Clostridium difficile triple detection card based on red nanospheres as well as preparation method and application of clostridium difficile triple detection card

Abstract

The invention relates to a clostridium difficile triple detection card based on red nanospheres, and a preparation method and application thereof, and belongs to the technical field of immunodetection. The clostridium difficile triple detection card is composed of three detection test paper strips, and the preparation method comprises the following steps: processing a sample pad and a combination pad, marking clostridium difficile toxin A, clostridium difficile toxin B, clostridium difficile glutamate dehydrogenase detection antibodies and chicken IgY antibodies by using red cellulose nanospheres, preparing a marking reconstitution fluid, spraying the marking reconstitution fluid on the combination pad, and detecting the clostridium difficile glutamate dehydrogenase. The detection card has the advantages that the detection limit of the clostridium difficile toxin A is as low as 0.1 ng / mL, the detection limit of the clostridium difficile toxin B is as low as 0.25 ng / mL, the detection limit of the clostridium difficile glutamate dehydrogenase is as low as 0.1 ng / mL, and the clostridium difficile glutamate dehydrogenase detection card has good specificity and sensitivity; the generation of a false positive phenomenon is greatly avoided, and the detection effect is further improved by virtue of the nanospheres based on an immunochromatography technology.

Description

Technical Field

[0001] The present invention relates to the technical field of Clostridium difficile detection, and in particular to a Clostridium difficile triple detection card based on red nanospheres, a preparation method and an application thereof. Background Art

[0002] In today's medical environment, Clostridium difficile infection has become one of the most common intestinal infectious diseases in hospitals, especially after patients use broad-spectrum antibiotics, the incidence rate increases significantly. This pathogen has the ability to produce enterotoxin A and cytotoxin B, which can cause serious damage to intestinal epithelial cells, leading to a series of symptoms such as diarrhea and pseudomembranous colitis. In severe cases, it can even pose a direct threat to the patient's life safety. Therefore, whether Clostridium difficile infection can be diagnosed quickly and accurately plays a vital role in subsequent timely treatment and disease control.

[0003] At present, the detection methods mainly include cell culture, cytotoxicity neutralization test, polymerase chain reaction (PCR) and immunoassay. However, cell culture and cytotoxicity neutralization tests have obvious limitations. They are not only time-consuming, but also have extremely high professional requirements for technicians, and it is difficult to meet the actual needs of rapid screening. Although polymerase chain reaction (PCR) has high accuracy, this method requires special equipment and professional technicians to operate, which undoubtedly leads to a significant increase in its cost. At the same time, the processing process is relatively long and the test results cannot be obtained in a short time. It can be seen that there is an urgent need to develop a new detection method that has the characteristics of high sensitivity, can quickly obtain test results, is easy to operate, and has a relatively low cost, so as to effectively meet the urgent needs in clinical practice.

[0004] Traditional test strips based on colloidal gold immunochromatography technology have limited sensitivity for low concentrations of target analytes, and it is difficult to detect trace amounts of biomarkers or pathogens, which may lead to missed diagnosis of the disease in the early stages of infection or failure to detect pathogens in time. In addition, the stability of colloidal gold markers is easily affected by environmental factors (such as temperature, humidity, light, etc.). Long-term storage may cause marker aggregation, denaturation or reduced activity, thereby reducing the detection performance of the test strips. The shelf life is relatively short, which limits its application in some environments that require long-term storage or unstable conditions. In addition, the existing colloidal gold test strips for Clostridium difficile do not detect the three related substances (Clostridium difficile toxin A, Clostridium difficile toxin B and Clostridium difficile glutamate dehydrogenase) separately, resulting in cross-reactions between antibodies and non-target substances, resulting in false positive results, thereby affecting the accuracy of diagnosis. For example, when detecting certain pathogens, other microorganisms or impurities in the sample may bind to the colloidal gold-labeled antibodies, resulting in a false positive result. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a triple detection card capable of simultaneously detecting Clostridium difficile toxin A, Clostridium difficile toxin B and Clostridium difficile glutamate dehydrogenase and a preparation method thereof. The preparation method comprises the following steps: using a sample pad treatment solution and a binding pad treatment solution containing specific components to soak a sample pad and a binding pad, respectively, and then drying and dehydrating the resulting mixture; taking a cellulose nanosphere suspension to react with Clostridium difficile toxins A and B, glutamate dehydrogenase detection antibodies and chicken IgY antibodies, respectively, and obtaining a labeled solution through centrifugation, redissolution and other operations, and mixing the resulting labeled solution into a labeled complex solution; spraying each labeled complex solution on the corresponding test strip binding pad, and at the same time diluting each corresponding capture antibody to a specific concentration with a coating diluent and then spraying the solution on the coating membrane detection line, spraying the diluted sheep anti-chicken IgY antibody on the quality control line, and finally arranging three test strips in parallel to obtain a detection card.

[0006] The first object of the present invention is to provide a method for preparing a Clostridium difficile triple test card based on red nanospheres, wherein the Clostridium difficile triple test card is composed of a Clostridium difficile toxin A test strip, a Clostridium difficile toxin B test strip, and a Clostridium difficile glutamate dehydrogenase test strip arranged in parallel and connected, and the preparation method specifically comprises the following steps:

[0007] soaking the sample pad in a sample pad treatment solution and then drying and dehydrating the sample pad, wherein the sample pad treatment solution includes Tris-HCl, casein or bovine serum albumin, a surfactant, a blocking agent and a preservative;

[0008] soaking the conjugate pad in a conjugate pad treatment solution and then drying and dehydrating the conjugate pad, wherein the conjugate pad treatment solution includes Tris-HCl, casein or bovine serum albumin, a surfactant, and a preservative;

[0009] Take the cellulose nanosphere suspension, add Clostridium difficile toxin A detection antibody, Clostridium difficile toxin B detection antibody and Clostridium difficile glutamate dehydrogenase detection antibody respectively, so that the final concentration of the three detection antibodies in each system is 5-15 μg / mL, centrifuge to obtain the precipitate, add the resolubilization buffer to the precipitate, and obtain Clostridium difficile toxin A detection antibody nanosphere labeling liquid, Clostridium difficile toxin B detection antibody nanosphere labeling liquid and Clostridium difficile glutamate dehydrogenase detection antibody nanosphere labeling liquid;

[0010] Take the cellulose nanosphere suspension, add chicken IgY antibody to make the final concentration of the chicken IgY antibody be 5-15 μg / mL, centrifuge to obtain the precipitate, add the reconstitution buffer to the precipitate, and obtain the chicken IgY antibody nanosphere labeling solution;

[0011] Mixing the chicken IgY antibody nanosphere labeling solution with the Clostridium difficile toxin A detection antibody nanosphere labeling solution to obtain a first labeling complex solution, mixing the chicken IgY antibody with the Clostridium difficile toxin B detection antibody nanosphere labeling solution to obtain a second labeling complex solution, and mixing the chicken IgY antibody with the Clostridium difficile glutamate dehydrogenase detection antibody nanosphere labeling solution to obtain a third labeling complex solution;

[0012] The Clostridium difficile toxin A test strip is provided with a sample pad, a conjugation pad, a coating membrane and a water-absorbing pad in sequence on the bottom plate, and a Clostridium difficile toxin A detection line and a quality control line are provided on the coating membrane. The first labeled complex solution is sprayed on the conjugation pad, and the Clostridium difficile toxin A capture antibody is diluted to a final concentration of 1-1.5 mg / mL using a coating diluent and then sprayed on the Clostridium difficile toxin A detection line;

[0013] The Clostridium difficile toxin B test strip is provided with a sample pad, a conjugate pad, a coating membrane and a water-absorbing pad in sequence on the bottom plate, and a Clostridium difficile toxin B detection line and a quality control line are provided on the coating membrane. The second labeled complex solution is sprayed on the conjugate pad, and the Clostridium difficile toxin B capture antibody is diluted to a final concentration of 1.4-2 mg / mL using a coating diluent and then sprayed on the Clostridium difficile toxin B detection line;

[0014] The Clostridium difficile toxin glutamate dehydrogenase test strip is provided with a sample pad, a conjugation pad, a coating membrane and a water-absorbing pad in sequence on the bottom plate, and a Clostridium difficile toxin glutamate dehydrogenase detection line and a quality control line are provided on the coating membrane. The third labeled complex solution is sprayed on the conjugation pad, and the Clostridium difficile toxin glutamate dehydrogenase capture antibody is diluted to a final concentration of 1.1-1.5 mg / mL using a coating diluent and then sprayed on the Clostridium difficile toxin glutamate dehydrogenase detection line;

[0015] The Clostridium difficile toxin A test strip, the Clostridium difficile toxin B test strip and the Clostridium difficile toxin glutamate dehydrogenase test strip are arranged in parallel to prepare the Clostridium difficile triple test card.

[0016] Furthermore, the amount of the sample pad treatment solution is 0.05-0.1 mL / cm 2 The amount of the conjugate pad treatment solution is 0.04-0.08 mL / cm 2 .

[0017] Furthermore, the sample pad treatment solution includes 40-80 mM Tris-HCl, 0.4-0.8 wt % casein or BSA, 0.8-1.5 wt % surfactant S9, 0.04-0.1 wt % Tween 20, 0.02-0.05 wt % sodium azide or ProClin 300 and 0.002-0.006 wt % heterophilic antibody blocking agent HBR.

[0018] Furthermore, the pH value of the sample pad treatment solution is 8-9.

[0019] Furthermore, the conjugate pad treatment solution includes 40-80 mM Tris-HCl, 0.4-0.8 wt % casein or BSA, 0.04-0.08 wt % Tween 20, and 0.02-0.06 wt % sodium azide or ProClin300.

[0020] Furthermore, the pH value of the binding pad treatment solution is 8-9.

[0021] Furthermore, the spraying amount of the first re-solution, the second re-solution and the third re-solution on the conjugate pad is 4.5-5.5 μL / cm.

[0022] Furthermore, the reconstitution buffer contains 30-60 mM boric acid, 0.1-0.5 wt % casein, and 10-30 wt % sucrose.

[0023] Furthermore, the pH value of the reconstitution buffer is 9-10.

[0024] Furthermore, the spraying amount of the diluted Clostridium difficile toxin A capture antibody, Clostridium difficile toxin B capture antibody and Clostridium difficile toxin glutamate dehydrogenase capture antibody on the detection line is 1-2 μL / cm.

[0025] Furthermore, the sheep anti-chicken IgY antibody was diluted to a final concentration of 0.8-1.2 mg / mL using a coating diluent and then sprayed onto the quality control lines of the Clostridium difficile toxin A test strip, the Clostridium difficile toxin B test strip and the Clostridium difficile toxin glutamate dehydrogenase test strip.

[0026] Furthermore, when cellulose nanospheres are used to label the detection antibody, a conjugate buffer and a blocking solution are also added to the system, and the blocking solution is composed of casein and boric acid.

[0027] Furthermore, when cellulose nanospheres are used to label Clostridium difficile toxin A detection antibodies, the conjugation buffer is a boric acid conjugation buffer; when cellulose nanospheres are used to label Clostridium difficile toxin B detection antibodies and Clostridium difficile glutamate dehydrogenase detection antibodies, the conjugation buffer is a MES conjugation buffer.

[0028] Furthermore, the reconstitution process also includes a step of washing away excess nanospheres using a washing buffer.

[0029] Furthermore, the washing buffer contains 30-60 mM boric acid.

[0030] Furthermore, the pH value of the washing buffer is 9.5-10.5.

[0031] The second object of the present invention is to provide a Clostridium difficile triple test card prepared by any of the above methods.

[0032] The third object of the present invention is to provide an auxiliary diagnosis product for Clostridium difficile infection comprising the above-mentioned Clostridium difficile triple test card.

[0033] Furthermore, it also includes a sample diluent, which includes 2-4wt% sodium chloride, 0.3-0.6wt% Tris, 0.5-1wt% bovine serum albumin, 0.03-0.06wt% Tween 20, and 0.02-0.05wt% sodium azide or ProClin300.

[0034] Furthermore, the pH value of the sample diluent is 7.5-8.

[0035] Beneficial effects of the present invention:

[0036] The Clostridium difficile triple test card provided by the present invention can detect three Clostridium difficile marker antigens at the same time, thereby improving the detection efficiency. Through reasonable antibody labeling, coating and test strip preparation processes, the detection of target pathogens can be achieved, which is helpful to accurately judge the infection of Clostridium difficile. The test card is easy to carry and store, and the operation process is relatively simple, does not require complex equipment and professional technicians, and reduces the detection threshold. The reagents and materials used in the preparation process are relatively conventional, low in cost, suitable for large-scale clinical promotion and application, and the results can be observed in a short time. The test results can be obtained quickly, meeting the needs of clinical rapid diagnosis, and being conducive to timely taking corresponding treatment measures for patients infected with Clostridium difficile. The detection limit of Clostridium difficile toxin A is as low as 0.1ng / mL, the detection limit of Clostridium difficile toxin B is as low as 0.25ng / mL, and the detection limit of Clostridium difficile glutamate dehydrogenase is as low as 0.1ng / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0038] Figure 1 This is a side view of the test strip provided by the present invention, wherein 1 is a sample pad, 2 is a conjugate pad, 3 is a nitrocellulose membrane, 31 is a test line, 32 is a quality control line, 4 is a water absorbent pad, and 5 is a bottom plate;

[0039] Figure 2This is a top view of the triple test card provided by the present invention after assembly, wherein A represents the Clostridium difficile toxin A test strip, B represents the Clostridium difficile toxin B test strip, GDH represents the Clostridium difficile glutamate dehydrogenase test strip, T represents the test line, C represents the quality control line, and 61, 62, and 63 correspond to the Clostridium difficile toxin A test strip, Clostridium difficile toxin B test strip, and Clostridium difficile glutamate dehydrogenase test strip sample holes on the triple card, respectively. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0041] The cellulose nanospheres used in the following Examples 1-2 and Comparative Examples 1-2 were red, had a concentration of 1 wt%, a particle size of 341 nm, and an OD value of 231;

[0042] The cellulose nanospheres used in the following comparative example 3 are blue in color, have a concentration of 1 wt %, a particle size of 338 nm, and an OD value of 265.

[0043] Example 1: Preparation of test strips

[0044] 1. Solution preparation

[0045] Sample pad treatment solution: 50 mM Tris-HCl, 0.5 wt% BSA, 0.8 wt% surfactant S9, 0.06 wt% Tween 20, 0.05 wt% ProClin 300, 0.004 wt% heterophilic antibody blocking agent HBR, solution pH 8.5;

[0046] Conjugate pad treatment solution: 50 mM Tris-HCl, 0.5 wt% BSA, 0.06 wt% Tween 20, 0.05 wt% ProClin 300, pH value of the solution is 8.5;

[0047] MES conjugation buffer: 10 mM MES buffer, pH adjusted to 5 with hydrochloric acid;

[0048] Boric acid conjugate buffer: 10 mM boric acid, pH adjusted to 9 with sodium hydroxide;

[0049] Blocking solution: 0.8 wt% casein, 100 mM boric acid, pH adjusted to 8.5 using sodium hydroxide;

[0050] Wash buffer: 40 mM boric acid, pH adjusted to 10 with sodium hydroxide;

[0051] Reconstitution buffer: 40 mM boric acid, 0.3 wt% casein, 15 wt% sucrose, pH adjusted to 9 with sodium hydroxide;

[0052] Coating diluent: 10 mM PBS, 1.5 wt% sucrose, 0.5 wt% BSA.

[0053] Sample diluent: 3wt% NaCl, 0.5wt% Tris, 1wt% bovine serum albumin, 0.05wt% Tween 20, 0.04wt% ProClin 300, pH value of the solution is 7.5.

[0054] 2. Preprocessing

[0055] 1. Add the sample pad treatment solution at 0.08mL / cm 2 Add the amount of into the soaking container, soak the sample pad for 5 minutes, then dehydrate, place the dehydrated sample pad in a 33-50℃ oven to dry for 7 hours, and store it in a sealed state under a humidity ≤30%.

[0056] 2. Add the conjugate pad treatment solution at 0.06 mL / cm 2 Add the amount of into the soaking container, soak the conjugate pad for 5 minutes, then dehydrate, place the dehydrated conjugate pad in a 33-50℃ oven to dry for 7 hours, and store it in a sealed state under a humidity ≤30%.

[0057] 3. Labeling and reconstitution of detection antibodies

[0058] 1. Clostridium difficile toxin A antibody detection

[0059] Take 10 μL of 1 wt% cellulose nanosphere suspension, add 80-100 μL of boric acid conjugate buffer, then add mouse anti-human Clostridium difficile toxin A detection antibody to a concentration of 10 μg / mL, mix well, and place in a 40°C water bath for 130 minutes;

[0060] Add 1.3 mL of blocking solution to the above solution, mix thoroughly, place in a 40°C water bath for 60 minutes, perform high-speed centrifugation at a centrifugal force of 15,000 g for 15 minutes, and then remove the supernatant;

[0061] 1.3 mL of washing buffer was added to the above precipitate from which the supernatant was removed, the precipitate was broken up and resuspended, ultrasonicated for 60 seconds using a bath sonicator, and subjected to high-speed centrifugation at a centrifugal force of 15,000 g for 15 minutes, after which the supernatant was removed;

[0062] 120 μL of reconstitution buffer was added to the precipitate from which the supernatant was removed, the precipitate was broken up and resuspended, and ultrasonicated for 60 seconds using a bath sonicator to obtain a mouse anti-human Clostridium difficile toxin A detection antibody cellulose nanosphere labeling liquid.

[0063] 2. Clostridium difficile toxin B antibody detection

[0064] Take 10 μL of 1 wt% cellulose nanosphere suspension, add 80-100 μL MES conjugation buffer, then add mouse anti-human Clostridium difficile toxin B detection antibody to a concentration of 10 μg / mL, mix well, and place in a 40°C water bath for 130 minutes;

[0065] Add 1.3 mL of blocking solution to the above solution, mix thoroughly, place in a 40°C water bath for 60 minutes, perform high-speed centrifugation at a centrifugal force of 15,000 g for 15 minutes, and then remove the supernatant;

[0066] 1.3 mL of washing buffer was added to the above precipitate from which the supernatant was removed, the precipitate was broken up and resuspended, ultrasonicated for 60 seconds using a bath sonicator, and subjected to high-speed centrifugation at a centrifugal force of 15,000 g for 15 minutes, after which the supernatant was removed;

[0067] 120 μL of reconstitution buffer was added to the precipitate from which the supernatant was removed, the precipitate was broken up and resuspended, and ultrasonicated for 60 seconds using a bath sonicator to obtain a mouse anti-human Clostridium difficile toxin B detection antibody cellulose nanosphere labeling solution.

[0068] 3. Detection of antibodies against Clostridium difficile glutamate dehydrogenase

[0069] Take 10 μL of 1 wt% cellulose nanosphere suspension, add 80-100 μL MES conjugation buffer, then add mouse anti-human Clostridium difficile glutamate dehydrogenase detection antibody to a concentration of 10 μg / mL, mix well, and place in a 40°C water bath for 130 minutes;

[0070] Add 1.3 mL of blocking solution to the above solution, mix thoroughly, place in a 40°C water bath for 60 minutes, perform high-speed centrifugation at a centrifugal force of 15,000 g for 15 minutes, and then remove the supernatant;

[0071] 1.3 mL of washing buffer was added to the above precipitate from which the supernatant was removed, the precipitate was broken up and resuspended, and then ultrasonicated for 60 seconds using a bath sonicator, and then high-speed centrifuged at a centrifugal force of 15000 g for 15 minutes, after which the supernatant was removed;

[0072] 120 μL of reconstitution buffer was added to the precipitate from which the supernatant was removed, the precipitate was broken up and resuspended, and then ultrasonicated for 60 seconds using a bath sonicator to obtain a mouse anti-human Clostridium difficile glutamate dehydrogenase detection antibody cellulose nanosphere labeling solution.

[0073] 4. Chicken IgY antibody

[0074] Take 10 μL of 1 wt% cellulose nanosphere suspension, add 80-100 μL of MES conjugation buffer, then add chicken IgY antibody to a concentration of 10 μg / mL, mix well, and place in a 40°C water bath for 130 minutes;

[0075] Add 1.3 mL of blocking solution to the above solution, mix thoroughly, place in a 40°C water bath for 60 minutes, perform high-speed centrifugation at a centrifugal force of 15,000 g for 15 minutes, and then remove the supernatant;

[0076] 1.3 mL of washing buffer was added to the above precipitate from which the supernatant was removed, the precipitate was broken up and resuspended, ultrasonicated for 60 seconds using a bath sonicator, and subjected to high-speed centrifugation at a centrifugal force of 15,000 g for 15 minutes, after which the supernatant was removed;

[0077] 80 μL of reconstitution buffer was added to the precipitate from which the supernatant was removed, the precipitate was broken up and resuspended, and ultrasonicated for 60 seconds using a bath sonicator to obtain a chicken IgY antibody cellulose nanosphere labeling solution.

[0078] 4. Capture Antibody Coating

[0079] 1. Clostridium difficile toxin A capture antibody

[0080] Dilute the mouse anti-human Clostridium difficile toxin A capture antibody to 1 mg / mL with a coating diluent to obtain a mouse anti-human Clostridium difficile toxin A capture antibody coating solution;

[0081] 2. Clostridium difficile toxin B capture antibody

[0082] Dilute the mouse anti-human Clostridium difficile toxin B capture antibody to 1.5 mg / mL with a coating diluent to obtain a mouse anti-human Clostridium difficile toxin B capture antibody coating solution;

[0083] 3. Clostridium difficile glutamate dehydrogenase capture antibody

[0084] Dilute the mouse anti-human Clostridium difficile glutamate dehydrogenase capture antibody to 1.2 mg / mL using a coating diluent to obtain a mouse anti-human Clostridium difficile glutamate dehydrogenase capture antibody coating solution;

[0085] 4. Sheep anti-chicken IgY antibody

[0086] The goat anti-chicken IgY antibody was diluted to 1 mg / mL with a coating diluent to obtain a goat anti-chicken IgY antibody coating solution.

[0087] 5. Assembly of test strips

[0088] 1. Take 20 μL of the chicken IgY antibody cellulose nanosphere labeling solution and add it to the mouse anti-human Clostridium difficile toxin A detection antibody cellulose nanosphere labeling solution, mouse anti-human Clostridium difficile toxin B detection antibody cellulose nanosphere labeling solution and mouse anti-human Clostridium difficile glutamate dehydrogenase detection antibody cellulose nanosphere labeling solution, then break up and resuspend the precipitate. The three solutions after mixing are recorded as labeling resolution A, labeling resolution B and labeling resolution GDH;

[0089] 2. Spray the labeled solution A, labeled solution B and labeled solution GDH on the three corresponding conjugate pads after treatment at a spray volume of 5 μL / cm, then bake the sprayed conjugate pads at 40°C for 6 hours, and then seal and store the dried conjugate pads in an environment with a humidity of ≤30%;

[0090] 3. Match the mouse anti-human Clostridium difficile toxin A capture antibody coating solution, mouse anti-human Clostridium difficile toxin B capture antibody coating solution, and mouse anti-human Clostridium difficile glutamate dehydrogenase capture antibody coating solution with the goat anti-chicken IgY antibody coating solution, and spray them on three corresponding nitrocellulose membranes at a spray volume of 1 μL / cm, forming a test line and a quality control line on each nitrocellulose membrane, and then bake the treated nitrocellulose membrane at 40°C for 6 hours, and then seal and store the dried nitrocellulose membrane in an environment with a humidity of ≤30%;

[0091] 4. Assemble the test strip. The positional relationship between the sample pad, conjugate pad, nitrocellulose membrane, water absorbent pad and bottom plate is as follows: the sample pad, conjugate pad, water absorbent pad and nitrocellulose membrane are all bonded to the bottom plate, wherein the nitrocellulose membrane is located near the middle of the bottom plate, one end of the nitrocellulose membrane is bonded to the water absorbent pad with a 2mm overlap, the other end is bonded to the conjugate pad with a 2mm overlap, and the other end of the conjugate pad is bonded to the sample pad with a 2mm overlap. The bottom plate is located at the bottom to support the entire test strip. The structure of the test strip is as follows: Figure 1 As shown;

[0092] 5. Arrange the three test strips in parallel to form a triple test card. The structure of the triple test card is as follows: Figure 2 shown.

[0093] Example 2: Detection method of triple detection card

[0094] (1) Dilute the Clostridium difficile toxin A antigen standard with the sample diluent provided in Example 1 to a standard solution with the following concentrations: 1 μg / mL, 0.5 ng / mL, 0.25 ng / mL, 0.1 ng / mL, 0.05 ng / mL; dilute the Clostridium difficile toxin B antigen standard with the sample diluent shown in Example 1 to a standard solution with the following concentrations: 2.33 μg / mL, 1 ng / mL, 0.5 ng / mL, 0.25 ng / mL, 0.1 ng / mL; dilute the Clostridium difficile glutamate dehydrogenase antigen standard with the sample diluent shown in Example 1 to a standard solution with the following concentrations: 1.78 μg / mL, 0.5 ng / mL, 0.25 ng / mL, 0.1 ng / mL, 0.05 ng / mL;

[0095] (2) Take 80 μL of the 12 concentrations of antigen standard solutions prepared in step (1) and place them on the 12 test strips of the four triple test cards prepared in Example 1. Due to capillary action, the sample will move along the test strip to the conjugate pad and the nitrocellulose membrane. Observe and display the results after 10 minutes (Note: color development is invalid after 15 minutes);

[0096] (3) Result observation: When the concentration of Clostridium difficile toxin A antigen is greater than or equal to 0.1 ng / mL, the test result is positive, and when it is less than 0.1 ng / mL, the test result is negative; when the concentration of Clostridium difficile toxin B antigen is greater than or equal to 0.25 ng / mL, the test result is positive, and when it is less than 0.25 ng / mL, the test result is negative; when the concentration of Clostridium difficile glutamate dehydrogenase antigen is greater than or equal to 0.1 ng / mL, the test result is positive, and when it is less than 0.1 ng / mL, the test result is negative;

[0097] (4) Repeat step (2), except that the result is observed and displayed after 5 minutes (the color development is invalid after 10 minutes). The result shows that when the concentration of Clostridium difficile toxin A antigen is greater than or equal to 0.2 ng / mL, the test result is positive, and when it is less than 0.2 ng / mL, the test result is negative; when the concentration of Clostridium difficile toxin B antigen is greater than or equal to 0.5 ng / mL, the test result is positive, and when it is less than 0.5 ng / mL, the test result is negative; when the concentration of Clostridium difficile glutamate dehydrogenase antigen is greater than or equal to 0.2 ng / mL, the test result is positive, and when it is less than 0.2 ng / mL, the test result is negative. This result reveals that the triple test card provided by the present invention can still maintain a low detection limit while shortening the color development time, thereby greatly improving the detection efficiency.

[0098] Comparative Example 1

[0099] (1) The test strips were prepared according to the method described in Example 1, except that: for antibody labeling and re-dissolution, 180 μL of re-dissolution buffer was added respectively to obtain mouse anti-human Clostridium difficile toxin A detection antibody cellulose nanosphere labeling solution, mouse anti-human Clostridium difficile toxin B detection antibody cellulose nanosphere labeling solution, and mouse anti-human Clostridium difficile glutamate dehydrogenase detection antibody cellulose nanosphere labeling solution;

[0100] According to the method described in Example 2 (10 minutes for color development), the Clostridium difficile toxin A antigen standard solution, the Clostridium difficile toxin B antigen standard solution, and the Clostridium difficile glutamate dehydrogenase antigen standard solution were detected respectively, and the minimum detection limits were 0.25 ng / mL, 0.5 ng / mL, and 0.25 ng / mL, respectively;

[0101] (2) A test strip was prepared according to the method described in Example 1, except that: the antibody coating solution was prepared, the mouse anti-human Clostridium difficile toxin A capture antibody was diluted to 0.5 mg / mL, the mouse anti-human Clostridium difficile toxin B capture antibody was diluted to 0.8 mg / mL, and the mouse anti-human Clostridium difficile glutamate dehydrogenase capture antibody was diluted to 0.8 mg / mL;

[0102] According to the method described in Example 2, the Clostridium difficile toxin A antigen standard solution, the Clostridium difficile toxin B antigen standard solution, and the Clostridium difficile glutamate dehydrogenase antigen standard solution were respectively detected, and the minimum detection limits were 0.5 ng / mL, 1 ng / mL, and 0.5 ng / mL, respectively;

[0103] It can be seen that reducing the amount of detection antibody on the conjugate pad or the amount of capture antibody on the nitrocellulose membrane will affect the detection performance of the reagent strip.

[0104] Comparative Example 2

[0105] A test strip was prepared according to the method described in Example 1, except that: the labeled complex solution A, the labeled complex solution B and the labeled complex solution GDH were evenly mixed and sprayed on the same conjugate pad at a spray volume of 15 μL / cm, and the mouse anti-human Clostridium difficile toxin A capture antibody coating solution, the mouse anti-human Clostridium difficile toxin B capture antibody coating solution, the mouse anti-human Clostridium difficile glutamate dehydrogenase capture antibody coating solution and the goat anti-chicken IgY antibody coating solution were sprayed on the same nitrocellulose membrane, respectively, to form a Clostridium difficile toxin A detection line, a Clostridium difficile toxin B detection line, a Clostridium difficile glutamate dehydrogenase detection line and a quality control line.

[0106] The sample dilution of Example 1 was tested according to the method described in Example 2, and it was found that the Clostridium difficile toxin A test line, the Clostridium difficile toxin B test line, and the Clostridium difficile glutamate dehydrogenase test line were all positive.

[0107] It can be seen from this that preparing multiple test lines for Clostridium difficile on the same test strip is prone to false positives, affecting the accuracy of the reagents, thereby significantly reducing the sensitivity and accuracy of the test.

[0108] Comparative Example 3

[0109] The test strips were prepared according to the method described in Example 1, except that the cellulose nanospheres used were blue, had a concentration of 1 wt%, a particle size of 338 nm, and an OD value of 265;

[0110] According to the method described in Example 2, the Clostridium difficile toxin A antigen standard solution, the Clostridium difficile toxin B antigen standard solution, and the Clostridium difficile glutamate dehydrogenase antigen standard solution were respectively detected, and the minimum detection limits were 0.25 ng / mL, 1 ng / mL, and 0.5 ng / mL, respectively;

[0111] It can be seen that using different types of cellulose nanospheres as detection antibody markers will affect the detection performance of the reagent strip.

[0112] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for preparing a Clostridium difficile triple test card based on red nanospheres, wherein the Clostridium difficile triple test card is composed of a Clostridium difficile toxin A test strip, a Clostridium difficile toxin B test strip, and a Clostridium difficile glutamate dehydrogenase test strip arranged in parallel and connected, characterized in that: The preparation method specifically comprises the following steps: soaking the sample pad in a sample pad treatment solution and then drying and dehydrating the sample pad, wherein the sample pad treatment solution includes Tris-HCl, casein or bovine serum albumin, a surfactant, a blocking agent and a preservative; soaking the conjugate pad in a conjugate pad treatment solution and then drying and dehydrating the conjugate pad, wherein the conjugate pad treatment solution includes Tris-HCl, casein or bovine serum albumin, a surfactant, and a preservative; Take the cellulose nanosphere suspension, add Clostridium difficile toxin A detection antibody, Clostridium difficile toxin B detection antibody and Clostridium difficile glutamate dehydrogenase detection antibody respectively, so that the final concentration of the three detection antibodies in each system is 5-15 μg / mL, centrifuge to obtain the precipitate, add the resolubilization buffer to the precipitate, and obtain Clostridium difficile toxin A detection antibody nanosphere labeling liquid, Clostridium difficile toxin B detection antibody nanosphere labeling liquid and Clostridium difficile glutamate dehydrogenase detection antibody nanosphere labeling liquid; Take the cellulose nanosphere suspension, add chicken IgY antibody to make the final concentration of the chicken IgY antibody be 5-15 μg / mL, centrifuge to obtain the precipitate, add the reconstitution buffer to the precipitate, and obtain the chicken IgY antibody nanosphere labeling solution; Mixing the chicken IgY antibody nanosphere labeling solution with the Clostridium difficile toxin A detection antibody nanosphere labeling solution to obtain a first labeling complex solution, mixing the chicken IgY antibody with the Clostridium difficile toxin B detection antibody nanosphere labeling solution to obtain a second labeling complex solution, and mixing the chicken IgY antibody with the Clostridium difficile glutamate dehydrogenase detection antibody nanosphere labeling solution to obtain a third labeling complex solution; The Clostridium difficile toxin A test strip is provided with a sample pad, a conjugation pad, a coating membrane and a water-absorbing pad in sequence on the bottom plate, and a Clostridium difficile toxin A detection line and a quality control line are provided on the coating membrane. The first labeled complex solution is sprayed on the conjugation pad, and the Clostridium difficile toxin A capture antibody is diluted to a final concentration of 1-1.5 mg / mL using a coating diluent and then sprayed on the Clostridium difficile toxin A detection line; The Clostridium difficile toxin B test strip is provided with a sample pad, a conjugate pad, a coating membrane and a water-absorbing pad in sequence on the bottom plate, and a Clostridium difficile toxin B detection line and a quality control line are provided on the coating membrane. The second labeled complex solution is sprayed on the conjugate pad, and the Clostridium difficile toxin B capture antibody is diluted to a final concentration of 1.4-2 mg / mL using a coating diluent and then sprayed on the Clostridium difficile toxin B detection line; The Clostridium difficile toxin glutamate dehydrogenase test strip is provided with a sample pad, a conjugation pad, a coating membrane and a water-absorbing pad in sequence on the bottom plate, and a Clostridium difficile toxin glutamate dehydrogenase detection line and a quality control line are provided on the coating membrane. The third labeled complex solution is sprayed on the conjugation pad, and the Clostridium difficile toxin glutamate dehydrogenase capture antibody is diluted to a final concentration of 1.1-1.5 mg / mL using a coating diluent and then sprayed on the Clostridium difficile toxin glutamate dehydrogenase detection line; The Clostridium difficile toxin A test strip, the Clostridium difficile toxin B test strip and the Clostridium difficile toxin glutamate dehydrogenase test strip are arranged in parallel to prepare the Clostridium difficile triple test card.

2. The preparation method according to claim 1, characterized in that: The amount of the sample pad treatment solution is 0.05-0.1 mL / cm 2 The amount of the conjugate pad treatment solution is 0.04-0.08 mL / cm 2 .

3. The preparation method according to claim 1, characterized in that: The spraying amount of the first re-solution, the second re-solution and the third re-solution on the conjugate pad is 4.5-5.5 μL / cm.

4. The preparation method according to claim 1, characterized in that: The spraying amount of the diluted Clostridium difficile toxin A capture antibody, Clostridium difficile toxin B capture antibody and Clostridium difficile toxin glutamate dehydrogenase capture antibody on the test line is 1-2 μL / cm.

5. The preparation method according to claim 1, characterized in that: Use coating diluent to dilute the sheep anti-chicken IgY antibody to a final concentration of 0.8-1.2 mg / mL and then spray it onto the quality control line of the Clostridium difficile toxin A test strip, the Clostridium difficile toxin B test strip and the Clostridium difficile toxin glutamate dehydrogenase test strip.

6. The preparation method according to claim 1, characterized in that: When the cellulose nanospheres are used to mark the detection antibody, a conjugate buffer and a blocking solution are also added to the system, and the blocking solution is composed of casein and boric acid.

7. The preparation method according to claim 6, characterized in that: When cellulose nanospheres are used to label Clostridium difficile toxin A detection antibodies, the conjugation buffer is a boric acid conjugation buffer; when cellulose nanospheres are used to label Clostridium difficile toxin B detection antibodies and Clostridium difficile glutamate dehydrogenase detection antibodies, the conjugation buffer is a MES conjugation buffer.

8. The Clostridium difficile triple test card prepared by any method of claims 1-7.

9. An auxiliary diagnostic product for Clostridium difficile infection comprising the Clostridium difficile triple test card as described in claim 8.

10. The auxiliary diagnosis product for Clostridium difficile infection according to claim 9, characterized in that: The invention also comprises a sample diluent, which comprises 2-4wt% sodium chloride, 0.3-0.6wt% Tris, 0.5-1wt% bovine serum albumin, 0.03-0.06wt% Tween 20, and 0.02-0.05wt% sodium azide or ProClin300.

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