the results, the semi-quantitative result performance of the human AD7c-NTP colloidal gold test strip is good, the correlation coefficient is high, the AUC predicted value is high, the CV value is low, the test result is accurate, and the test strip has the advantages of simplicity and convenience in operation, rapidness in reaction, accuracy and credibility in result, suitability for field test and the like.
In conclusion, by means of the technical scheme, the AD7c-NTP protein monoclonal antibodies A and B and the goat anti-mouse IgG are selected, so that the rapid and simple detection of the human AD7c-NTP protein can be realized, the sensitivity and the specificity are high, and the method has the advantages of convenience, rapidness, simplicity in operation, accurate result and the like, and is suitable for clinical rapid detection.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Claims (7)
1. The utility model provides a human AD7C-NTP colloidal gold test paper strip, its characterized in that, includes PVC bottom plate and adsorbed layer, the adsorbed layer comprises sample pad, glass fiber membrane, nitrocellulose membrane and the pad that absorbs water overlap joint in proper order, the last AD7C-NTP albumen monoclonal antibody A that contains the colloidal gold mark of glass fiber membrane, be provided with quality control line C and detection line T on the nitrocellulose membrane, the parcel has goat anti mouse IgG's antibody on quality control line C, the parcel has AD7C-NTP albumen monoclonal antibody B on the detection line T.
2. The human AD7C-NTP colloidal gold test strip of claim 1, further comprising an outer packaging shell, wherein the outer packaging shell comprises an upper shell and a lower shell, the upper shell and the lower shell are clamped and fixed, a fixing buckle is arranged in the lower shell, the PVC bottom plate and the adsorption layer are connected with the lower shell through the fixing buckle, an observation window of a quality control line C and a detection line T is arranged at the position of the upper shell corresponding to the nitrocellulose membrane, and a sample adding hole is arranged at the position of the upper shell corresponding to the sample pad.
3. The human AD7c-NTP colloidal gold test strip of claim 2, wherein the outer casing is made of polyvinyl chloride material.
4. The human AD7c-NTP colloidal gold test strip of claim 1, wherein the absorbent pad is made of a paper material.
5. The human AD7c-NTP colloidal gold test strip of claim 1, wherein the side of the nitrocellulose membrane is coated with an adhesive.
6. A method for preparing the human AD7c-NTP colloidal gold test strip according to any one of claims 1-5, which comprises the following steps:
s1, preparing a colloidal gold solution by adopting a sodium citrate reduction method: adding 1mL of chloroauric acid into 100mL of deionized water, heating to boil, quickly adding 10mL of trisodium citrate, continuously boiling for 15min, cooling, and supplementing with deionized water to the original volume to obtain a colloidal gold solution;
s2, carrying out colloidal gold labeling on the AD7c-NTP protein monoclonal antibody A under the conditions that the pH value is 8.0 and the concentration of the AD7c-NTP protein monoclonal antibody A is 20 mu g/mL: adding lmL and 0.lM K into 10mL of colloidal gold solution 2 CO 3 Stirring the solution for 30min, adding 200 mu g of AD7c-NTP protein monoclonal antibody A, stirring for 30min, adding lmL 10% BSA, stirring for 30min, centrifuging for 30min at the temperature of 4 ℃ under the condition of 10000g, removing supernatant, and carrying out heavy suspension precipitation by using 0.01M sodium phosphate buffer solution to obtain the AD7c-NTP protein monoclonal antibody A marked by colloidal gold particles;
s3, soaking, spraying and drying the glass cellulose membrane to obtain a marker-combined glass fiber membrane;
s4, respectively spraying the AD7C-NTP protein monoclonal antibody B and goat anti-mouse IgG on a nitrocellulose membrane, and drying to obtain the nitrocellulose membrane coated with a detection line T and a quality control line C;
s5, pasting a sample pad, a glass fiber membrane, a nitrocellulose membrane and a water absorption pad on the PVC base plate, wherein the sample pad, the glass fiber membrane, the nitrocellulose membrane and the water absorption pad are sequentially overlapped.
7. Use of a human AD7c-NTP colloidal gold test strip according to any one of claims 1-5 for non-diagnostic purposes in the detection of urine AD7c-NTP proteins.
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