A high-sensitivity colloidal gold immunochromatography kit and its application

Abstract

The present invention belongs to the field of biological detection technology, and specifically relates to a highly sensitive colloidal gold immunochromatographic kit and its application. The present invention improves upon a conventional colloidal gold immunochromatographic kit by loading glucose and a redox indicator or acid-base indicator onto the sample pad of a test strip. The invention utilizes the principle that colloidal gold catalyzes an oxidation reaction of glucose to produce hydrogen peroxide and gluconic acid, causing the redox indicator or acid-base indicator to change color. When the colloidal gold method is used for detection, the indicator changes color due to the oxidation reaction at the locations where the colloidal gold nanoparticles in the kit aggregate, thereby increasing the original signal intensity and greatly improving the sensitivity and accuracy of the colloidal gold immunochromatographic detection.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and particularly relates to a high-sensitivity colloidal gold immunochromatography kit and application thereof. Background Art

[0002] Colloidal gold immunochromatography kits are widely used in various testing fields such as food safety and clinical medical testing due to their advantages of low cost, easy operation and fast detection speed.

[0003] However, the optical signal of colloidal gold is limited, and the detection sensitivity of the colloidal gold immunochromatography kit is low, which can easily lead to missed detection during the detection process. How to further improve the sensitivity of the colloidal gold immunochromatography kit and improve the accuracy of detection has become a research hotspot in recent years.

[0004] Helicobacter pylori is a Gram-negative bacterium that mainly parasitizes in the human stomach and duodenum, and is closely related to the occurrence and development of various gastric diseases.

[0005] At present, the rapid urease test and the fecal antigen test are commonly used methods for rapid detection of Helicobacter pylori. Among them, the rapid urease test relies on the property of Helicobacter pylori to produce a large amount of urease, which can decompose urea. The person to be tested swallows a urea pill containing carbon or nitrogen radionuclides, and the amount of radionuclides in the exhaled breath is detected to determine whether the person is infected with Helicobacter pylori. The fecal antigen test is based on the property that Helicobacter pylori can be excreted from the body with feces. It uses specific antibodies to Helicobacter pylori to detect Helicobacter pylori antigens in fecal samples, and presents corresponding signals through the detection system to determine whether the person is infected with Helicobacter pylori.

[0006] Although the colloidal gold method has been studied for typing Helicobacter pylori antibodies, this method is limited by methodology and its detection sensitivity needs to be improved. Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides a high-sensitivity colloidal gold immunochromatography kit and its application.

[0008] The colloidal gold immunochromatography kit provided by the present invention includes a test strip, which includes a PVC base plate, a nitrocellulose membrane, a sample pad, a conjugation pad, and a water-absorbing pad; the sample pad, conjugation pad, nitrocellulose membrane, and water-absorbing pad are sequentially adhered to the PVC base plate and connected in an overlapping manner. The above features are common to conventional test strips and will not be explained in detail here. The present invention improves the test strip in the colloidal gold immunochromatography kit as follows:

[0009] First, glucose and an indicator are loaded on the sample pad of the test strip, and the indicator is selected from any one of a redox indicator and an acid-base indicator.

[0010] In addition, the sample pad is also loaded with a buffer system of a conventional test strip to ensure that the detection process is carried out within a normal and reasonable pH range. This is existing technology and will not be described in detail.

[0011] It should be noted that in the present invention, the buffer system loaded on the sample pad only provides a suitable pH environment for the antigen-antibody reaction. The setting of the buffer system does not affect the color development of the acid-base indicator or redox indicator in the present invention. The main reasons are:

[0012] First, the acid-base indicator used in the present invention itself does not develop color or is the same color as the background under these conditions;

[0013] Secondly, when the sample is tested, the chromatography speed of the sample is relatively fast. Although a small amount of glucose may be oxidized to produce gluconic acid during the chromatography process, the amount of gluconic acid is negligible and is evenly distributed on the nitrocellulose membrane. Therefore, the amount of gluconic acid produced during the chromatography process will not change the pH environment provided by the buffer system. As the reaction proceeds, colloidal gold gradually accumulates at the C line and T line, and gluconic acid and hydrogen peroxide also gradually accumulate at the C line and T line. Only when the buffering capacity of the buffer system is exceeded will the pH environment of the system at the C line and T line be changed, causing an obvious color change of the acid-base indicator or the redox indicator. At this time, the water content in the system is relatively balanced, and the position of the colloidal gold on the nitrocellulose membrane is relatively stable. In summary, the present invention has verified through a large number of experiments that the color change of the indicator is consistent with the signal of colloidal gold. When the colloidal gold is distributed in a planar manner on the nitrocellulose membrane for chromatography, even if the color of the indicator changes slightly, it is consistent with the background color and will not be prominently colored on the C line and T line. Only after the colloidal gold accumulates at the C line and T line will the color signal on the C line and T line be enhanced without affecting the signal of the original reaction system.

[0014] Secondly, the conjugate pad of the test strip is loaded with antibodies or antigens labeled with colloidal gold nanoparticles, and the particle size of the colloidal gold nanoparticles is 20-40 nm.

[0015] In the above improvements, preferably, the mass fraction of the glucose is 1%-2%.

[0016] Preferably, the mass fraction of the indicator is 0.001%-0.05%.

[0017] Preferably, the redox indicator is selected from at least one of sodium diphenylamine sulfonate, o-phenanthroline ferrous acid, diphenylamine, o-phenylaminobenzoic acid, methylene blue, quinoline yellow, and methyl blue.

[0018] Preferably, the acid-base indicator is at least one selected from litmus, methyl red, methyl orange, thymol blue, neutral red, and Congo red.

[0019] As further preferred, the acid-base indicator is methyl orange.

[0020] In addition, the application of the highly sensitive colloidal gold immunochromatographic kit in detecting Helicobacter pylori is also the focus of protection of the present invention.

[0021] The working principle of the kit provided by the present invention is as follows Figure 1 shown.

[0022] Attachment Figure 1 In the figure, (a) is the working principle of a conventional colloidal gold immunochromatography kit, and (b) is the working principle of the improved colloidal gold immunochromatography kit of the present invention.

[0023] According to the attached Figure 1 The mechanism of action of the kit before and after the improvement of the present invention is described as follows: When a conventional colloidal gold immunochromatographic kit is used for detection, the signal of the T line is only the signal generated by the aggregation of colloidal gold, that is, Figure (a);

[0024] After the improvement of the present invention, as shown in Figure (b), glucose and an indicator (redox reaction indicator, acid-base indicator) are added to the reaction system of the sample pad. When the glucose and the indicator move forward rapidly with the chromatography of the sample, a layer of glucose and the indicator will be spread on the nitrocellulose membrane. When the glucose and the indicator reach the T line with the chromatography of the sample, the colloidal gold gathers at the T line to generate a signal. At this time, the glucose coming from the chromatography is fully contacted with the oxygen in the air under the catalytic action of the colloidal gold, and an oxidation reaction occurs to produce gluconic acid and hydrogen peroxide, which triggers the color change of the redox indicator or the acid-base indicator, thereby causing secondary color development at the aggregation point of the nanogold particles, thereby improving the original signal intensity of the colloidal gold, and thus improving the detection sensitivity of the colloidal gold immunochromatography kit.

[0025] The beneficial effects of the present invention are:

[0026] By improving the test strip in the colloidal gold immunochromatography kit, that is, loading glucose and a redox indicator or an acid-base indicator on the sample pad of the test strip, and using colloidal gold nanoparticles as a catalyst for glucose oxidation, glucose is oxidized to gluconic acid through a catalytic reaction while producing hydrogen peroxide. During the detection process of the colloidal gold immunochromatography kit, the colloidal gold aggregation area can be secondary colored, thereby amplifying the detection signal and effectively improving the accuracy and sensitivity of the detection.

[0027] In addition, the colloidal gold immunochromatography kit provided by the present invention has universal applicability and can be applied to the colloidal gold immunochromatography detection process of various diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Diagrams showing the mechanism of action of the colloidal gold immunochromatography kit provided by the present invention, wherein (a) is a diagram showing the mechanism of action of a conventional colloidal gold immunochromatography kit, and (b) is a diagram showing the mechanism of action of the improved colloidal gold immunochromatography kit of the present invention;

[0029] Figure 2 The figures show the test results of a conventional colloidal gold immunochromatography kit and the colloidal gold immunochromatography kit provided by the present invention in the detection of Helicobacter pylori antibody typing; wherein (a) is the test result figure of the improved colloidal gold immunochromatography kit in Example 2 of the present invention, and (b) is the test result figure of the conventional colloidal gold immunochromatography kit. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the present invention, the present invention will be further explained in conjunction with specific embodiments.

[0031] The present invention only takes Helicobacter pylori antibody typing detection as an example to verify the effect of the colloidal gold immunochromatography kit provided by the present invention, but this does not limit the present invention.

[0032] Example 1

[0033] This embodiment specifically applies the provided colloidal gold immunochromatography kit to the typing detection of Helicobacter pylori antibodies, providing a highly sensitive Helicobacter pylori antibody typing detection kit.

[0034] In this embodiment, the conjugate pad uses 40 nm colloidal gold nanoparticles as antibody markers.

[0035] In this embodiment, sodium diphenylamine sulfonate was used as an indicator, and 1% glucose and 0.03% sodium diphenylamine sulfonate were added to the original sample pad raw material system.

[0036] The other structures, preparation processes, and pH system of the sample pad of the Helicobacter pylori antibody typing detection kit are exactly the same as those of the conventional Helicobacter pylori antibody typing detection kit, and will not be described in detail here.

[0037] During the test, the sample to be tested was added according to the normal sample addition operation, and the test results were observed after 15-20 min. In this embodiment, samples with the determined minimum detection limit concentrations of Ure antibody, CagA antibody, and VacA antibody were selected, and 3 batches of test kits were used for continuous detection for three days, one round of detection per day, and each batch of kits was tested 20 times for each sample in each round. In this way, for each detection limit sample, 3 batches of kits were tested a total of 60 times per day, and a total of 180 times in three days. The same detection limit concentration of 3 parallel samples was detected 540 times in total, and the detection rate was calculated to verify the detection effect of the test kit of the present invention. The test results are as follows:

[0038] Table 1 Results of the Ure antibody minimum detection limit test

[0039]

[0040] Table 2 Results of the validation test for the minimum detection limit of CagA antibodies

[0041]

[0042] Table 3 Results of the validation test for the minimum detection limit of VacA antibodies

[0043]

[0044] After 540 tests, the detection rate of Ure antibodies was 98.9%, the detection rate of CagA antibodies was 98.7%, and the detection rate of VacA antibodies was 98.1%. The average detection rate of the three items was 98.6%.

[0045] The three batch numbers 20221023, 20221101, and 20221109 described in the table refer to test kits obtained at different production times under the material and test kit preparation conditions described in this example.

[0046] Example 2

[0047] This embodiment specifically applies the provided colloidal gold immunochromatography kit to the typing detection of Helicobacter pylori antibodies, providing a highly sensitive Helicobacter pylori antibody typing detection kit.

[0048] In this embodiment, the conjugate pad uses 30 nm colloidal gold nanoparticles as antibody markers.

[0049] In this embodiment, sodium diphenylamine sulfonate was used as an indicator, and 1.5% glucose and 0.03% sodium diphenylamine sulfonate were added to the original sample pad raw material system.

[0050] The other structures, preparation processes, and pH system of the sample pad of the Helicobacter pylori antibody typing detection kit are exactly the same as those of the conventional Helicobacter pylori antibody typing detection kit, and will not be described in detail here.

[0051] During the test, add the sample to be tested according to the normal sample addition operation and observe the test results after 15-20 minutes.

[0052] Samples with the determined minimum detection limit concentrations of Ure antibodies, CagA antibodies, and VacA antibodies were selected and tested continuously for three days using three batches of test kits, with one test round per day. Each batch of kits tested each sample 20 times in each round. Thus, for each detection limit sample, three batches of kits were tested 60 times per day, for a total of 180 tests over three days. Compared to three parallel samples tested 540 times at the same detection limit concentration, the detection rate was calculated to verify the detection effect of the test kit of the present invention. The test results are as follows:

[0053] Table 4 Results of the Ure antibody minimum detection limit test

[0054]

[0055] Table 5 Results of the validation test for the minimum detection limit of CagA antibodies

[0056]

[0057] Table 6 Results of the minimum detection limit validation test for VacA antibodies

[0058]

[0059] The three batch numbers 20221025, 20221103, and 20221112 described in the table refer to test kits obtained at different production times under the material and test kit preparation conditions described in this example.

[0060] Example 3

[0061] This embodiment specifically applies the provided colloidal gold immunochromatography kit to the typing detection of Helicobacter pylori antibodies, providing a highly sensitive Helicobacter pylori antibody typing detection kit.

[0062] In this embodiment, the conjugate pad uses 20 nm colloidal gold nanoparticles as antibody markers.

[0063] In this embodiment, sodium diphenylamine sulfonate was used as an indicator, and 2% glucose and 0.03% sodium diphenylamine sulfonate were added to the original sample pad raw material system.

[0064] The other structures, preparation processes, and pH system of the sample pad of the Helicobacter pylori antibody typing detection kit are exactly the same as those of the conventional Helicobacter pylori antibody typing detection kit, and will not be described in detail here.

[0065] During the test, add the sample to be tested according to the normal sample addition operation and observe the test results after 15-20 minutes.

[0066] Samples with the determined minimum detection limit concentrations of Ure antibodies, CagA antibodies, and VacA antibodies were selected and tested continuously for three days using three batches of test kits, with one test round per day. Each batch of kits tested each sample 20 times in each round. Thus, for each detection limit sample, three batches of kits were tested 60 times per day, for a total of 180 tests over three days. Compared to three parallel samples tested 540 times at the same detection limit concentration, the detection rate was calculated to verify the detection effect of the test kit of the present invention. The test results are as follows:

[0067] Table 7 Ure antibody minimum detection limit test results

[0068]

[0069] Table 8 Results of the validation test for the minimum detection limit of CagA antibodies

[0070]

[0071] Table 9 Results of the validation test for the minimum detection limit of VacA antibodies

[0072]

[0073] The three batch numbers 20221028, 20221108, and 20221115 described in the table refer to test kits obtained at different production times under the material and test kit preparation conditions described in this example.

[0074] Example 4

[0075] This embodiment specifically applies the provided colloidal gold immunochromatography kit to the typing detection of Helicobacter pylori antibodies, providing a highly sensitive Helicobacter pylori antibody typing detection kit.

[0076] In this embodiment, the conjugate pad uses 30 nm colloidal gold nanoparticles as antibody markers.

[0077] In this embodiment, sodium diphenylamine sulfonate was used as an indicator, and 1.5% glucose and 0.03% methyl orange were added to the original sample pad raw material system.

[0078] The other structures, preparation processes, and pH system of the sample pad of the Helicobacter pylori antibody typing detection kit are exactly the same as those of the conventional Helicobacter pylori antibody typing detection kit, and will not be described in detail here.

[0079] During the test, add the sample to be tested according to the normal sample addition operation and observe the test results after 15-20 minutes.

[0080] Samples with the determined minimum detection limit concentrations of Ure antibodies, CagA antibodies, and VacA antibodies were selected and tested continuously for three days using three batches of test kits, with one test round per day. Each batch of kits tested each sample 20 times in each round. Thus, for each detection limit sample, three batches of kits were tested 60 times per day, for a total of 180 tests over three days. Compared to three parallel samples tested 540 times at the same detection limit concentration, the detection rate was calculated to verify the detection effect of the test kit of the present invention. The test results are as follows:

[0081] Table 10 Results of the Ure antibody minimum detection limit test

[0082]

[0083] Table 11 Results of the validation test for the minimum detection limit of CagA antibodies

[0084]

[0085] Table 12 Results of the validation test for the minimum detection limit of VacA antibodies

[0086]

[0087] The three batch numbers 20221030, 20221116, and 20221119 described in the table refer to test kits obtained at different production times under the material and test kit preparation conditions described in this example.

[0088] The differences between the kits in Examples 1 to 4 and the test results are summarized in Table 13 below.

[0089] Table 13 Helicobacter pylori antibody typing test results after different improvements

[0090]

[0091] It can be seen from the experimental results of the above embodiments that as the colloidal gold particle size decreases, although the signal of the colloidal gold particles themselves is weakened, the ability of the colloidal gold particles to catalyze glucose oxidation is enhanced. The increase or decrease of the final test kit T-line signal must comprehensively consider the superposition of the two. From the results of the above embodiments, when the colloidal gold particle size is 30 nm and the glucose concentration is 1.5%, the comprehensive signal is the strongest.

[0092] Comparative Example 1

[0093] A conventional Helicobacter pylori antibody typing detection kit was used without any modification. The colloidal gold particle size was 40 nm, the same as in Example 1. The average detection rate of the kit for the reference substances with the minimum detection limits of Ure antibody, CagA antibody, and VacA antibody was only 95.0%.

[0094] Table 14 Results of the validation test for the minimum detection limit of Ure antibodies

[0095]

[0096] Table 15 Results of the validation test for the minimum detection limit of CagA antibodies

[0097]

[0098] Table 16 Results of the validation test for the minimum detection limit of VacA antibodies

[0099]

[0100] The three batch numbers 20221019, 20221026, and 20221029 described in the table refer to test kits obtained at different production times under the material and test kit preparation conditions described in this example.

[0101] Attachment Figure 2 The results of the kit in Example 2 and the kit in Comparative Example 1 for Helicobacter pylori antibody typing detection with the same detection limit reference are shown respectively. Figure 2 In the figure, (a) is the test result of the kit of Example 2, and (b) is the test result of the conventional kit of Comparative Example 1.

[0102] Attachment Figure 2 It can be seen that after the kit is improved in the present invention, the signal of the T line of the kit is significantly enhanced compared with the T line signal of the conventional kit, and the sensitivity and accuracy of the kit are significantly improved.

[0103] The specific information of the specifications, batch numbers and manufacturers of the conventional kit provided by the present invention are as follows:

[0104] Product Name: Helicobacter pylori Antibody Typing Detection Kit (Colloidal Gold Method);

[0105] Packaging specifications: 25 servings / box;

[0106] Product batch:

[0107] In Example 1: 20221023, 20221101, 20221109; in Example 2: 20221025, 20221103, 20221112; in Example 3: 20221028, 20221108, 20221115; in Example 4: 20221030, 20221116, 20221119; in Comparative Example 1: 20221019, 20221026, 20221029;

[0108] Manufacturer: Jinan Jiufang Biotechnology Co., Ltd.

Claims

1. A high-sensitivity colloidal gold immunochromatography kit, comprising a test strip comprising a PVC base, a nitrocellulose membrane, a sample pad, a conjugation pad, and a water-absorbing pad; the sample pad, conjugation pad, nitrocellulose membrane, and water-absorbing pad are sequentially adhered to the PVC base and connected in an overlapping manner, characterized in that: The sample pad is loaded with glucose and an indicator, wherein the indicator is selected from any one of sodium diphenylamine sulfonate and methyl orange; the conjugate pad is loaded with antibodies or antigens labeled with colloidal gold nanoparticles, wherein the particle size of the colloidal gold nanoparticles is 20-40 nm; the mass fraction of the glucose is 1%-2%; and the mass fraction of the indicator is 0.001%-0.05%.

2. Use of the highly sensitive colloidal gold immunochromatographic kit according to claim 1 in detecting Helicobacter pylori.

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