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CN-121114433-B - Combined detection kit for pepsinogen I and pepsinogen II and application thereof

CN121114433BCN 121114433 BCN121114433 BCN 121114433BCN-121114433-B

Abstract

The application relates to the technical field of biological detection, and particularly discloses a combined detection kit for pepsinogen I and pepsinogen II and application thereof. The application discloses a pepsinogen I and pepsinogen II combined detection kit which comprises a sample pad, a combination pad, a coating film and a water absorption pad, wherein the coating film is NC film coating antibody, the combination pad is prepared by treating a fiber film by pretreatment liquid and drying, then spraying time-resolved fluorescent microsphere labeled antibody, and the pretreatment liquid 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 combined detection of pepsinogen I/pepsinogen II by using the detection kit and the detection method provided by the application has higher sensitivity, accuracy and repeatability.

Inventors

  • WANG XUNKUN
  • JIANG WENQING
  • Lou Shanmei
  • TIAN ZHEN

Assignees

  • 青岛华晶生物技术有限公司

Dates

Publication Date
20260505
Application Date
20251023

Claims (8)

  1. 1. The combined detection kit for pepsinogen I and pepsinogen II is characterized by comprising a sample pad, a binding pad, a coating film and a water absorption pad; The coating film is NC film coated antibody, wherein in the preparation method of NC film coated antibody, the coating liquid is 0.8-1.2% mannitol, 0.05-0.09% cocoyl glutamic acid, 0.3-0.7% sodium caseinate and 8-12mM PBS solution, and the pH value is 7.2-7.6; The preparation method of the bonding pad comprises the steps of treating a fiber membrane by pretreatment liquid, drying, and then spraying time-resolved fluorescent microsphere labeled antibody; The pretreatment solution is a solution of 8-12mM borax-boric acid buffer solution, 0.8-1.2% BSA, 0.8-1.2% PEG4000, 0.02-0.03% surfactant and 4-6% sucrose, the pH value is 8.0-8.4, and the surfactant is formed by mixing Tween-20, CHAPS and poloxamer 188 with the weight ratio of 10 (3-7) to 0.1-0.5.
  2. 2. The combined detection kit of pepsinogen I and pepsinogen II according to claim 1, wherein the NC membrane coated antibody is formed by sequentially arranging a detection line T1 coated with PGI monoclonal antibody II with the concentration of 0.4-0.6mg/mL, a detection line T2 coated with PGII monoclonal antibody II with the concentration of 0.8-1.2mg/mL and a quality control line C coated with sheep anti-chicken IgY antibody with the concentration of 0.8-1.2mg/mL on an NC membrane.
  3. 3. The kit for combined detection of pepsinogen I and pepsinogen II according to claim 1, wherein the coating solution comprises 0.9-1.1% mannitol, 0.06-0.08% cocoyl glutamic acid, 0.4-0.6% sodium caseinate and 9-11mM PBS solution, and has a pH of 7.3-7.5.
  4. 4. The kit for combined detection of pepsinogen I and pepsinogen II according to claim 3, wherein the coating solution comprises 0.9-1.1% mannitol, 0.065-0.075% cocoylglutamic acid, 0.45-0.55% sodium caseinate and 9-11mM PBS solution, and has a pH of 7.3-7.5.
  5. 5. The kit for combined detection of pepsinogen I and pepsinogen II according to claim 1, wherein the pretreatment solution for the binding pad is a solution of 9-11mM borax-boric acid buffer solution, 0.9-1.1% bsa, 0.9-1.1% PEG4000, 0.02-0.03% surfactant and 4.5-5.5% sucrose, and the pH is 8.1-8.3.
  6. 6. The combined detection kit for pepsinogen I and pepsinogen II according to claim 1, wherein the surfactant is prepared by mixing tween-20, CHAPS and poloxamer 188 in a weight ratio of 10 (4-6) to 0.2-0.4.
  7. 7. The combined pepsinogen I and pepsinogen II detection kit according to claim 1, further comprising a sample diluent of 47-52mM Tris, 1.9-2.1% bsa, 0.9-1.1% nacl, and 0.045-0.055% Proclin300, at a pH of 7.3-7.5.
  8. 8. The combined detection kit for pepsinogen I and pepsinogen II according to claim 1, wherein the preparation method of the time-resolved fluorescence microsphere labeled antibody is as follows: (1) Preparing a PGI monoclonal antibody I marked by time-resolved fluorescent microballoons: activating, namely uniformly mixing 40-60 mu L of fluorescent microspheres, 80-120 mu L of 20-30mM MES solution with pH of 5.8-6.2, 5-8 mu L of 0.8-1.2mg/mL EDC and 5-8 mu L of 0.8-1.2mg/mL NHS, vibrating for activation, centrifuging, and discarding the supernatant; Labeling, namely adding 40-60 mu L of 20-30mM borax-boric acid buffer solution with pH of 8.0-8.4, adding 20-30 mu g PGI monoclonal antibody I, coupling for 20-40min, centrifuging, and discarding the supernatant; Sealing by adding 40-60 μl of sealing solution, shaking for sealing for 2-4 hr, wherein the sealing solution is solution of 8-12mM borax-boric acid buffer solution and 1.8-2.2% BSA, pH is 8.0-8.4, shaking, centrifuging, and discarding supernatant; preserving by adding 40-60 mu L of preserving fluid which is a solution of 20-30mM borax-boric acid buffer solution, 0.8-1.2% BSA, 0.04-0.06% proclin-300 and 7-9% sucrose, wherein the pH value is 7.2-7.6; (2) Preparing a PGII monoclonal antibody I marked by time-resolved fluorescent microspheres: Activating, namely uniformly mixing 40-60 mu L of fluorescent microspheres, 80-120 mu L of 20-30mM MES solution with pH of 5.8-6.2, 5-8 mu L of 0.8-1.2mg/mL EDC and 5-8 mu L of 0.8-1.2mg/mL NHS, vibrating for activation, centrifuging, and discarding the supernatant; Labeling, namely adding 40-60 mu L of 20-30mM borax-boric acid buffer solution with pH of 7.0-7.4, adding 20-30 mu g PGII monoclonal antibody I, coupling for 20-40min, centrifuging, and discarding the supernatant; Sealing by adding 40-60 μl of sealing solution, shaking and sealing for 2-4 hr, wherein the sealing solution is solution of 8-12mM borax-boric acid buffer solution with pH of 7.2-7.6 and 1.8-2.2% BSA, shaking, centrifuging, and discarding supernatant; preserving by adding 40-60 mu L of preserving fluid which is a solution of 20-30mM borax-boric acid buffer solution, 0.8-1.2% BSA, 0.04-0.06% proclin-300 and 7-9% sucrose, wherein the pH value is 7.2-7.6; (3) Preparing a chicken IgY antibody marked by time-resolved fluorescent microspheres: Activating, namely uniformly mixing 40-60 mu L of fluorescent microspheres, 80-120 mu L of 20-30mM MES solution with pH of 5.8-6.2, 5-8 mu L of 0.8-1.2mg/mL EDC and 5-8 mu L of 0.8-1.2mg/mL NHS, vibrating for activation, centrifuging, and discarding the supernatant; Labeling, namely adding 40-60 mu L of 20-30mM MES with pH of 5.8-6.2, adding 20-30 mu g of chicken IgY antibody, coupling for 40-80min, centrifuging, and discarding the supernatant; Blocking by adding 8-12 μl of blocking solution, shaking for 2-4 hr, wherein the blocking solution is solution of 20-30mM borax-boric acid buffer solution and 8-12% BSA, pH is 7.2-7.6, shaking, centrifuging, and discarding supernatant; preserving by adding 40-60 mu L of preserving solution which is a solution of 20-30mM borax-boric acid buffer solution, 0.8-1.2% BSA, 0.04-0.06% proclin-300 and 7-9% sucrose, and the pH value is 7.2-7.6.

Description

Combined detection kit for pepsinogen I and pepsinogen II and application thereof Technical Field The application relates to the technical field of biological detection, in particular to a combined detection kit of pepsinogen I and pepsinogen II and application thereof. Background Pepsinogen (PG) is an inactive precursor of pepsin in gastric juice and is divided into two subgroups PGI and PGII according to its biochemical properties and immunogenicity. PGI is secreted mainly by the main cells of the basal glands of the stomach and by the cervical mucus cells, and is closely related to gastric acid content. PGII is derived from the whole gastric glands (gastric cardia gland, gastric basal gland, antral pylorus gland) and duodenal glands. PGI and PGII reflect the secretion function of different parts of gastric mucosa, most of synthesized PG enters the gastric cavity, and the synthesized PG is activated into pepsin under the action of acidic gastric juice, and only a small amount of PG enters the blood circulation through gastric mucosa capillaries. The serum PG concentration reflects the morphology and function of gastric mucosa at different positions, PGI is a pointer for detecting the function of gastric acid secretion gland cells, PGI is increased due to gastric acid secretion, PGI is decreased due to gastric acid secretion or gastric mucosa gland atrophy, PGII is more relevant to gastric fundus mucosa lesions (relative to antral mucosa), and the increase of PGI is related to gastric fundus gland atrophy, gastric epithelium metaplasia or pseudo pylorogland metaplasia and abnormal hyperplasia. Progressive decrease in the PGI/PGII ratio correlates with progression of gastric mucosal atrophy. The current methods for detecting PGI and PGII in clinic and laboratory include enzyme-linked immunosorbent assay, fluorescence immunochromatography, latex turbidimetry, chemiluminescence and the like. However, the existing detection method has the defects of low sensitivity, low accuracy and low repeatability. Disclosure of Invention In order to solve the technical problems, the application provides a combined detection kit for pepsinogen I and pepsinogen II and application thereof. In a first aspect, the application provides a combined detection kit of pepsinogen I and pepsinogen II, which is characterized by comprising a sample pad, a binding pad, a coating film and a water absorption pad; The coating film is NC film coating antibody, wherein in the preparation method of NC film coating antibody, the coating liquid is PBS solution with pH of 7.2-7.6+0.8-1.2% mannitol+0.05-0.09% cocoyl glutamic acid+0.3-0.7% sodium caseinate+8-12 mM; The preparation method of the bonding pad comprises the steps of treating a fiber membrane by pretreatment liquid, drying, and then spraying time-resolved fluorescent microsphere labeled antibody; The pretreatment solution is a solution of pH 8.0-8.4+8-12mM BS+0.8-1.2% BSA+0.8-1.2% PEG 4000+0.02-0.03% surfactant+4-6% sucrose, and the surfactant is formed by mixing Tween-20, CHAPS and poloxamer 188 in a weight ratio of 10:3-7:0.1-0.5. The pepsinogen I and pepsinogen II combined detection kit provided by the application has the principle that the content of pepsinogen I (PGI) and pepsinogen II (PGII) in a sample (comprising whole blood, serum and plasma) is quantitatively detected based on a fluorescent immune double-antibody sandwich method. And (3) dripping a sample to be detected into a sample adding hole of the detection card, and reacting the PGI or PGII with a time-resolved fluorescence microsphere marked antibody I in the binding pad to form an antigen-antibody complex when the sample to be detected contains a certain amount of PGI or PGII, wherein the reaction complex moves forward along the nitrocellulose membrane under the action of chromatography and is captured by a detection area on the nitrocellulose membrane coated with an antibody II in advance. The more analytes in the sample, the more complexes accumulate on the detection zone and the signal intensity of the fluorescent antibody reflects the amount of captured analytes. In addition, mannitol in the coating liquid can protect the activity of the antibody and improve the detection sensitivity, can form a glassy state structure in the drying process to wrap PGI/PGII monoclonal antibody molecules, can reduce the damage of moisture crystallization to the protein space structure, avoid the denaturation of the active site of the antibody, ensure the efficient combination of the antigen and the antibody during detection, and directly improve the detection sensitivity. The cocoyl glutamic acid can optimize the fixing efficiency, ensure the detection accuracy, regulate the surface tension of a coated carrier, slightly change the surface charge of the PGI/PGII antibody, help the antibody to be adsorbed on the surface of the carrier more uniformly and stably, and avoid the active site from being shielded due to excessive adsorption, e