CN115327108A - A kind of human AD7c-NTP colloidal gold detection test strip and preparation method and application thereof - Google Patents

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 bottom plate and an adsorption layer, and the adsorption layer is composed of a sample pad, a glass fiber membrane and a nitrocellulose membrane. It is formed by overlapping with the absorbent pad in turn, the glass fiber membrane contains colloidal gold-labeled AD7c-NTP protein monoclonal antibody A, and the nitrocellulose membrane is provided with a quality control line C and a detection line T. The quality control line Line C is coated with goat anti-mouse IgG antibody, and the detection line T is coated with AD7c-NTP protein monoclonal antibody B. The human AD7c-NTP colloidal gold detection test strip of the present invention can realize the rapid and simple detection of human AD7c-NTP protein by selecting AD7c-NTP protein monoclonal antibodies A, B and goat anti-mouse IgG, with high sensitivity and specificity , has the advantages of convenience, quickness, simple operation, accurate results, etc., and is suitable for clinical rapid detection.

Description

A kind of human AD7c-NTP colloidal gold detection test strip and its preparation method and application

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 and its preparation method and application.

Background technique

Alzheimer's disease-associated neurofilament protein (AD7c-NTP) is a transmembrane phosphoprotein expressed in neuronal cell bodies. In 1996, researchers isolated AD7c-NTP cDNA from the brains of AD patients for the first time, and detected clinical specimens and found that the concentration of AD7c-NTP in the cerebrospinal fluid of early AD patients was significantly higher than that of the control group and the non-AD neurological disease control group. There has been widespread interest in AD7c-NTP as a potential AD biomarker. Up to now, a large number of studies have shown that AD7c-NTP is an AD biomarker whose expression level is specifically increased in brain tissue, cerebrospinal fluid and urine of AD patients, and the detection results of urine and cerebrospinal fluid AD7c-NTP have similar Specificity and sensitivity, the levels of AD7c-NTP in urine and CSF were positively correlated with the severity of dementia.

AD7c-NTP is a transmembrane protein that can easily pass through cell barriers; its molecular weight is only 41KDa, and it can enter urine through the kidney; its isoelectric point is 9.89, and it is a basic protein that is tightly combined with most acidic plasma proteins in plasma. After the kidney, due to a layer of negatively charged sticky glycoprotein on the glomerular basement membrane, the positively charged AD7c-NTP was concentrated into the primary urine by ultrafiltration. Under the dual balance system of charge and concentration, the protein content of AD7c-NTP in urine is stable, and the detection is relatively accurate and reliable.

At present, the detection method of AD7c-NTP protein in human urine is mainly based on ELISA detection method, but the ELISA method takes a long time to operate, requires incubator shaker, microplate reader and other equipment, and is not suitable for rapid on-site detection. Therefore, it is necessary to establish a fast and simple detection method.

Contents of the invention

Aiming at the above-mentioned technical problems in the related art, the present invention proposes a human AD7c-NTP colloidal gold detection test strip and its preparation method and application, which can overcome the above-mentioned deficiencies in the prior art.

For realizing above-mentioned technical purpose, technical scheme of the present invention is realized like this:

A kind of human AD7c-NTP colloidal gold detection test strip, comprises PVC bottom plate and adsorption layer, and described adsorption layer is made up of sample pad, glass fiber membrane, nitrocellulose membrane and water-absorbing pad successively overlapped, and on described glass fiber membrane Containing colloidal gold-labeled AD7c-NTP protein monoclonal antibody A, the nitrocellulose membrane is provided with a quality control line C and a detection line T, and the quality control line C is coated with a goat anti-mouse IgG antibody, so The detection line T is coated with AD7c-NTP protein monoclonal antibody B.

Preferably, the human AD7c-NTP colloidal gold detection test strip also includes an outer casing, the outer casing includes an upper casing and a lower casing, the upper casing and the lower casing are clamped and fixed, and the lower casing is provided with There is a fixed buckle, the PVC bottom plate and the adsorption layer are connected to the lower shell through the fixed buckle, and the position of the upper shell corresponding to the nitrocellulose membrane is provided with an observation window of a quality control line C and a detection line T. The upper shell is provided with a sample injection hole corresponding to the position of the sample pad.

Preferably, the outer packaging shell is made of polyvinyl chloride material.

Preferably, the absorbent pad is made of paper material.

Preferably, the side of the nitrocellulose membrane is coated with an adhesive.

According to another aspect of the present invention, a kind of preparation method of human AD7c-NTP colloidal gold detection test strip is provided, and the method comprises the steps:

S1 adopts sodium citrate reduction method to prepare colloidal gold solution: add 1mL 1% chloroauric acid to 100mL deionized water, heat to boiling, add 10mL 1% trisodium citrate rapidly, continue to boil for 15min, replenish with deionized water after cooling Qi to the original volume, to obtain colloidal gold solution;

S2 Colloidal gold labeling of AD7c-NTP protein monoclonal antibody A under the conditions of pH value 8.0 and AD7c-NTP protein monoclonal antibody A concentration of 20 μg/mL: take 10 mL of colloidal gold solution, add 1 mL, 0.1M K ₂ CO ₃ solution, stirred for 30min, added 200μg AD7c-NTP protein monoclonal antibody A, stirred for 30min, added 1mL 10% BSA, stirred for 30min, centrifuged at 10000g at 4°C for 30min, removed the supernatant, buffered with 0.01M sodium phosphate The precipitate was resuspended in liquid solution to obtain AD7c-NTP protein monoclonal antibody A labeled with colloidal gold particles;

S3 soaking, spraying and drying the glass cellulose membrane to obtain a marker-bound glass fiber membrane;

S4 Spray the AD7c-NTP protein monoclonal antibody B and goat anti-mouse IgG on the nitrocellulose membrane respectively, and dry to obtain the nitrocellulose membrane coated with the detection line T and the quality control line C;

S5 Paste the sample pad, the glass fiber membrane, the nitrocellulose membrane and the water-absorbing pad on the PVC bottom plate, and the sample pad, the glass fiber membrane, the nitrocellulose membrane and the water-absorbing pad are sequentially overlapped.

The present invention also provides the application of the human AD7c-NTP colloidal gold detection test strip in non-diagnostic detection of urine AD7c-NTP protein.

Beneficial effects of the present invention: the human AD7c-NTP colloidal gold detection test strip of the present invention can realize the quick and easy detection of human AD7c-NTP protein by selecting AD7c-NTP protein monoclonal antibody A, B and goat anti-mouse IgG, and is sensitive Higher in performance and specificity, the detection linear range is 10ng/ml-2000ng/ml, it has the advantages of convenience, quick operation, simple operation, and accurate results, and is suitable for rapid clinical detection.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without any creative effort.

Fig. 1 is a schematic structural view of a test strip according to an embodiment of the present invention, wherein: 1, a sample pad, 2, a glass fiber membrane, 3, a nitrocellulose membrane, 4, an absorbent pad, 5, a PVC bottom plate, 6, Test line T, 7, quality control line C;

Fig. 2 is a schematic diagram of theoretical detection results of test strips according to an embodiment of the present invention;

Fig. 3 is according to the AD7c-NTP antigen standard product semi-quantitative experimental result described in the embodiment of the present invention;

Fig. 4 is the result of a semi-quantitative experiment on a clinical urine sample according to an embodiment of the present invention.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention belong to the protection scope of the present invention.

Example 1

As shown in Figure 1, a kind of human AD7c-NTP colloidal gold detection test strip according to the embodiment of the present invention comprises PVC bottom plate 5 and adsorption layer, and described adsorption layer is made of sample pad 1, glass fiber membrane 2, nitric acid The cellulose membrane 3 and the water-absorbing pad 4 are sequentially overlapped to form, the glass fiber membrane 2 contains colloidal gold-labeled AD7c-NTP protein monoclonal antibody A, and the nitrocellulose membrane 3 is provided with a quality control line C7 and detection Line T6, the quality control line C7 is coated with goat anti-mouse IgG antibody, and the detection line T6 is coated with AD7c-NTP protein monoclonal antibody B. AD7c-NTP protein monoclonal antibodies A and B recognize different epitopes of AD7c-NTP protein.

The absorbent pad 4 is made of paper material, and the side of the nitrocellulose membrane 3 is coated with an adhesive.

In some other embodiments of the present invention, the human AD7c-NTP colloidal gold detection test strip also includes an outer packaging shell, the outer packaging shell includes an upper shell and a lower shell, and the upper shell and the lower shell are clamped and fixed , the lower shell is provided with a fixed buckle, the PVC bottom plate and the adsorption layer are connected with the lower shell through the fixed buckle, and the position of the upper shell corresponding to the nitrocellulose membrane is provided with a quality control line C and a detection line The observation window of T, the upper shell is provided with a sample injection hole corresponding to the position of the sample pad, and the outer shell is made of polyvinyl chloride material.

The human AD7c-NTP colloidal gold detection test strip of the present invention detects urine AD7c-NTP protein by a double-antibody sandwich method. During the test, the AD7c-NTP protein antigen in the sample is first combined with the AD7c-NTP protein monoclonal antibody A labeled with 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, it 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 band. Next, combine with goat anti-mouse IgG at the quality control line C to develop color, as the basis for quality control of whether the sample is sufficient and whether the test strip is working normally. The theoretical detection results are shown in Figure 2.

Example 2

The preparation method of human AD7c-NTP colloidal gold detection test strip described in embodiment 1, the method may further comprise the steps:

Preparation of S1 colloidal gold solution:

Colloidal gold was prepared by sodium citrate reduction method. Add 1mL 1% chloroauric acid to 100mL deionized water, heat to boiling, quickly add 10mL 1% trisodium citrate, continue to boil for 15min, make up to the original volume (100mL) with deionized water after cooling, evaporate when returning to boiling volume of.

Colloidal gold labeling of S2 monoclonal antibody A:

Determination of the optimal conditions for the binding of S21 monoclonal antibody A to colloidal gold:

The classic antibody labeling method of colloidal gold is mainly electrostatic adsorption. By adjusting the pH value of the solution, the labeled protein is negatively charged as a whole. The electrostatic adsorption method is easily affected by factors such as the ionic strength of the labeled solution, pH value, the molecular weight of the labeled protein, and the isoelectric point of the protein. interference. Therefore, it is necessary to explore the best labeling conditions for different labeled proteins.

Screening of optimal concentration of S211 monoclonal antibody A combined with colloidal gold:

Add the antibody solution to be labeled into a 40kD ultrafiltration tube, centrifuge at 10,000g for 10min, resuspend with colloidal gold solution, and make the concentration of the antibody solution be 5-50μg/mL (as shown in Table 1), vortex and mix well, and then stand at room temperature 10min. Add 100 μL of 10% NaCl to each tube, vortex and mix well, and let stand at room temperature for 10 min. The minimum antibody concentration whose color is red is the optimal concentration, and the optimal concentration of AD7c-NTP protein monoclonal antibody A is finally determined to be 20 μg/mL.

Table 1. Optimum concentration screening of monoclonal antibody A combined with colloidal gold

S212 monoclonal antibody A combined with colloidal gold for optimal pH screening:

Use K ₂ CO ₃ solution to adjust the pH of the colloidal gold solution to 3-10 (as shown in Table 2). Take 50 μL of 20 μg/mL AD7c-NTP protein monoclonal antibody A and add it to each colloidal gold solution. Set for 10min. Add 100 μL of 10% NaCl to each tube, vortex and mix well, and let stand at room temperature for 10 min. The lowest pH with a red color is the optimal pH, and the optimal pH is finally determined to be 8.

Table 2. Optimum pH screening of monoclonal antibody A combined with colloidal gold

pH 3 4 5 6 7 8 9 10 Monoclonal Antibody A (μg) 1 1 1 1 1 1 1 1

Colloidal gold labeling of S213 monoclonal antibody A:

Take 10 mL of colloidal gold solution, add 1 mL of 0.1M K ₂ CO ₃ solution, and stir for 30 min. Add 200 μg AD7c-NTP protein monoclonal antibody A, and stir for 30 minutes. Add 1 mL of 10% BSA and stir for 30 min. Centrifuge at 10,000 g for 30 min at 4°C, remove the supernatant, and resuspend the pellet with 0.01 M sodium phosphate buffer to obtain colloidal gold particle-labeled AD7c-NTP protein monoclonal antibody A.

Preparation of S3 glass fiber membrane:

The glass fiber membrane was soaked in 0.01 M PBS buffer solution with a pH value of 8.0 containing 3% BSA and 0.1% Tween20 for 1 hour, and dried at room temperature. Colloidal gold particle-labeled AD7c-NTP protein monoclonal antibody A was sprayed on the glass fiber membrane using a gold sprayer, and dried in an oven at 37°C overnight to obtain a glass fiber membrane bound with the marker.

Preparation of S4 nitrocellulose membrane:

The nitrocellulose membrane was soaked in 0.01M PBS buffer solution with a pH value of 8.0 containing 3% BSA and 0.1% Tween20 for 1 h, and dried at room temperature. AD7c-NTP protein monoclonal antibody B was prepared at 1 mg/ml using 0.01M PBS with a pH value of 8.0, and goat anti-mouse IgG was prepared at 1.5 mg/ml. Use a film sprayer to streak the AD7c-NTP protein monoclonal antibody B on the nitrocellulose membrane at a parameter of 20 μL/cm, coat the detection line T, and streak the goat anti-mouse IgG at a parameter of 1.0 μL/cm, Coated quality control line C. Dry in an oven at 24°C to obtain a nitrocellulose membrane coated with a detection line T and a quality control line C.

Assembly of S5 test strips

The test strip is composed of sample pad, glass fiber membrane, nitrocellulose membrane, and water-absorbing pad, which are overlapped sequentially. Take a PVC bottom plate, paste the nitrocellulose membrane in the center, and paste the water-absorbing pad and glass fiber membrane on the nitrocellulose respectively. Above and below the membrane, overlap 2mm; paste the sample pad under the glass fiber membrane. Cut the test paper into 8cm*0.5cm strips.

Among them, goat anti-mouse IgG antibody was purchased from Zhengzhou Dening Biotechnology Co., Ltd., product number DK239; AD7c-NTP protein monoclonal antibody A was purchased from Beijing Boaosen Company, product number is V3003; AD7c-NTP protein monoclonal antibody B was purchased from Beijing Boaosen Company, the product number is V3005.

Example 3

The method for detecting urine AD7c-NTP protein using the human AD7c-NTP colloidal gold detection test strip described in Example 1 of the present invention is compared with other methods, as shown in Table 3.

Table 3. Comparison of technical indicators of different methods

Urine samples were collected from 30 patients diagnosed with Alzheimer's disease and 30 healthy controls. Take the urine sample with a disposable straw and drop 2 to 3 drops vertically on the sample pad of the test strip described in Example 1, react for 10 minutes, and determine the detection result. At the same time, the above-mentioned clinical samples were tested with a kit that has obtained the National Medical Device Registration Certificate - Alzheimer's-associated neurofilament protein (AD7c-NTP) detection kit (enzyme-linked immunosorbent assay), and the test results were analyzed. It shows that the test results of the two methods are well correlated, and the correlation coefficient R ² =0.99.

From the above results, it can be judged that the human AD7c-NTP colloidal gold detection test strip of the present invention has good performance, and at the same time has the advantages of simple operation, fast response, accurate and reliable results, and is suitable for on-site detection.

Example 4

The test strip described in Embodiment 1 of the present invention can be judged by the naked eye through qualitative detection results. If the gray value is further combined with a scanner and analysis software, semi-quantitative detection can also be realized.

The test strip described in Example 1 of the present invention detects AD7c-NTP antigen standard substances with different concentrations, and the experimental results are shown in Table 4 and FIG. 3 .

Table 4, AD7c-NTP antigen standard semi-quantitative experimental results

Collect 30 cases of urine samples from mild cognitive impairment (MCI) patients, AD dementia patients, and healthy controls (HC), and use a disposable straw to draw urine samples and add 2 to 3 drops vertically to the test sample described in Example 1. Put the sample pad on the paper strip, react for 10 minutes, and judge the test result. A scanner combined with computer analysis software was used to count the gray value, and the experimental results are shown in Table 5.

Table 5. Semi-quantitative test results of clinical urine samples

At the same time, the above-mentioned clinical samples were tested with the kit that has obtained the national medical device registration certificate - Alzheimer's-associated neurofilament protein (AD7c-NTP) detection kit (enzyme-linked immunoassay), the test results were analyzed, and compared Negative and positive diagnostic coincidence rate. Calculate the positive diagnostic coincidence rate (AD 86.67%; MCI 56.67%) and negative diagnostic coincidence rate (90%) of the test strip described in Example 1 of the present invention. Draw the ROC curve, the test strip described in Example 1 of the present invention has an AUC value of 0.8811 for the quantitative detection of AD patients, and an AUC value of 0.6450 for the early detection of MCI patients (see Figure 4).

It can be judged by the above results that the semi-quantitative result performance of the human AD7c-NTP colloidal gold detection test strip of the present invention is good, the correlation coefficient is high, the AUC prediction value is high, the CV value is low, the detection result is accurate, and it has easy operation and reaction Fast, accurate and reliable results, suitable for on-site testing and other advantages.

In summary, by means of the above technical scheme of the present invention, by selecting AD7c-NTP protein monoclonal antibodies A, B and goat anti-mouse IgG, the rapid and easy detection of human AD7c-NTP protein can be realized, and the sensitivity and specificity are relatively high. High, convenient and fast, simple operation, accurate results, etc., suitable for rapid clinical detection.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (7)

1. a kind of people's AD7c-NTP colloidal gold detects test strip, it is characterized in that, comprises PVC base plate and adsorption layer, described adsorption layer is formed by lapping successively of sample pad, glass fiber membrane, nitrocellulose membrane and water-absorbing pad, The glass fiber membrane contains colloidal gold-labeled AD7c-NTP protein monoclonal antibody A, the nitrocellulose membrane is provided with a quality control line C and a detection line T, and the quality control line C is coated with goat antibody Mouse IgG antibody, the detection line T is coated with AD7c-NTP protein monoclonal antibody B. 2. people's AD7c-NTP colloidal gold detection test strip according to claim 1, is characterized in that, also comprises outer packing shell, and described outer packing shell comprises upper shell and lower shell, and described upper shell and lower shell clamp The lower shell is provided with a fixed buckle, the PVC bottom plate and the adsorption layer are connected with the lower shell through the fixed buckle, and the upper shell is provided with a quality control line C and a position corresponding to the nitrocellulose membrane. The observation window of the detection line T, the upper shell is provided with a sample injection hole corresponding to the position of the sample pad. 3. people's AD7c-NTP colloidal gold detection test strip according to claim 2, is characterized in that, described outer packing shell is made of polyvinyl chloride material. 4. people's AD7c-NTP colloidal gold detection test strip according to claim 1, is characterized in that, described absorbent pad is made of paper material. 5. people's AD7c-NTP colloidal gold detection test strip according to claim 1, is characterized in that, the side of described nitrocellulose membrane is coated with adhesive. 6. a preparation method of human AD7c-NTP colloidal gold detection test paper strip as described in any one of claim 1-5, is characterized in that, comprises the steps: S1 Prepare colloidal gold solution by sodium citrate reduction method: add 1mL 1% chloroauric acid to 100mL deionized water, heat to boiling, quickly add 10mL 1% trisodium citrate, continue to boil for 15min, and replenish with deionized water after cooling Qi to the original volume, to obtain colloidal gold solution; S2 Colloidal gold labeling of AD7c-NTP protein monoclonal antibody A under the condition that the pH value is 8.0 and the concentration of AD7c-NTP protein monoclonal antibody A is 20 μg/mL: take 10 mL of colloidal gold solution, add 1 mL, 0.1M K ₂ CO ₃ solution, stirred for 30min, added 200μg AD7c-NTP protein monoclonal antibody A, stirred for 30min, added 1mL 10% BSA, stirred for 30min, centrifuged at 10000g at 4°C for 30min, removed the supernatant, buffered with 0.01M sodium phosphate The precipitate was resuspended in liquid solution to obtain AD7c-NTP protein monoclonal antibody A labeled with colloidal gold particles; S3 soaking, spraying, and drying the glass fiber membrane to obtain a marker-bound glass fiber membrane; S4 Spray AD7c-NTP protein monoclonal antibody B and goat anti-mouse IgG on the nitrocellulose membrane respectively, and dry to obtain the nitrocellulose membrane coated with detection line T and quality control line C; S5 Paste the sample pad, the glass fiber membrane, the nitrocellulose membrane and the water-absorbent pad on the PVC bottom plate, and the sample pad, the glass fiber membrane, the nitrocellulose membrane and the water-absorbent pad are sequentially overlapped. 7. The application of a human AD7c-NTP colloidal gold detection test strip as described in any one of claims 1-5 in the detection of urine AD7c-NTP protein for non-diagnostic purposes.

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