WO2021218661A1 - G-17, pgi and pgii combined detection device and preparation method therefor - Google Patents

Abstract

A gastrin 17 (G-17), pepsinogen I (PGI) and pepsinogen II (PGII) combined detection device and a preparation method therefor. The combined detection device is prepared by adhering a nitrocellulose membrane (3) with a solid phase containing high-specificity G-17, PGI and PGII antibodies and goat anti-mouse IgG polyclonal antibodies, a glass fiber membrane (2) adsorbed with colloidal gold labeled G-17, PGI and PGII antibodies, a sample pad (1), absorbent paper (4) and other auxiliary materials. According to the combined detection device, the complete release of the immune colloidal gold is ensured, the sensitivity of reaction is effectively improved, the dosage of immune colloidal gold can be reduced under the same threshold, the cost is saved, the cost is saved, three gastric functions and gastric cancer risk markers of gastrin 17, pepsinogen I and pepsinogen II in a specimen can be detected at the same time, and the complexity of production operation is not increased. The device is high in sensitivity, strong in specificity, simple and convenient to operate, time-saving and strong in practicability.

Description

G-17, PGI, PGII combined detection device and preparation method thereof

This application claims the priority of a Chinese patent application filed with the Chinese Patent Office on April 30, 2020, the application number is 202010370096.6, and the invention title is "G-17, PGI, PGII joint detection device and its preparation method", and its entire content Incorporated in this application by reference.

Technical field

The present invention relates to the field of medical detection equipment, in particular to a gastrin 17, pepsin I, and pepsin II combined detection device and a preparation method thereof. The method uses colloidal gold immunochromatography technology and the principle of double antibody sandwich method to quantitatively detect whole blood, The detection device and preparation method of human gastrin 17 (G-17), pepsinogen I (PGI), and pepsinogen II (PGII) in serum and plasma samples can realize the sensitivity, specificity, and sensitivity of gastric cancer risk markers. Quick check.

Background technique

Gastric mucosal lesions are caused by many factors, including drugs, alcohol, abnormal gastric acid secretion, and Helicobacter pylori infection, among which Helicobacter pylori infection is the main factor; after the gastric mucosa is damaged, its function will also change. Gastric cancer is a group of progressive malignant lesions of epithelial cells involving multiple factors, mostly from chronic atrophic gastritis. About 7% of chronic atrophic gastritis eventually progress to carcinoma in situ. Abnormal cell proliferation and HP infection can accelerate the development of atrophic gastritis. China is an area with a high incidence of gastric cancer, and more than 200,000 residents die of gastric cancer each year. Although radical surgery combined with radiotherapy and chemotherapy can improve the five-year survival rate of gastric cancer patients, it has no obvious effect on reducing mortality. Studies have shown that early screening is the key to reducing the incidence and mortality of gastric cancer.

Gastrin (gastrin, GAS) is a type of gastrointestinal hormone secreted by the G cells of the gastric antrum, duodenum and upper jejunum. Edkins was first discovered in a dog's gastric antrum in 1905 and was later known. Gastrin in human serum mainly includes two isomers: gastrin-17 (gastrin-17, G-17) and gastrin-34 (gastrin-34, G-34), of which gastrin- 17 accounts for 85%-90%. Therefore, people usually choose gastrin-17 to represent the serum gastrin level. Under physiological conditions, gastrin mainly acts by affecting the secretion of gastric acid. On the one hand, gastrin can directly stimulate parietal cells to secrete gastric acid. Gastric acid activates pepsinogen into active pepsin, thereby decomposing protein. The decomposition products will in turn stimulate the secretion of gastrin; on the other hand, gastrin can also act on the cholecystokinic receptors on enterochromaffin-like cells to promote the secretion of histamine from enterochromaffin-like cells, and then through histamine Stimulate parietal cells to secrete gastric acid. While gastrin affects the secretion of gastric acid, it can also reduce the tension of the lower esophageal sphincter. Some recent studies have pointed out that gastrin can be used as a marker of gastric mucosal function: the level of gastrin-17 in chronic atrophic gastritis is significantly higher than that of normal mucosa, and some people think that the serum level of gastrin-17 may be higher than that of serum. Total gastrin can more accurately reflect the severity of antral atrophy.

Pepsinogen (PG) is a protein polypeptide chain secreted by the main cells in the gastric mucosa. Human pepsinogen is mainly divided into two biochemical and immunologically different isoenzymes (PGI and PGII). Pepsinogen I is secreted by gastric gland main cells and neck cells, and pepsinogen II can also be secreted by cardia, pyloric and proximal duodenal glands. Pepsinogen itself does not have biological activity. It can be activated into digestive pepsin under the action of gastric acid, thereby decomposing the protein ingested by the body into easily absorbed small molecule peptides and amino acids. The vast majority of pepsinogen is secreted into the gastric cavity, and only a small part can be absorbed into the blood. We usually detect the pepsin in the serum which originally represents the pepsin level of the human body. According to reports, the levels of pepsinogen I and pepsinogen II in the serum can accurately reflect the function and histological status of the gastric mucosa. Therefore, serum pepsinogen is also called "serological biopsy". The detection of serum pepsinogen I and pepsinogen II combined with sophisticated endoscopy can effectively identify gastric diseases, especially early gastric mucosal lesions. Existing studies have shown that serum pepsinogen I and pepsinogen II levels are significantly abnormal in erosive gastritis, atrophic gastritis, gastric ulcer, gastric cancer and other gastric diseases, and can provide valuable screening methods for these diseases .

At present, there is still a lack of uniform clinical screening methods for early gastric cancer. The detection of tumor markers and related proteins can effectively reflect abnormal cell proliferation, and is suitable for early cancer screening and postoperative follow-up. Gastrointestinal angiography and chest fluoroscopy are mainly used to observe gastric ulcers and advanced gastric cancer, but they have low sensitivity and accuracy for carcinoma in situ. Microscopic biopsy is a traumatic operation and has many contraindications, making it difficult to carry out. Gastrin 17, pepsinogen I, and pepsinogen II are digestive enzymes secreted by gastric mucosal tissues. Pathological changes in gastric mucosa such as the fundus of the stomach and antrum can cause their levels to change. The combined detection of gastrin 17, pepsinogen Ⅰ and Ⅱ is effective in early gastric cancer screening, which can effectively improve the diagnosis rate of gastric cancer and is worthy of clinical application and promotion.

Colloidal gold immunochromatography assay (GICA) is a solid-phase membrane immunoassay technique that combines colloidal gold labeling technology and protein chromatography technology with a microporous membrane as a carrier. Colloidal gold immunochromatography technology is a commonly used immunochromatographic detection method. Because of its simple operation, time-saving, low manufacturing cost, and easy-to-interpret results, it is very suitable for on-site testing and is widely used in biology, medicine, and food. And other fields. Since the colloidal gold immunochromatography technique completes the detection in one step, there are many interference factors in the detection process, and its low sensitivity is the main factor that limits the application scope of colloidal gold immunochromatography. The detection limit of the traditional colloidal gold immunochromatography technique is higher than ELISA and other methods.

In colloidal gold immunochromatographic detection, the protein is fixed on the nitrocellulose membrane (NC membrane) as a capture reagent for the sample to be tested. Since the detection result completely depends on whether the capture reagent achieves a good adsorption effect on the membrane, the uniform and good adsorption of the protein on the membrane is very important for the detection result of colloidal gold. If the amount of protein bound on the NC membrane is insufficient or the protein binding force is not strong enough, there will be quite a lot of problems, which are very obvious on the detection line of the test results. If the amount of protein bound on the membrane is too low, the color of the detection line will be weak and the detection sensitivity will be reduced in the result. If the protein cannot be firmly adsorbed on the NC membrane, then the protein will diffuse before it is adsorbed on the NC membrane, resulting in a wider detection line and weaker color rather than bright and clear, making the detection result difficult to interpret. Under extreme conditions, if the physical adsorption of the protein on the NC membrane is too weak, the flowing protein detection substance and surfactant solution may wash off the fixed protein from the NC membrane, thus showing a wide or unclear detection at all Line, it is difficult to interpret the test results.

Summary of the invention

The invention provides a G-17, PGI and PGII combined detection device and a preparation method thereof, which solves the problems of insufficient amount of NC membrane adsorbed protein and weak binding force existing in the prior art. The three-in-one combined detection device for human gastrin 17, pepsinogen I and pepsinogen II prepared by the present invention can realize the sensitive, specific and rapid detection of gastric cancer risk markers, and increase the risk of gastric cancer for patients with gastric diseases Reasonable and comprehensive judgments can quickly and accurately carry out early warning of gastric cancer and disease risk judgment.

The technical scheme adopted by the present invention is: a G-17, PGI and PGII combined detection device, the sample pad 1, the immune colloidal gold glass fiber membrane 2, the immune nitrocellulose membrane 3, and the absorption pad 4 are respectively pasted on the plastic plate 5. The two ends of the immune nitrocellulose membrane 3 are overlapped with the absorbent pad 4 and the immune colloidal gold glass fiber membrane 2 respectively, and the other end of the immune colloidal gold glass fiber membrane 2 is overlapped with the sample pad 1; the immune nitric acid The cellulose membrane 3 is provided with a first detection line T1, a second detection line T2, a third detection line T3, and a quality control line C; the first detection line T1 has a solid phase with high specific gastrin 17 antibody; The second detection line T2 has a high specific pepsinogen I antibody in the solid phase; the third detection line T3 has a high specific pepsinogen II antibody in the solid phase; the immune nitrocellulose membrane 3 The detection lines T1, T2, T3 set on the above can be arranged longitudinally on the same immune nitrocellulose membrane 3 to form a combined detection device; the detection lines T1, T2, T3 set on the immune nitrocellulose membrane 3 can also be They are respectively arranged on three immune nitrocellulose membranes 3 and arranged side by side to form a combined detection device; the quality control line C is sprayed with goat anti-mouse IgG polyclonal antibody.

The present invention also provides a method for preparing a G-17, PGI, and PGII combined detection device, which includes the following steps:

(a) Preparation of colloidal gold using trisodium citrate reduction method;

(b) Using the colloidal gold prepared in step (a) to label gastrin 17, pepsinogen I and pepsinogen II antibodies to obtain immune colloidal gold;

(c) Dilute the immune colloidal gold in step (b) with a spray gold buffer to obtain an immune colloidal gold solution, and spray the immune colloidal gold solution on a glass fiber mat to prepare an immune colloidal gold glass fiber membrane;

(d) After pre-treatment of nitrocellulose membrane with polyethylene glycol glycerol treatment solution, spray dots of gastrin 17, pepsinogen I and pepsinogen II antibodies bound to oleic acid-modified zinc sulfide nanoparticles As the detection line, spray the goat anti-mouse IgG antibody as the quality control line to prepare immune nitrocellulose membrane;

(e) Paste the pretreated sample pad, the immune colloidal gold glass fiber membrane prepared in step (c), the immune nitrocellulose membrane prepared in step (d), and absorbent paper in sequence on the glue plate, and cut to prepare the detection reagent Finally, put the test reagent strip into the plastic case.

The colloidal gold particles prepared by the trisodium citrate reduction method described in step (a) of the present invention have a particle size of 20-60 nm.

The gold spraying buffer described in step (c) of the present invention is composed of Tris-HCl solution, sucrose, trehalose, and bovine serum albumin BSA, with a pH value of 8.5, wherein the concentration of Tris-HCl is 0.02 mol/L, and the concentration of sucrose is 5 ~20%, trehalose concentration is 1~5%, bovine serum albumin BSA concentration is 0.5~1%.

The pretreatment of the nitrocellulose membrane with polyethylene glycol glycerol treatment solution in step (d) of the present invention is: soak the nitrocellulose membrane with the polyethylene glycol glycerol treatment solution for 1 hour, and then shake it slowly After taking it out, wash it with distilled water 3 times, and finally dry it in a vacuum drying oven.

The zinc sulfide nanoparticles described in step (d) of the present invention respectively bind gastrin 17, pepsinogen I and pepsinogen II antibodies: take oleic acid-modified ZnS as a carrier, take 1 mL of gastrin 17, pepsin The original I and pepsinogen II antibody solutions were stirred for 1 hour and collected by centrifugation at 12000 rpm, 8500 rpm and 7000 rpm for 10 minutes, respectively, and washed with deionized water twice each.

The polyethylene glycol glycerol treatment solution described in step (d) of the present invention is composed of polyethylene glycol glycerol diluted to a concentration of 0.5%, filtered through a 0.22 μm filter membrane, and ready for use.

The polyethylene glycol glycerol treatment liquid described in step (d) of the present invention is composed of a mixture of polyethylene glycol glycerol and polylysine (SIGMA, 150KD～300KD), wherein the concentration of polyethylene glycol glycerol is The concentration of polylysine is 0.5%, and the concentration of polylysine is 0.5%. It is filtered through a 0.22μm filter membrane for use.

The polyethylene glycol glycerol treatment solution described in step (d) of the present invention is composed of a mixture of polyethylene glycol glycerol, polylysine (SIGMA, 150KD～300KD), and PEG20000, wherein polyethylene glycol propylene The concentration of triol is 0.5%, the concentration of polylysine is 0.5%, and the concentration of PEG20000 is 0.1%, filtered through a 0.22 μm filter membrane for use.

The method for preparing oleic acid-modified zinc sulfide nanoparticles described in step (d) of the present invention: Take 15ml of oleic acid in absolute ethanol and add 15ml of zinc acetate aqueous solution with a concentration of 0.3mol/L, stir in a water bath at 40°C, and use ammonia Adjust the pH value, and then add 15ml of sodium sulfide aqueous solution with a concentration of 0.3mol/L. After reacting for 5 minutes, add 5ml of SDS aqueous solution. After the mixture is evenly mixed, the reaction solution is poured into a 90ml hydrothermal kettle. After the hydrothermal kettle is airtight, it is placed in a constant temperature drying box and reacted at a constant temperature for a certain time. After the reaction is over, the temperature is lowered to 50°C, and the product is taken out. Wash with acetone, deionized water, ethanol, centrifuge, and vacuum dry at 50°C for 2 hours to obtain powder ZnS, which is stored for later use.

The sample pad treatment liquid used in the pretreated sample pad described in step (e) of the present invention is composed of Tris-HCl solution, bovine serum albumin BSA, casein, and surfactant (alkylphenol polyoxyethylene ether), wherein The concentration of Tris-HCl solution is 0.1 mol/L, the concentration of bovine serum albumin BSA is 0.5-1%, the concentration of casein is 0.1-0.2%, and the concentration of surfactant is 0.5-1%.

The reaction of ethylene glycol and epichlorohydrin is catalyzed by alkali, and the product is neutralized with dilute hydrochloric acid, extracted with carbon tetrachloride, and distilled under reduced pressure to obtain polyethylene glycol glycerol (PEGG), which is a pale yellow viscous substance. PEGG can be miscible with water in any ratio, and can also be dissolved in common organic solvents such as ethanol, acetone, tetrahydrofuran, chloroform, and has a certain surface activity. The structure of polyethylene glycol glycerol contains multiple hydroxyl groups for coupling, and the activation process is simple, which facilitates the immobilization of proteins on the surface of the NC membrane. Under normal conditions, the number of antibodies bound to the NC membrane per unit area is limited. After treatment with polyethylene glycol glycerol, the number of antibodies bound to the NC membrane per unit area can be increased, thereby achieving higher detection sensitivity. .

On the basis of improving the adsorption of NC membrane to protein, exploring the adsorption effect of protein to NC membrane is another way to improve the sensitivity of colloidal gold. The ZnS modified with oleic acid/sodium lauryl sulfate not only has a nanometer-scale particle size, but also has good water solubility and biocompatibility. Using the functional groups on its outer surface, it can be uniformly dispersed in an aqueous medium. Can be combined with biological macromolecules. Zinc sulfide nanoparticles have the advantages of good stability, easy preparation, good biocompatibility and low immunogenicity, etc., and they are widely studied in the field of biomedicine. However, there is no reported application in colloidal gold immunochromatography. This study explored the effect of zinc sulfide nanoparticles on NC membrane-coated antibodies. Firstly, the antibody used for marking was combined with zinc sulfide nanoparticles, blocked, and purified by centrifugation. Unbound antibodies were removed, and then reconstituted to a certain ratio before marking. Membrane, such a zinc sulfide particle can bind multiple antibodies, thereby increasing the efficiency of coating antibodies, and the sensitivity will be greatly improved.

In order to improve the sensitivity of the colloidal gold immunochromatographic technique, the present invention pretreated the nitrocellulose membrane with polyethylene glycol glycerol treatment solution to combine the NC-coated antibody with the zinc sulfide nanoparticles to achieve improved The purpose of the test paper sensitivity.

The positive effects of the present invention are:

1. The detection device of the present invention has a simple structure and a novel concept. The gastrin 17, pepsinogen I and pepsinogen II antibodies are coated on the nitrocellulose membrane, which has strong specificity and can simultaneously detect gastric secretions in specimens. Peptin 17, pepsinogen I and pepsinogen II did not increase the complexity of production operations.

2. In the preparation step of immune colloidal gold, by cooperating with suitable gold spraying buffer and sample pad treatment solution, it can ensure the complete release of immune colloidal gold, and effectively improve the sensitivity of the reaction. Under the same threshold, it can also reduce The amount of immune colloidal gold saves costs.

3. The present invention pretreats the nitrocellulose membrane, modifies the antibody coating the nitrocellulose membrane, and improves the sensitivity and specificity of the detection test paper.

4. The detection device of the present invention does not require any special equipment, and the detection cost is low.

5. The detection device of the present invention is easy to operate, does not require professionals to operate, and has strong practicability.

Description of the drawings

Figure 1 is a schematic diagram of the structure of the present invention. In the figure, the detection lines T1, T2, and T3 arranged on the immune nitrocellulose membrane are arranged longitudinally on the same immune nitrocellulose membrane;

Figure 2 is a cross-sectional view taken along line A-A of Figure 1;

Figure 3 is a schematic diagram of another structure of the present invention. In the figure, the detection lines T1, T2, and T3 are respectively arranged on three immune nitrocellulose membranes, arranged side by side to form a joint detection device;

Figure 4 is a B-B cross-sectional view of Figure 3;

Figure 5 is a C-C cross-sectional view of Figure 3;

Fig. 6 is a cross-sectional view taken along the line D-D in Fig. 3.

Detailed ways

The present invention will be further described below in conjunction with embodiments and drawings.

Example 1

The sample pad 1, the immune colloidal gold glass fiber membrane 2, the immune nitrocellulose membrane 3, and the absorbent pad 4 are respectively pasted on the plastic plate 5. The two ends of the immune nitrocellulose membrane 3 are respectively connected to the absorbent pad 4 and the immune colloidal gold glass The fiber membrane 2 is overlapped, and the other end of the immune colloidal gold glass fiber membrane 2 is overlapped with the sample pad 1; the immune nitrocellulose membrane 3 is provided with a first detection line T1, a second detection line T2, and a third detection Line T3, and quality control line (C); the first test line T1 has high specific gastrin 17 antibody in the solid phase; the second test line T2 has high specific pepsinogen in the solid phase I antibody; the third detection line T3 has a high-specificity pepsinogen II antibody in the solid phase; as shown in Figures 1 and 2, the detection lines T1, T2, T3 are set on the immune nitrocellulose membrane 3 It can be arranged longitudinally on the same immune nitrocellulose membrane 3 to form a combined detection device; as shown in Figures 3, 4, 5 and 6, the detection lines T1, T2, T3 are provided on the immune nitrocellulose membrane 3. It can also be separately arranged on three immune nitrocellulose membranes 3 and arranged side by side to form a combined detection device; the quality control line C is sprayed with goat anti-mouse IgG polyclonal antibody.

The preparation method of the G-17, PGI, and PGII combined detection device of the present invention includes solid-phase purified high-specificity gastrin 17, pepsinogen I and pepsinogen II antibodies (detection lines T1, T2, T3) Nitrocellulose membrane with goat anti-mouse IgG polyclonal antibody (control line), glass fiber membrane with colloidal gold-labeled gastrin 17, pepsinogen I and pepsinogen II antibodies, sample pads, absorbent paper, etc. The auxiliary materials are thus made by pasting. The specific steps are as follows:

(a) Preparation of colloidal gold using trisodium citrate reduction method

Quickly add gold chloride to the heated 100ml purified water, after the solution boils again, quickly add trisodium citrate, gold chloride: trisodium citrate 1:0.5, continue to boil, observe the color of the solution changes from yellow to black and then to purple , After finally turning into a stable wine red, keep heating for 10 minutes at a time, and the colloidal gold particles are 20nm;

(b) Preparation of immune colloidal gold

1) Take 100ml of 20nm colloidal gold solution, add 140μl of pH regulator, and mix well; let stand for 5min;

2) In the 20nm colloidal gold solution, add gastrin 17, pepsinogen I and pepsinogen II at the ratio of 14 μg per milliliter of colloidal gold solution, respectively, 1.4 mg in total, and mix; let stand for 5 min;

3) Add 0.4 ml of colloidal gold stabilizer in the proportion of 0.4% respectively, mix well, and let stand for 5 minutes;

4) Centrifuge at 10,000, 12,000, and 14,000 rpm for 10 min, collect the precipitates respectively, and combine the precipitates collected three times;

(c) Use the optimized spray gold buffer to dilute the immune colloidal gold to obtain the immune colloidal gold solution, and spray the immune colloidal gold solution on the glass fiber mat to prepare the immune colloidal glass fiber membrane; the spray gold buffer includes: the concentration is 20mM Tris-HCl solution, sucrose concentration is 5%, trehalose concentration is 1%, BSA concentration is 1%, pH is 8.5;

(d) Solid phase nitrocellulose membrane

1) Pretreatment of nitrocellulose membrane with polyethylene glycol glycerol treatment solution

Prepare polyethylene glycol glycerol treatment solution: the concentration of polyethylene glycol glycerol is 0.5%, filtered through a 0.22μm filter membrane for use;

Pretreatment of nitrocellulose membrane with polyethylene glycol glycerol treatment solution: Put the nitrocellulose membrane in the polyethylene glycol glycerol treatment solution to soak for 1 hour, shake it slowly, take it out, and wash it with distilled water 3 times. Finally, it is dried in a vacuum drying oven;

2) Zinc sulfide nanoparticles modified gastrin 17, pepsinogen I and pepsinogen II antibodies

Preparation of oleic acid modified zinc sulfide nanoparticles: Take 15ml of oleic acid in absolute ethanol and add it to 15ml of 0.3mol/L zinc acetate aqueous solution, stir in a water bath at 40℃, adjust the pH with ammonia, and then add 15ml to a concentration of 0.3 mol/L sodium sulfide aqueous solution, after reacting for 5 minutes, add 5ml SDS aqueous solution, after mixing uniformly, pour the reaction solution into a 90ml hydrothermal kettle. After the hydrothermal kettle is airtight, it is placed in a constant temperature drying box and reacted at a constant temperature for a certain time. After the reaction is over, the temperature is lowered to 50°C, and the product is taken out. Wash with acetone, deionized water, ethanol, centrifuge, and vacuum dry at 50°C for 2 hours to obtain powdered ZnS, which is stored for later use.

Zinc sulfide nanoparticles modified gastrin 17, pepsinogen I and pepsinogen II antibodies:

Using oleic acid-modified ZnS as a carrier, take 1ml of gastrin 17, pepsinogen I and pepsinogen II antibody solution, stir for 1 hour, centrifuge at 12000rpm, 8500rpm and 7000rpm for 10 minutes to collect, wash each with deionized water 2 times.

3) Coating of nitrocellulose membrane detection line and quality control line antibody

When the spray volume is 1.4μl/cm, dilute zinc sulfide nanoparticles-gastrin 17 antibody, zinc sulfide nanoparticles-pepsinogen I antibody, zinc sulfide nanoparticles-pepsinogen II antibody to 1.5mg/ml, The quality control line goat anti-mouse IgG antibody is diluted to 1mg/ml, and the test line and the quality control line of the nitrocellulose membrane are respectively coated, dried overnight at room temperature, and stored for later use;

4) Sample pad pretreatment

Soak the glass fiber in the sample pad treatment solution for 10 minutes. The sample pad treatment solution includes: Tris-HCl solution concentration of 0.1M, bovine serum albumin BSA concentration of 0.5%, casein concentration of 0.1%, and surfactant concentration of 0.5%, dried at 37°C for use, the sample pad after treatment can improve the sensitivity of the reaction;

e. Assembly

The pre-treated sample pad, immune colloidal gold glass fiber membrane, immune nitrocellulose membrane, and absorbent paper are sequentially pasted on the rubber sheet, and cut to obtain a detection reagent strip, and finally the detection reagent strip is put into a plastic shell.

f. Quantitative detection: The minimum detection concentration of human gastrin 17 detected by this combined detection device is 0.5pg/ml, the minimum detection concentration of pepsinogen I is 5ng/ml, and the minimum detection concentration of pepsinogen II is detected by the colloidal gold detector. It is 5ng/ml.

Example 2

The prepared colloidal gold particles are 40nm;

The gold spray buffer includes: a concentration of 20mM Tris-HCl solution, a sucrose concentration of 12%, a trehalose concentration of 3%, a BSA concentration of 0.7%, and a pH of 8.5;

The preparation of polyethylene glycol glycerol treatment solution is composed of a mixture of polyethylene glycol glycerol and polylysine (SIGMA, 150KD～300KD), wherein the concentration of polyethylene glycol glycerol is 0.5%, and polylysine The acid concentration is 0.5%, filtered through a 0.22μm filter membrane, ready for use;

The cup pad treatment solution includes: Tris-HCl solution concentration of 0.1M, bovine serum albumin BSA concentration of 0.7%, casein concentration of 0.15%, and surfactant concentration of 0.7%;

The rest is the same as in Example 1.

Example 3

The preparation of colloidal gold particles is 60nm;

The gold spray buffer includes: a concentration of 20mM Tris-HCl solution, a sucrose concentration of 20%, a trehalose concentration of 5%, a BSA concentration of 1%, and a pH of 8.5;

Preparation of polyethylene glycol glycerol treatment solution: it is composed of polyethylene glycol glycerol, polylysine (SIGMA, 150KD～300KD) and PEG2000, in which the concentration of polyethylene glycol glycerol is 0.5%, The concentration of polylysine is 0.5%, and the concentration of PEG20000 is 0.1%, filtered through a 0.22μm filter membrane for use;

Cup mat treatment solution includes: Tris-HCl solution concentration of 0.1M, bovine serum albumin BSA concentration of 1%, casein concentration of 0.2%, and surfactant concentration of 1%;

The rest is the same as in Example 1.

The following experiments will further illustrate the effect of the present invention.

Experimental example 1:

1. Comparison of the adsorption capacity of polyethylene glycol glycerol treatment solution on nitrocellulose membrane protein

1.1 Materials and methods

1.1 Material: nitrocellulose membrane, pore size 4.5μm, purchased from General Electric Company

1.2 Nitrocellulose membrane treatment method

1.2.1 Preparation of polyethylene glycol glycerol treatment solution

Prepare three kinds of polyethylene glycol glycerol treatment liquid: polyethylene glycol glycerol group, the concentration of polyethylene glycol glycerol is 0.5%; polyethylene glycol glycerol treatment liquid polylysine group , The concentration of polyethylene glycol glycerol is 0.5%, and the concentration of polylysine is 0.5%; the group of polyethylene glycol glycerol, polylysine and PEG20000, in which the concentration of polyethylene glycol glycerol is 0.5%, the concentration of polylysine is 0.5%, the concentration of PEG20000 is 0.1%, the three groups of treatment liquids are filtered through 0.22μm filter membrane for use.

1.2.2 Nitrocellulose membrane treatment method

The nitrocellulose membrane was soaked in the polyethylene glycol glycerol treatment solution for 1 hour, and then shaken slowly. After taking it out, it was washed with distilled water 3 times, and finally dried in a vacuum drying oven.

1.3 Experimental method

The untreated and treated nitrocellulose membranes were prepared according to the process flow of the above examples to prepare gastrin 17, pepsinogen I and pepsinogen II combined detection test papers. The test process refers to the test paper instructions to compare the nitrocellulose membranes. Adsorption and stability index difference after treatment and treatment.

1.4 Results

1.4.1 Comparison of protein adsorption capacity

Take 3 groups of test papers for the treatment group and the untreated group, respectively add the samples to be tested, and judge the protein adsorption ability of the treated membrane by observing the color development. The results are shown in Table 1. The results show that the treated nitrocellulose membrane is significantly better than the untreated membrane in terms of solution wettability. After the treatment, the positive stripe of the membrane is slightly darker, especially when the concentration is low, which improves the sensitivity of the reaction and indicates the protein adsorption capacity Significantly enhanced and improved response sensitivity. The adsorption effect of the polyethylene glycol glycerol-polylysine-PEG20000 treatment group was significantly better than that of the polyethylene glycol glycerol group and the polyethylene glycol glycerol-polylysine group.

Table 1 Comparison of adsorption capacity of nitrocellulose membrane

1.4.2 Comparison of stability of nitrocellulose membrane

Take 3 groups of test papers for the treatment group and the untreated group, and observe the color development through the 37°C accelerated experiment to judge the stability of the protein adsorption on the nitrocellulose membrane after treatment. The results are shown in Table 2. Comparing the results in Table 2 with the results in Table 1, it is found that the color change of the nitrocellulose membrane after treatment is basically the same as that of 10 days ago, and the stability is good.

Table 2 Accelerated stability comparison of nitrocellulose membrane (at 37°C for 10 days)

Experimental example 2:

2. Oleic acid modified zinc sulfide nanoparticles modified gastrin 17, pepsinogen I and pepsinogen II antibodies

2.1 Materials and methods

2.1.1 Material: nitrocellulose membrane, pore size 4.5μm, purchased from General Electric Company

2.1.2 Preparation of oleic acid modified zinc sulfide nanoparticles

Take 15ml of oleic acid absolute ethanol solution and add it to 15ml of 0.3mol/L zinc acetate aqueous solution, stir in a water bath at 40℃, adjust the pH value with ammonia water, then add 15ml of 0.3mol/L sodium sulfide aqueous solution, react for 5min Then, 5ml of SDS aqueous solution was added, and after mixing uniformly, the reaction solution was poured into a 90ml hydrothermal kettle. After the hydrothermal kettle is airtight, it is placed in a constant temperature drying box and reacted at a constant temperature for a certain time. After the reaction is over, the temperature is lowered to 50°C, and the product is taken out. Wash with acetone, deionized water and ethanol successively, centrifuge, and vacuum dry at 50°C for 2 hours to obtain powdered ZnS, which is stored for later use.

2.1.3 Zinc sulfide nanoparticles modified gastrin 17, pepsinogen I and pepsinogen II antibodies

Using oleic acid-modified ZnS as a carrier, take 1 mL of gastrin 17, pepsinogen I and pepsinogen II antibody solutions, stir for 1 hour, centrifuge at 12000 rpm, 8500 rpm and 7000 rpm for 10 minutes to collect, wash each with deionized water 2 times.

2.2 Experimental method

The gastrin 17, pepsinogen I and pepsinogen II antibodies modified by zinc sulfide nanoparticles and the unmodified gastrin 17, pepsinogen I and pepsinogen II antibodies were prepared according to the process flow of the above-mentioned embodiment. Secretin 17, pepsinogen I and pepsinogen II combined detection test paper. The test procedure refers to the test paper instructions to compare the difference in protein adsorption and stability indexes between treatment and untreated zinc sulfide nanoparticles.

2.3 Results

2.3.1 Comparison of protein adsorption capacity

Take the zinc sulfide nanoparticle-treated group and the untreated group of test papers, respectively add the samples to be tested, and judge the protein adsorption ability of the treated membrane by observing the color development. The results are shown in Table 3. The results showed that the color of the positive bands in the zinc sulfide nanoparticle modified group was slightly darker, especially when the concentration was low, which improved the sensitivity of the reaction, indicating that the protein adsorption capacity was significantly enhanced, and the sensitivity of the reaction was improved.

Table 3 Comparison of protein adsorption capacity of zinc sulfide nanoparticle modification

2.3.2 Stability comparison

Take the zinc sulfide nanoparticle-treated group and the untreated group test paper, and observe the color development by 37°C accelerated experiment to judge the stability of the protein adsorption on the nitrocellulose membrane after zinc sulfide modification. The results are shown in Table 4. Comparing the results in Table 4 with the results in Table 3, it is found that the color change of the nitrocellulose membrane after treatment is basically the same as that of 10 days ago, and the stability is good.

Table 4 Accelerated stability comparison (at 37°C for 10 days)

The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.

Claims (11)

1. A G-17, PGI, and PGII combined detection device, which is characterized in that: the sample pad (1), the immune colloidal gold glass fiber membrane (2), the immune nitrocellulose membrane (3) and the absorbent pad (4) are respectively pasted on Plastic board (5), the two ends of the immune nitrocellulose membrane (3) are respectively overlapped with the absorbent pad (4) and the immune colloidal gold glass fiber membrane (2), the immune colloidal gold glass fiber membrane (2) The other end is overlapped with the sample pad (1); the immune nitrocellulose membrane (3) is provided with a first detection line T1, a second detection line T2, a third detection line T3, and a quality control line C; The first detection line T1 has a high-specificity gastrin 17 antibody in the solid phase; the second detection line T2 has a high-specificity pepsinogen I antibody in the solid phase; the third detection line T3 has a solid phase There is a highly specific pepsinogen II antibody; the first detection line T1, the second detection line T2 and the third detection line T3 set on the immune nitrocellulose membrane (3) are arranged longitudinally on the same immune nitrocellulose membrane (3), a combined detection device is formed; or the first detection line T1, the second detection line T2, and the third detection line T3 set on the immune nitrocellulose membrane (3) are respectively set on three nitrocellulose membranes (3) Above, arranged side by side to form a combined detection device; on the quality control line C, a goat anti-mouse IgG polyclonal antibody is sprayed.
2. The preparation method of the G-17, PGI, and PGII combined detection device according to claim 1, characterized in that it comprises the following steps:

   (a) Preparation of colloidal gold using trisodium citrate reduction method;

   (b) Using the colloidal gold prepared in step (a) to label gastrin 17, pepsinogen I, and pepsinogen II antibodies to obtain immune colloidal gold;

   (c) Dilute the immune colloidal gold in step (b) with a spray gold buffer to obtain an immune colloidal gold solution, and spray the immune colloidal gold solution on a glass fiber mat to prepare an immune colloidal gold glass fiber membrane;

   (d) After pre-treatment of the nitrocellulose membrane with polyethylene glycol glycerol treatment solution, spray the gastrin 17, pepsinogen I, and pepsinogen II antibodies combined with oleic acid-modified zinc sulfide nanoparticles As the first test line T1, the second test line T2, and the third test line T3, respectively, the goat anti-mouse IgG polyclonal antibody was sprayed as the quality control line C to prepare the immune nitrocellulose membrane;

   (e) Paste the pretreated sample pad, the immune colloidal gold glass fiber membrane prepared in step (c), the immune nitrocellulose membrane prepared in step (d), and absorbent paper in sequence on the plastic plate, and cut to prepare the detection reagent Finally, put the test reagent strip into the plastic case.
3. The method for preparing a G-17, PGI, and PGII combined detection device according to claim 2, wherein the particle size of the colloidal gold in step (a) is 20-60 nm.
4. The method for preparing a G-17, PGI, and PGII combined detection device according to claim 2, wherein the gold spray buffer in step (c) is composed of Tris-HCl, sucrose, trehalose and bovine serum albumin It is composed of BSA with a pH value of 8.5, in which the concentration of Tris-HCl is 0.02 mol/L, the concentration of sucrose is 5-20%, the concentration of trehalose is 1-5%, and the concentration of bovine serum albumin BSA is 0.5-1%.
5. The method for preparing a G-17, PGI, and PGII combined detection device according to claim 2, wherein the pretreatment of the nitrocellulose membrane with polyethylene glycol glycerol treatment solution in step (d) is : Soak the nitrocellulose membrane with polyethylene glycol glycerol treatment solution for 1 hour, shake it slowly, take it out and wash it with distilled water 3 times, and finally dry it in a vacuum drying oven;

   The gastrin 17, pepsin I, and pepsin II antibodies bound to oleic acid-modified zinc sulfide nanoparticles described in step (d) are: use oleic acid-modified ZnS as a carrier, and take 1 mL of gastrin 17, respectively. The pepsin I and pepsin II antibody solutions were stirred for 1 hour, and the precipitates were collected by centrifugation at 12000 rpm, 8500 rpm and 7000 rpm for 10 minutes, respectively, and washed with deionized water twice.
6. The method for preparing a G-17, PGI, and PGII combined detection device according to claim 2 or 5, wherein the polyethylene glycol glycerol treatment solution in step (d) is made of polyethylene glycol glycerin Alcohol is diluted to a concentration of 0.5% to obtain, filtered through a 0.22μm filter membrane, and set aside.
7. The method for preparing a G-17, PGI, and PGII combined detection device according to claim 2 or 5, wherein the polyethylene glycol glycerol treatment solution in step (d) is made of polyethylene glycol glycerin Alcohol and polylysine are mixed and composed, wherein the concentration of polyethylene glycol glycerol is 0.5%, and the concentration of polylysine is 0.5%, filtered through a 0.22 μm filter membrane for use.
8. The method for preparing a G-17, PGI, and PGII combined detection device according to claim 7, wherein the polylysine is purchased from SIGMA and has a molecular weight of 150KD to 300KD.
9. The method for preparing a G-17, PGI, and PGII combined detection device according to claim 2 or 5, wherein the polyethylene glycol glycerol treatment solution in step (d) is made of polyethylene glycol glycerin A mixture of alcohol, polylysine, and PEG20000, wherein the concentration of polyethylene glycol glycerol is 0.5%, the concentration of polylysine is 0.5%, and the concentration of PEG20000 is 0.1%, filtered through a 0.22μm filter membrane for use.
10. The preparation method of the G-17, PGI, and PGII combined detection device according to claim 2 or 5, characterized in that: the preparation method of oleic acid modified zinc sulfide nanoparticles in step (d): take oleic acid anhydrous Add 15ml of ethanol solution to 15ml of 0.3mol/L zinc acetate aqueous solution, stir in a water bath at 40℃, adjust the pH value with ammonia water, then add 15ml of 0.3mol/L sodium sulfide aqueous solution, and after reacting for 5 minutes, add 5ml SDS After mixing the aqueous solution, the reaction solution is poured into a 90ml hydrothermal kettle. After the hydrothermal kettle is sealed, it is placed in a constant temperature drying oven, and reacted at a constant temperature for a certain period of time. After the reaction is completed, the temperature is reduced to 50°C and taken out The product was washed with acetone, deionized water, ethanol, centrifuged, and vacuum dried at 50°C for 2 hours to obtain powdered oleic acid modified ZnS, which was stored for later use.
11. The preparation method of the G-17, PGI, and PGII combined detection device according to claim 2, characterized in that: the sample pad treatment liquid used in the pretreated sample pad in step (e) is Tris-HCl solution, cattle Serum albumin BSA, casein, surfactant alkylphenol polyoxyethylene ether composition, in which the concentration of Tris-HCl solution is 0.1 mol/L, the concentration of bovine serum albumin BSA is 0.5-1%, and the concentration of casein is 0.1- 0.2%, the surfactant concentration is 0.5-1%.

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