A combined detection kit for pepsinogen i and pepsinogen ii and application thereof

Abstract

This application relates to the technical field of bioassay, specifically disclosing a combined detection kit for pepsinogen I and pepsinogen II and its application. The disclosed combined detection kit for pepsinogen I and pepsinogen II includes: a sample pad, a conjugate pad, a coating membrane, and an absorbent pad; the coating membrane is an NC membrane coated with antibody; the conjugate pad is prepared by pretreating and drying a fiber membrane, followed by spraying time-resolved fluorescent microspheres labeled with antibody; the pretreating solution is a solution of pH 8.0-8.4 + 8-12mM BS + 0.8-1.2% BSA + 0.8-1.2% PEG4000 + 0.02-0.03% surfactant + 4-6% sucrose. Using the detection kit and method provided in this application for the combined detection of pepsinogen I / pepsinogen II exhibits high sensitivity, accuracy, and repeatability.

Description

Technical Field

[0001] This application relates to the technical field of biological detection, specifically to a combined detection kit for pepsinogen I and pepsinogen II and its application. Background Technology

[0002] Pepsinogen (PG) is an inactive precursor of pepsin in gastric juice. Based on its biochemical properties and immunogenicity, it is divided into two subgroups: PGI and PGII. PGI is mainly secreted by the chief cells and neck mucous cells of the fundic glands and is closely related to gastric acid content. PGII, on the other hand, originates from all gastric glands (cardiac glands, fundic glands, and pyloric glands of the antrum) and duodenal glands.

[0003] PGI and PGII reflect the secretory function of different parts of the gastric mucosa. Most synthesized PG enters the gastric lumen and is activated into pepsin under the action of acidic gastric juice. Only a small amount of PG permeates the gastric mucosal capillaries into the bloodstream. Serum PG concentration reflects the morphology and function of different parts of the gastric mucosa: PGI is an indicator of gastric acid-secreting gland function; increased gastric acid secretion leads to elevated PGI, while decreased gastric acid secretion or gastric mucosal gland atrophy leads to decreased PGI. PGII is more correlated with lesions of the gastric fundus mucosa (relative to the antral mucosa); its elevation is associated with atrophy of the fundic glands, gastric metaplasia or pseudopyloric gland metaplasia, and dysplasia. A progressively decreasing PGI / PGII ratio is associated with the progression of gastric mucosal atrophy.

[0004] Currently, clinical and laboratory methods for detecting PGI and PGII include enzyme-linked immunosorbent assay (ELISA), fluorescence immunochromatography, latex immunoturbidimetry, and chemiluminescence immunoassay. However, existing detection methods suffer from low sensitivity, poor accuracy, and poor repeatability. Summary of the Invention

[0005] To address the aforementioned technical problems, this application provides a combined detection kit for pepsinogen I and pepsinogen II and its application.

[0006] In a first aspect, this application provides a combined detection kit for pepsinogen I and pepsinogen II, characterized in that it comprises the following components: a sample pad, a conjugate pad, a coating membrane, and an absorbent pad;

[0007] The coating membrane is an NC membrane coated with antibody; in the preparation method of the NC membrane coated antibody, the coating solution is pH 7.2-7.6 + 0.8-1.2% mannitol + 0.05-0.09% cocoyl glutamate + 0.3-0.7% sodium caseinate + 8-12mM PBS solution;

[0008] The method for preparing the binding pad is as follows: the fiber membrane is treated with a pretreatment solution and dried, and then time-resolved fluorescent microspheres labeled with antibodies are sprayed on it.

[0009] The pretreatment solution is a solution of pH 8.0-8.4 + 8-12mM BS + 0.8-1.2% BSA + 0.8-1.2% PEG4000 + 0.02-0.03% surfactant + 4-6% sucrose; the surfactant is a mixture of Tween-20, CHAPS and poloxamer 188 in a weight ratio of 10:3-7:0.1-0.5.

[0010] The principle of the pepsinogen I and pepsinogen II combined detection kit provided in this application is based on the quantitative detection of pepsinogen I (PGI) and pepsinogen II (PGII) content in samples (including whole blood, serum, and plasma) using a fluorescent immunoassay double antibody sandwich method. When the sample to be tested is added to the sample well of the test card, if the sample contains a certain amount of PGI or PGII, PGI or PGII reacts with time-resolved fluorescent microsphere-labeled antibody I in the conjugate pad to form an antigen-antibody complex. This complex moves forward along the nitrocellulose membrane under chromatography and is captured by antibody II, which is pre-coated in the detection area of ​​the nitrocellulose membrane. The more analyte in the sample, the more complex accumulates in the detection area, and the signal intensity of the fluorescent antibody reflects the content of the captured analyte.

[0011] Furthermore, in the coating solution provided in this application, mannitol can protect antibody activity and improve detection sensitivity; it can form a glassy structure during the drying process, encapsulating PGI / PGII monoclonal antibody molecules; it can reduce the damage to the protein spatial structure caused by water crystallization, avoid denaturation of antibody active sites, and ensure that the antigen can bind efficiently with the antibody during detection, directly improving detection sensitivity. Cocoyl glutamic acid can optimize immobilization efficiency, ensure detection accuracy, regulate the surface tension of the coating carrier, and slightly change the surface charge of PGI / PGII antibodies; on the one hand, it helps the antibody to adsorb more uniformly and stably on the carrier surface, and on the other hand, it avoids the masking of active sites due to excessive antibody adsorption, ensuring that the binding of antigen and antibody is specific, reducing non-specific reactions, and thus ensuring detection accuracy. Sodium caseinate can block non-specific sites, reduce background interference, and preferentially bind to blank sites on the carrier surface not occupied by PGI / PGII antibodies, preventing antigens or other miscellaneous proteins in the detection system from non-specifically adsorbing onto the carrier, avoiding the problems of "false positives" or "excessively high background signals," ensuring that the detection signal comes only from "antigen-antibody specific binding," improving accuracy and repeatability. By using the above components at specific concentrations to form a coating solution, high sensitivity, high accuracy, and high repeatability of PGI / PGII detection results can be achieved.

[0012] The purpose of the pretreatment solution designed in this application is to pretreat the conjugate pad. The conjugate pad is the portion of the test strip on which the "detection antibody-label complex" (i.e., the conjugate of monoclonal antibodies of PGI and PGII with fluorescent microspheres) is immobilized. The role of the pretreatment solution is to ensure that these "detection antibody-label complexes" are stably and dryly preserved on the conjugate pad, and that they are rapidly, uniformly, and completely redissolved as the sample flows through them, flowing forward with the liquid, while minimizing non-specific binding and ensuring the sensitivity and accuracy of the detection.

[0013] In the pretreatment solution, BS (borax-boric acid buffer) maintains a relatively stable pH, creating an optimal microenvironment for antibody labeling. BSA (bovine serum albumin) fills the space around antibody molecules during the drying process, preventing denaturation and inactivation, thus acting as a protective agent. PEG4000 increases the viscosity of the liquid, helping to regulate the flow rate of the sample on the nitrocellulose membrane, making it more stable and uniform; it also forms a protective film during the drying process. The surfactant, composed of Tween-20, CHAPS, and poloxamer 188, can disrupt protein aggregates or lipids in the sample, preventing them from occupying reaction sites on the binding pad and reducing false negatives or signal attenuation. It also reduces non-specific adsorption between the sample and the binding pad surface, minimizing background noise and allowing accurate capture of even low concentrations of PGI / PGII, thus improving sensitivity. Furthermore, it prevents antibody denaturation and aggregation during drying or storage, maintaining the activity of its binding sites and ensuring binding only to PGI / PGII, reducing false positives. Its mild dissolving properties do not damage the antibody structure and can remove any residual denatured proteins on the binding pad, further reducing interference and ensuring accurate test results. This surfactant can also regulate the hydrophilicity and permeability of the binding pad, ensuring uniform diffusion of the sample on the pad and avoiding signal differences caused by uneven diffusion, thus guaranteeing consistency in test results across different batches and samples. Simultaneously, it creates a mild buffer environment, reducing the impact of external factors such as temperature and humidity on the antibody-antigen binding reaction, allowing each test to be performed under similar conditions, further improving repeatability. The three components work together to solve the functional limitations of a single surfactant and avoid antagonism between components, ultimately achieving detection results with high sensitivity, high accuracy and high repeatability.

[0014] Preferably, the NC membrane is coated with antibodies by sequentially setting a detection line T1 coated with PGI monoclonal antibody II at a concentration of 0.4-0.6 mg / mL, a detection line T2 coated with PGII monoclonal antibody II at a concentration of 0.8-1.2 mg / mL, and a control line C coated with goat anti-chicken IgY antibody at a concentration of 0.8-1.2 mg / mL on the NC membrane.

[0015] Preferably, the coating solution is a pH 7.3-7.5 solution containing 0.9-1.1% mannitol, 0.06-0.08% cocoyl glutamic acid, 0.4-0.6% sodium caseinate, and 9-11 mM PBS.

[0016] Preferably, the coating solution is a pH 7.3-7.5 solution containing 0.9-1.1% mannitol, 0.065-0.075% cocoyl glutamic acid, 0.45-0.55% sodium caseinate, and 9-11 mM PBS.

[0017] Preferably, the conjugate pad pretreatment solution is a solution with pH 8.1-8.3 + 9-11 mM BS + 0.9-1.1% BSA + 0.9-1.1% PEG4000 + 0.02-0.03% surfactant + 4.5-5.5% sucrose.

[0018] Preferably, the surfactant is composed of a mixture of Tween-20, CHAPS, and poloxamer 188 in a weight ratio of 10:4-6:0.2-0.4.

[0019] In one specific embodiment, the weight ratio of Tween-20, CHAPS, and poloxamer 188 in the surfactant can be 10:3:0.1, 10:4:0.1, 10:5:0.1, 10:6:0.1, 10:7:0.1, 10:3:0.2, 10:4:0.2, 10:5:0.2, 10:6:0.2, or 10:7:0.2. 10:3:0.3, 10:4:0.3, 10:5:0.3, 10:6:0.3, 10:7:0.3, 10:3:0.4, 10:4:0.4, 10:5:0.4, 10:6:0.4, 10:7:0.4, 10:3:0.5, 10:4:0.5, 10:5:0.5, 10:6:0.5, 10:7:0.5.

[0020] Experimental analysis shows that the surfactant composed of Tween-20, CHAPS, and poloxamer 188 in the above weight ratio, as the raw material for the binding pad pretreatment solution, can further improve the application performance of the detection kit and detection method.

[0021] Preferably, the sample diluent is a solution with pH 7.3-7.5, 47-52 mM Tris, 1.9-2.1% BSA, 0.9-1.1% NaCl, and 0.045-0.055% Proclin 300.

[0022] Preferably, the method for preparing the time-resolved fluorescent microsphere-labeled antibody is as follows:

[0023] (1) Preparation of time-resolved fluorescent microsphere-labeled PGI monoclonal antibody I:

[0024] Activation: Mix 40-60 μL of fluorescent microspheres, 80-120 μL of pH 5.8-6.2 + 20-30 mM MES solution, 5-8 μL of 0.8-1.2 mg / mL EDC, and 5-8 μL of 0.8-1.2 mg / mL NHS, shake to activate, centrifuge, and discard the supernatant;

[0025] Labeling: Add 40-60 μL of 20-30 mM BS at pH 8.0-8.4, add 20-30 μg of PGI monoclonal antibody I, conjugate for 20-40 min, centrifuge, and discard the supernatant;

[0026] Blocking: Add 40-60 μL of blocking solution and shake to block for 2-4 hours; the blocking solution is a solution with pH 8.0-8.4 + 8-12 mM BS + 1.8-2.2% BSA. Shake, centrifuge, and discard the supernatant.

[0027] Preservation: Add 40-60 μL of preservation solution; the preservation solution is a solution with pH 7.2-7.6 + 20-30 mM BS + 0.8-1.2% BSA + 0.04-0.06% proclin-300 + 7-9% sucrose;

[0028] (2) Preparation of time-resolved fluorescent microsphere-labeled PGII monoclonal antibody I:

[0029] Activation: Mix 40-60 μL of fluorescent microspheres, 80-120 μL of pH 5.8-6.2 + 20-30 mM MES solution, 5-8 μL of 0.8-1.2 mg / mL EDC, and 5-8 μL of 0.8-1.2 mg / mL NHS, shake to activate, centrifuge, and discard the supernatant;

[0030] Labeling: Add 40-60 μL of 20-30 mM BS at pH 7.0-7.4, add 20-30 μg of PGII monoclonal antibody I, conjugate for 20-40 min, centrifuge, and discard the supernatant;

[0031] Blocking: Add 40-60 μL of blocking solution and shake to block for 2-4 hours; the blocking solution is a solution with pH 7.2-7.6 + 8-12 mM BS + 1.8-2.2% BSA. Shake, centrifuge, and discard the supernatant.

[0032] Preservation: Add 40-60 μL of preservation solution; the preservation solution is a solution with pH 7.2-7.6 + 20-30 mM BS + 0.8-1.2% BSA + 0.04-0.06% proclin-300 + 7-9% sucrose;

[0033] (3) Preparation of time-resolved fluorescent microsphere-labeled chicken IgY antibody:

[0034] Activation: Mix 40-60 μL of fluorescent microspheres, 80-120 μL of pH 5.8-6.2 + 20-30 mM MES solution, 5-8 μL of 0.8-1.2 mg / mL EDC, and 5-8 μL of 0.8-1.2 mg / mL NHS, shake to activate, centrifuge, and discard the supernatant;

[0035] Labeling: Add 40-60 μL of 20-30 mM MES at pH 5.8-6.2, add 20-30 μg of chicken IgY antibody, conjugate for 40-80 min, centrifuge, and discard the supernatant;

[0036] Blocking: Add 8-12 μL of blocking solution and shake to block for 2-4 hours; the blocking solution is a solution with pH 7.2-7.6 + 20-30 mM BS + 8-12% BSA. Shake, centrifuge, and discard the supernatant.

[0037] Preservation: Add 40-60 μL of preservation solution; the preservation solution is a solution with pH 7.2-7.6, 20-30 mM BS, 0.8-1.2% BSA, 0.04-0.06% proclin-300, and 7-9% sucrose.

[0038] Secondly, this application also provides a method for the combined detection of pepsinogen I and pepsinogen II, using the combined detection kit for pepsinogen I and pepsinogen II; specifically, it includes the following steps in sequence:

[0039] Add the sample to be tested to the sample diluent at a dilution ratio of 1:1-2, then add it to the sample well of the test card, wait for the sample to spread for 8-12 minutes, and then detect the fluorescence value.

[0040] Preferably, the fluorescence value is detected using a fluorescence immunochromatographic analyzer, wherein the fluorescence excitation wavelength is 365 nm and the emission wavelength is 610 nm.

[0041] Thirdly, this application also provides a diagnostic kit for the progression of gastric mucosal atrophy, including the combined detection kit for pepsinogen I and pepsinogen II.

[0042] In summary, the technical solution of this application has the following effects:

[0043] The kit provided in this application, based on a combination of immunofluorescence and immunochromatography techniques, offers a method for the combined quantitative detection of pepsinogen I and pepsinogen II. This technology utilizes fluorescent microspheres to bind with antibodies to form fluorescently labeled antibodies, which are then detected through an antigen-antibody reaction. This approach retains the advantages of colloidal gold immunochromatography—simple operation, rapid detection, and high portability—while also achieving accurate quantification of the results through fluorescence tracing enhancement technology.

[0044] The kit provided in this application designs the composition and concentration of raw materials in the coating solution of the NC membrane-coated antibody preparation method to maximize the retention of the activity and specificity of PGI / PGII antibodies, while optimizing the detection reaction environment and improving the sensitivity, accuracy and repeatability of the detection method.

[0045] The kit provided in this application prepares a binding pad by pretreating and drying a fiber membrane with a pretreatment solution, followed by spraying time-resolved fluorescent microspheres labeled with antibodies. The precisely designed pretreatment solution formulation helps the subsequently dried labeled complex to be rapidly, uniformly, and completely reconstituted as the sample flows through; at the same time, it minimizes non-specific binding and ensures the sensitivity and accuracy of the detection. Detailed Implementation

[0046] The present application will be further described in detail below with reference to embodiments, comparative examples and performance test results. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0047] Information on the raw materials used in this application is shown in Table 1.

[0048] Table 1 Raw Material Information

[0049]

[0050] Example

[0051] Example 1

[0052] Example 1 provides a combined detection kit and detection method for pepsinogen I and pepsinogen II.

[0053] In this embodiment, the kit consists of a test card, a sample diluent, and an IC card; the test card consists of a test strip and a plastic card shell; the test strip includes the following components: a sample pad, a conjugate pad, a coating membrane, and an absorbent pad.

[0054] In this embodiment, the test strip includes a PVC base plate, which supports a chromatographic structure consisting of a sample pad, a coating membrane, a conjugate pad, and an absorbent pad. The chromatographic structure, extending from the sample application end towards the sample chromatography direction, comprises the sample pad, conjugate pad, coating membrane, and absorbent pad; these components overlap sequentially; the conjugate pad presses against the lower end of the coating membrane, the sample pad presses against the conjugate pad, and the absorbent pad presses against the upper end of the coating membrane, with an overlap of 1-2 mm.

[0055] The preparation method of the test strip in this embodiment is as follows:

[0056] (1) Preparation of coating membrane

[0057] Coating solution: pH 7.4 + 1% mannitol + 0.07% cocoyl glutamic acid (purchased from Yuan Ye, catalog number S25479) + 0.5% sodium caseinate + 10mM PBS. The coating concentrations were: PGI: 0.5 mg / mL; PGII: 1.0 mg / mL; C: 1.0 mg / mL.

[0058] Using coating buffer, PGI monoclonal antibody II, PGII monoclonal antibody II, and goat anti-chicken IgY antibody were diluted to 0.5 mg / mL, 1.0 mg / mL, and 1.0 mg / mL, respectively. Then, using a gold-sprayed membrane scribing instrument, PGI antibody was streaked onto the detection line T1 of the NC membrane at a flow rate of 1 μL / cm; PGII antibody was streaked onto the detection line T2 of the NC membrane; and C antibody was streaked onto the control line C of the NC membrane. The membrane was then dried in an oven at 37°C for 24 h to obtain the coated membrane.

[0059] (2) Preparation of binding pad

[0060] (2.1) Pretreatment of the bonding pad

[0061] The pretreatment solution was a solution of pH 8.2 + 10mM BS + 1% BSA + 1% PEG4000 + 0.025% surfactant + 5% sucrose; the surfactant was composed of Tween-20, CHAPS (3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonic acid) and poloxamer 188 in a weight ratio of 10:5:0.3.

[0062] The glass fiber membrane was immersed in the pretreatment solution and reacted at room temperature for 30 minutes. Then the glass fiber membrane was removed and placed in an oven at 37°C for drying for 16 hours.

[0063] (2.2) Preparation of time-resolved fluorescent microsphere labeled antibody

[0064] (2.2.1) Preparation of time-resolved fluorescent microsphere-labeled PGI monoclonal antibody I

[0065] Activation: Mix 50 μL of fluorescent microspheres, 100 μL of pH 6.0 + 25 mM MES solution, 6 μL of 1 mg / mL EDC, and 6 μL of 1 mg / mL NHS. Shake at 150 rpm for 30 min, centrifuge at 14000 rpm at 10℃ for 10 min, and discard the supernatant.

[0066] Labeling: Add 50 μL of 25 mM BS at pH 8.2, add 25 μg of antibody, couple at 150 rpm for 30 min, centrifuge at 12000 rpm and 10℃ for 10 min, and discard the supernatant;

[0067] Blocking: Add 50 μL of blocking solution and shake at 150 rpm for 3 h; the blocking solution is a solution with pH 8.2 + 10 mM BS + 2% BSA, centrifuged at 12000 rpm at 10℃ for 10 min, and the supernatant was discarded;

[0068] Preservation: Add 50 μL of preservation solution; the preservation solution is a solution with pH 7.4 + 25 mM BS + 1% BSA + 0.05% proclin-300 + 8% sucrose.

[0069] (2.2.2) Preparation of time-resolved fluorescent microsphere-labeled PGII monoclonal antibody I

[0070] Activation: Mix 50 μL of fluorescent microspheres, 100 μL of pH 6.0 + 25 mM MES solution, 6 μL of 1 mg / mL EDC, and 6 μL of 1 mg / mL NHS, shake at 150 rpm for 30 min, centrifuge at 14000 rpm at 10℃ for 10 min, and discard the supernatant.

[0071] Labeling: Add 50 μL of 25 mM BS at pH 7.2, add 25 μg of antibody, couple at 150 rpm for 30 min, centrifuge at 12000 rpm and 10℃ for 10 min, and discard the supernatant;

[0072] Blocking: Add 50 μL of blocking solution and shake at 150 rpm for 3 h; the blocking solution is a solution with pH 7.4 + 10 mM BS + 2% BSA, centrifuged at 12000 rpm at 10℃ for 10 min, and the supernatant was discarded;

[0073] Preservation: Add 50 μL of preservation solution; the preservation solution is a solution with pH 7.4 + 25 mM BS + 1% BSA + 0.05% proclin-300 + 8% sucrose.

[0074] (2.2.3) Preparation of time-resolved fluorescent microsphere-labeled chicken IgY antibody

[0075] Activation: Mix 50 μL of fluorescent microspheres, 50 μL of pH 6.0 + 25 mM MES solution, 6 μL of 1 mg / mL EDC, and 6 μL of 1 mg / mL NHS, shake at 150 rpm for 30 min, centrifuge at 14000 rpm at 10℃ for 10 min, and discard the supernatant.

[0076] Labeling: Add 50 μL of 25 mM MES at pH 6.0, add 25 μg of antibody, couple at 150 rpm for 1 h, centrifuge at 12000 rpm at 10 °C for 10 min, and discard the supernatant;

[0077] Blocking: Add 10 μL of blocking solution and shake at 150 rpm for 3 h; the blocking solution is a solution with pH 7.4 + 10 mM BS + 10% BSA, centrifuged at 12000 rpm at 10℃ for 10 min, and the supernatant was discarded;

[0078] Preservation: Add 50 μL of preservation solution; the preservation solution is a solution with pH 7.4 + 25 mM BS + 1% BSA + 0.05% proclin-300 + 8% sucrose.

[0079] (2.3) Spray pad

[0080] The time-resolved fluorescent microsphere-labeled PGI antibody, time-resolved fluorescent microsphere-labeled PGII antibody, and time-resolved fluorescent microsphere-labeled chicken IgY antibody prepared in step (2.2) were mixed at a volume ratio of 8:10:2. The mixture was then sprayed onto the conjugate pads pretreated in step (2.1) using a three-dimensional streak sprayer at a spray rate of 3 μL / cm. The mixture was then placed in an oven at 37°C and dried for 5 hours to obtain the conjugate pads.

[0081] (3) Detection method

[0082] Turn on the instrument, verify the IC card information, and ensure that the IC card batch number matches the test card batch number. The machine will then read the IC card information.

[0083] Dilute the sample to be tested with sample diluent at a dilution ratio of 1:1, then use a pipette to add 100 μL of the sample solution (serum / plasma / whole blood) into the sample well of the test card, and wait for the sample to spread for 10 minutes;

[0084] The sample dilution solution was a pH 7.4 + 50mM Tris + 2% BSA + 1% NaCl + 0.05% Proclin 300 solution.

[0085] Insert the test card into the fluorescence immunochromatographic analyzer (FA8000 fluorescence immunochromatographic analyzer manufactured by Qingdao Huajing Biotechnology Co., Ltd.), click "Test" to detect the fluorescence signal intensity of the test card. The IC card contains calibration curve information. The machine can directly output concentration information, that is, the output results are the concentration of PGI and PGII in the sample to be tested, and the PGI / PGII ratio.

[0086] Examples 2-5

[0087] Examples 2-5 provide a combined detection kit and detection method for pepsinogen I and pepsinogen II, respectively.

[0088] The difference between the above embodiments and Embodiment 1 is that the composition of the coating liquid is different.

[0089] In Example 2: Coating solution: pH 7.4 + 1% mannitol + 0.05% cocoyl glutamic acid + 0.5% sodium caseinate + 10mM PBS.

[0090] In Example 3: Coating solution: pH 7.4 + 1% mannitol + 0.09% cocoyl glutamic acid + 0.5% sodium caseinate + 10mM PBS.

[0091] In Example 4: Coating solution: pH 7.4 + 1% mannitol + 0.07% cocoyl glutamic acid + 0.3% sodium caseinate + 10mM PBS.

[0092] In Example 5: Coating solution: pH 7.4 + 1% mannitol + 0.07% cocoyl glutamic acid + 0.7% sodium caseinate + 10mM PBS.

[0093] All other process parameters in the above embodiments are the same as those in Embodiment 1.

[0094] Examples 6-7

[0095] Examples 6-7 provide a combined detection kit and detection method for pepsinogen I and pepsinogen II, respectively.

[0096] The difference between the above embodiments and Embodiment 1 is that the composition of the pretreatment solution is different.

[0097] In Example 6: The surfactant in the pretreatment solution was a mixture of Tween-20, CHAPS, and poloxamer 188 in a weight ratio of 10:3:0.5.

[0098] In Example 7: The pretreatment solution contained a surfactant composed of Tween-20, CHAPS, and poloxamer 188 in a weight ratio of 10:7:0.1.

[0099] All other process parameters in the above embodiments are the same as those in Embodiment 1.

[0100] Comparative Example

[0101] Comparative Examples 1-4

[0102] Comparative Examples 1-4 provide a combined detection kit and detection method for pepsinogen I and pepsinogen II, respectively.

[0103] The differences between the above comparative examples and Example 1 are as follows.

[0104] In Comparative Example 1, the pretreatment solution consisted of a pH 7.4 solution containing 1% mannitol, 0.03% cocoyl glutamic acid, 0.5% sodium caseinate, and 10 mM PBS.

[0105] In Comparative Example 2, the pretreatment solution consisted of a pH 7.4 solution containing 1% mannitol, 0.07% cocoyl glutamic acid, 1% sodium caseinate, and 10 mM PBS.

[0106] In Comparative Example 3: Poloxamer 188 was replaced with an equal amount of Pluronic F127 in the pretreatment solution.

[0107] In Comparative Example 4: the pretreatment solution contained a surfactant composed of Tween-20, CHAPS, and poloxamer 188 in a weight ratio of 10:0.3:5.

[0108] All other process parameters in the above comparative examples are the same as those in Example 1.

[0109] Performance testing

[0110] (1) Limit of detection and linear range

[0111] Using the detection kits and detection methods of the examples and comparative examples, the limits of detection and correlation coefficients of linear range for PGI and PGII were determined, respectively.

[0112] Limit of Detection (LOD): Dilute the test sample standard to six concentrations at a certain ratio (PGI: 15 ng / mL, 10 ng / mL, 5 ng / mL, 2 ng / mL, 1 ng / mL, 0.5 ng / mL; PGII: 10 ng / mL, 5 ng / mL, 2 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.2 ng / mL). Repeat the test five times for each concentration. Determine the LOD based on whether the corresponding concentration can be detected.

[0113] Correlation coefficient of linear range: The test sample standard was diluted to 5 concentrations according to a certain ratio (PGI: 200 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 2 ng / mL; PGII: 100 ng / mL, 50 ng / mL, 10 ng / mL, 5 ng / mL, 1 ng / mL). Each concentration was measured 3 times, and the average value was calculated. The average value of the measured concentration was fitted to the theoretical concentration using the least squares method, and the linear correlation coefficient r value was calculated.

[0114] (2) Accuracy

[0115] Using the detection kits and detection methods of the examples and comparative examples, pepsinogen I and pepsinogen II standards with a concentration of 100 ng / mL were used as test samples for detection. The results were substituted into the standard curve of the corresponding examples to calculate the detection value and the accuracy. Each sample was measured 6 times, and the relative deviation of the accuracy was calculated.

[0116] (3) Repeatability

[0117] Using the detection kits and detection methods described in the examples and comparative examples, pepsinogen I and pepsinogen II standards at a concentration of 100 ng / mL were used as test samples to detect the intra-batch and inter-batch coefficients of variation, in order to examine the repeatability of the detection kits and detection methods in this application.

[0118] Intra-batch coefficient of variation: The intra-batch coefficient of variation of the detection method in this application was calculated by repeatedly testing each sample 6 times using the same batch of reagent kits.

[0119] Inter-batch coefficient of variation: Each sample was tested using 6 different batches of reagent kits, and the inter-batch coefficient of variation of the detection method in this application was calculated.

[0120] Test results are shown in Table 2.

[0121] Table 2. Detection results in the examples and comparative examples.

[0122]

[0123] Analysis of the test results in Table 2 shows that the detection kit and method provided in this application exhibit good performance indicators for the combined detection of pepsinogen I and pepsinogen II, specifically: 1) Limit of detection: the limit of detection for pepsinogen I (PGI) is not higher than 2 ng / mL; the limit of detection for pepsinogen II (PGII) is not higher than 1 ng / mL; 2) The correlation coefficient r for the linear range of PGI is ≥0.9930; the correlation coefficient r for the linear range of PGII is ≥0.9920; 3) Accuracy: the relative deviations of PGI and PGII are both within 9.0%; 4) Repeatability: the intra-assay coefficient of variation and inter-assay coefficient of variation are both less than 12%. The above test results indicate that the detection kit and method provided in this application have high sensitivity, accuracy, and repeatability for the combined detection of pepsinogen I and pepsinogen II.

[0124] (4) Specificity

[0125] The detection kit and detection method provided in this application have good specificity when detecting pepsinogen I and pepsinogen II together. Specifically, when measuring a PGII sample with a concentration of not less than 200 ng / mL, the PGI measurement result is not higher than 1.0 ng / mL; when measuring a PGI sample with a concentration of not less than 100 ng / mL, the PGII measurement result is not higher than 0.5 ng / mL.

[0126] (5) Anti-interference

[0127] The detection kit and detection method provided in this application also have good anti-interference properties when jointly detecting pepsinogen I and pepsinogen II. Specifically, when the bilirubin in the sample is 0.2 mg / mL, hemoglobin is 5 mg / mL, triglycerides are 2 mg / mL, RF is 1000 IU / mL, roxithromycin is 80 mg / L, and aspirin is 10 μg / mL, there is no interference with the test.

[0128] (6) HOOK effect

[0129] When the detection kit and detection method provided in this application are used to detect pepsinogen I and pepsinogen II in combination, no hook effect is observed when the PGI concentration reaches 500.000 ng / mL; no hook effect is observed when the PGII concentration reaches 400.000 ng / mL.

[0130] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A combined detection kit for pepsinogen I and pepsinogen II, characterized in that, It includes the following components: sample pad, conjugate pad, coating membrane, and absorbent pad; The coating membrane is an NC membrane coated antibody; in the preparation method of the NC membrane coated antibody, the coating solution is 0.8-1.2% mannitol, 0.05-0.09% cocoyl glutamic acid, 0.3-0.7% sodium caseinate and 8-12mM PBS solution, with a pH of 7.2-7.6; The method for preparing the binding pad is as follows: the fiber membrane is treated with a pretreatment solution and dried, and then time-resolved fluorescent microspheres labeled with antibodies are sprayed on it. The pretreatment solution is a solution of 8-12mM borax-boric acid buffer, 0.8-1.2% BSA, 0.8-1.2% PEG4000, 0.02-0.03% surfactant and 4-6% sucrose, with a pH of 8.0-8.4; the surfactant is a mixture of Tween-20, CHAPS and poloxamer 188 in a weight ratio of 10:(3-7):(0.1-0.5).

2. The combined detection kit for pepsinogen I and pepsinogen II according to claim 1, characterized in that, The NC membrane is coated with antibodies by sequentially setting a detection line T1 coated with PGI monoclonal antibody II at a concentration of 0.4-0.6 mg / mL, a detection line T2 coated with PGII monoclonal antibody II at a concentration of 0.8-1.2 mg / mL, and a control line C coated with goat anti-chicken IgY antibody at a concentration of 0.8-1.2 mg / mL on the NC membrane.

3. The combined detection kit for pepsinogen I and pepsinogen II according to claim 1, characterized in that, The coating solution is 0.9-1.1% mannitol, 0.06-0.08% cocoyl glutamic acid, 0.4-0.6% sodium caseinate and 9-11 mM PBS solution, with a pH of 7.3-7.

5.

4. The combined detection kit for pepsinogen I and pepsinogen II according to claim 3, characterized in that, The coating solution is 0.9-1.1% mannitol, 0.065-0.075% cocoyl glutamic acid, 0.45-0.55% sodium caseinate and 9-11 mM PBS solution, with a pH of 7.3-7.

5.

5. The combined detection kit for pepsinogen I and pepsinogen II according to claim 1, characterized in that, The conjugate pad pretreatment solution is a solution of 9-11 mM borax-boric acid buffer, 0.9-1.1% BSA, 0.9-1.1% PEG4000, 0.02-0.03% surfactant and 4.5-5.5% sucrose, with a pH of 8.1-8.

3.

6. The combined detection kit for pepsinogen I and pepsinogen II according to claim 1, characterized in that, The surfactant is composed of a mixture of Tween-20, CHAPS, and poloxamer 188 in a weight ratio of 10:(4-6):(0.2-0.4).

7. The combined detection kit for pepsinogen I and pepsinogen II according to claim 1, characterized in that, It also includes a sample diluent, which is a solution of 47-52 mM Tris, 1.9-2.1% BSA, 0.9-1.1% NaCl and 0.045-0.055% Proclin 300, with a pH of 7.3-7.

5.

8. The combined detection kit for pepsinogen I and pepsinogen II according to claim 1, characterized in that, The preparation method of the time-resolved fluorescent microsphere labeled antibody is as follows: (1) Preparation of time-resolved fluorescent microsphere-labeled PGI monoclonal antibody I: Activation: Mix 40-60 μL of fluorescent microspheres, 80-120 μL of 20-30 mM MES solution (pH 5.8-6.2), 5-8 μL of 0.8-1.2 mg / mL EDC, and 5-8 μL of 0.8-1.2 mg / mL NHS, shake to activate, centrifuge, and discard the supernatant; Labeling: Add 40-60 μL of 20-30 mM borax-boric acid buffer (pH 8.0-8.4), add 20-30 μg of PGI monoclonal antibody I, conjugate for 20-40 min, centrifuge, and discard the supernatant; Blocking: Add 40-60 μL of blocking solution and shake to block for 2-4 hours; the blocking solution is a solution of 8-12 mM borax-boric acid buffer and 1.8-2.2% BSA, pH 8.0-8.

4. Shake, centrifuge, and discard the supernatant. Preservation: Add 40-60 μL of preservation solution; the preservation solution is a solution of 20-30 mM borax-boric acid buffer, 0.8-1.2% BSA, 0.04-0.06% proclin-300 and 7-9% sucrose, with a pH of 7.2-7.6; (2) Preparation of time-resolved fluorescent microsphere-labeled PGII monoclonal antibody I: Activation: Mix 40-60 μL of fluorescent microspheres, 80-120 μL of 20-30 mM MES solution at pH 5.8-6.2, 5-8 μL of 0.8-1.2 mg / mL EDC, and 5-8 μL of 0.8-1.2 mg / mL NHS, shake to activate, centrifuge, and discard the supernatant; Labeling: Add 40-60 μL of 20-30 mM borax-boric acid buffer (pH 7.0-7.4), add 20-30 μg of PGII monoclonal antibody I, couple for 20-40 min, centrifuge, and discard the supernatant; Blocking: Add 40-60 μL of blocking solution and shake to block for 2-4 hours; the blocking solution is a solution of 8-12 mM borax-boric acid buffer and 1.8-2.2% BSA with pH 7.2-7.

6. Shake, centrifuge, and discard the supernatant. Preservation: Add 40-60 μL of preservation solution; the preservation solution is a solution of 20-30 mM borax-boric acid buffer, 0.8-1.2% BSA, 0.04-0.06% proclin-300 and 7-9% sucrose, with a pH of 7.2-7.6; (3) Preparation of time-resolved fluorescent microsphere-labeled chicken IgY antibody: Activation: Mix 40-60 μL of fluorescent microspheres, 80-120 μL of 20-30 mM MES solution at pH 5.8-6.2, 5-8 μL of 0.8-1.2 mg / mL EDC, and 5-8 μL of 0.8-1.2 mg / mL NHS, shake to activate, centrifuge, and discard the supernatant; Labeling: Add 40-60 μL of 20-30 mM MES at pH 5.8-6.2, add 20-30 μg of chicken IgY antibody, conjugate for 40-80 min, centrifuge, and discard the supernatant; Blocking: Add 8-12 μL of blocking solution and shake to block for 2-4 hours; the blocking solution is a solution of 20-30 mM borax-boric acid buffer and 8-12% BSA, pH 7.2-7.

6. Shake, centrifuge, and discard the supernatant. Preservation: Add 40-60 μL of preservation solution; the preservation solution is a solution of 20-30 mM borax-boric acid buffer, 0.8-1.2% BSA, 0.04-0.06% proclin-300 and 7-9% sucrose, with a pH of 7.2-7.6.

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