Search

CN-121978337-A - Kit and detection method for tumor marker Legumain detection

CN121978337ACN 121978337 ACN121978337 ACN 121978337ACN-121978337-A

Abstract

The invention provides a kit for detecting a tumor marker Legumain, which comprises a biosensor combined with an antibody Anti-Legumian-Ab1 and a colloidal gold labeled Legumain antibody solution. The invention also provides a method for detecting the tumor marker Legumain by using the kit, which comprises the steps of exposing the biosensor combined with the antibody Anti-Legumian-Ab1 to a human serum sample, transferring the biosensor combined with a target molecule into a colloidal gold-labeled Legumain antibody solution, determining the amount of the colloidal gold-labeled Anti-Legumain-Ab2 antibody deposited on the biosensor by using a biological film interferometry method, and according to the deposition amount of the colloidal gold-labeled Anti-Legumain-Ab2 antibody. The method has higher detection sensitivity.

Inventors

  • TANG CAIFENG
  • LI CHUNYING
  • LI WEI

Assignees

  • 苏州品赛医疗科技有限公司

Dates

Publication Date
20260505
Application Date
20231228

Claims (10)

  1. 1. A kit for detecting a tumor marker Legumain is characterized by comprising a biosensor combined with an antibody Anti-Legumian-Ab1, wherein the biosensor is a suspension prepared by immersing the optical fiber biosensor with a silanized surface in a mixed solution of an antibody Anti-Legumian-Ab1, EDC and NHS, and incubating, a colloidal gold-labeled Legumain antibody solution, wherein the antibody is a suspension prepared by blocking with bovine serum albumin after the antibody Anti-Legumain-Ab2 is labeled with the colloidal gold, and a PBST solution, wherein the concentration of the PBST solution is 0.01mol/L, and the pH is 7.2.
  2. 2. The kit for detecting a tumor marker Legumain according to claim 1, wherein the optical fiber biosensor is prepared by adopting a multimode optical fiber, taking a distal end surface of the optical fiber as a biological layer carrying surface, coating a high refractive index layer on the biological layer carrying surface, coating a transparent silicon dioxide film layer on the high refractive index layer by using a physical vapor deposition method, and coupling a light source and analysis equipment on the other end surface of the optical fiber biosensor.
  3. 3. The kit for tumor marker Legumain detection according to claim 2, wherein the length of the multi-film optical fiber is 10 mm-30 mm, and the diameter is 200 μm-1000 μm.
  4. 4. The kit for tumor marker Legumain detection according to claim 2, wherein the coating thickness of the high refractive index layer is 10-50 nm, and the coating thickness of the transparent silicon dioxide film layer is 500-800 nm.
  5. 5. The kit for tumor marker Legumain detection according to claim 1, wherein the surface silanized optical fiber biosensor is prepared by immersing the optical fiber biosensor in a 2% aptes ethanol solution after plasma surface activation.
  6. 6. The kit for tumor marker Legumain detection according to claim 1, wherein the mixed solution of Anti-Legumian-Ab1 antibody and EDC and NHS is prepared by adding 10mM EDC and 10mM Sulfo-NHS to an activation buffer, mixing the mixture into 5mM/5mM EDC/Sulfo-NHS mixture in equal volume, and adding Anti-Legumian-Ab1 antibody to a final concentration of 4ug/ml.
  7. 7. The kit for tumor marker Legumain detection according to claim 1, wherein the incubation step is performed for a period of 2 hours.
  8. 8. The kit for tumor marker Legumain detection according to claim 1, wherein the colloidal gold is prepared by placing a rotor in ultrapure water, heating on a heatable magnetic stirrer until bubbles emerge uniformly, rapidly adding 1ml of 1% chloroauric acid solution, immediately adding 1ml of 1.5% trisodium citrate after heating to boil, and continuing heating for 7min.
  9. 9. The kit for tumor marker Legumain detection according to claim 1, wherein the PBST solution has a Na 2 HPO 4 ·12H 2 O mass content of 0.26%, a NaH 2 PO 4 ·2H 2 O mass content of 0.044%, a sodium chloride concentration of 300mm, and a triton X-100 concentration of 0.05%.
  10. 10. A method of detecting a tumor marker Legumain using the kit of claim 1, wherein the method comprises exposing the biosensor bound with the antibody Anti-Legumian-Ab1 to a human serum sample, binding the biosensor to a target molecule, transferring the biosensor bound with the target molecule into a colloidal gold-labeled Legumain antibody solution, binding the biosensor bound with the target molecule to the Anti-Legumain-Ab2 antibody, and determining the amount of the target molecule on the biosensor based on the amount of deposition of the colloidal gold-labeled Anti-Legumain-Ab2 antibody by using the biofilm interferometry method with the biosensor bound with the antibody Anti-Legumian-Ab1 after equilibration in a PBST solution as a control.

Description

Kit and detection method for tumor marker Legumain detection Technical Field The invention relates to the field of biological detection, in particular to a detection method and a detection kit for a tumor marker Legumain. Background Ovarian cancer is a common gynecological malignant tumor, no diagnostic marker with good specificity exists at present, and the death rate of the ovarian cancer is the first of all gynecological malignant tumors, so that the female health is endangered. Since ovarian cancer is usually asymptomatic in early stages and lacks a simple and effective early diagnosis method, about 70% of patients have advanced stages at the time of symptomatic visit, the 5-year survival rate of advanced patients is 20% -30%, and the 5-year survival rate of early patients is 70-90%. Thus, early detection of ovarian cancer has an important role in the selection of its treatment regimen and in improving patient prognosis. At present, the clinical common methods for early detection of ovarian cancer mainly comprise gynecological examination, imaging examination, tumor marker detection and the like. The method and the method can accurately diagnose the ovarian cancer when being matched with each other, but have higher requirements on equipment and operators, improve the detection cost and simultaneously prevent the popularization and popularization of early detection of the ovarian cancer. The detection of tumor markers is widely used for early detection of tumors, and saccharide antigen125 (carbohydrate antigen, CA 125) is a tumor marker commonly used in diagnosis of ovarian cancer, but has low specificity when being used for diagnosis of ovarian cancer, and is easy to cause false positive and other problems, so that a new tumor marker needs to be found to replace or be complemented with the tumor marker. Disclosure of Invention The invention aims to solve the problems that the existing early screening method for the ovarian cancer tumor markers is low in specificity, easy to cause false positive and the like, and provides a kit and a detection method for detecting the tumor markers Legumain so as to achieve the effect of improving detection sensitivity. In order to achieve the aim, the invention provides a kit for detecting a tumor marker Legumain, which comprises a biosensor combined with an antibody Anti-Legumian-Ab1, wherein the biosensor is prepared by immersing a surface silanized optical fiber biosensor in a mixed solution of an Anti-Legumian-Ab1 antibody and EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), and incubating the obtained suspension, a colloidal gold labeled Legumain antibody solution, wherein the antibody is prepared by adding bovine serum albumin to the suspension after labeling the antibody Anti-Legumain-Ab2 with the colloidal gold, and a PBST (phosphate buffer) solution, wherein the concentration of the PBST solution is 0.01mol/L, and the pH is 7.2. The optical fiber biosensor is preferably prepared by adopting a multimode optical fiber, taking the distal end surface of the optical fiber as a biological layer carrying surface, coating a high refractive index layer on the biological layer carrying surface, coating a transparent silicon dioxide film layer on the high refractive index layer by using a physical vapor deposition method, and coupling a light source and analysis equipment on the other end surface of the optical fiber biosensor. Preferably, the length of the multi-film optical fiber is 10 mm-30 mm, and the diameter is 200 mu m-1000 mu m. Preferably, the coating thickness of the high refractive index layer is 10-50 nm, and the coating thickness of the transparent silicon dioxide film layer is 500-800 nm. Preferably, the surface silanized optical fiber biosensor is prepared by immersing the optical fiber biosensor in 2% APTES (aminopropyl triethoxysilane) ethanol solution after plasma surface activation. Preferably, the mixed solution of the Anti-Legumian-Ab1 antibody and EDC and NHS is prepared by adding 10mM EDC and 10mM sulfosuccinimide to an activation buffer, mixing the mixture with equal volume to 5mM/5mM EDC/sulfosuccinimide, and adding the Anti-Legumian-Ab1 antibody to obtain a final concentration of 4ug/ml. Preferably, the incubation step is performed for a period of 2 hours. Preferably, the colloidal gold is prepared by placing a rotor in ultrapure water, heating on a heatable magnetic stirrer until bubbles emerge uniformly, rapidly adding 1mL of 1% chloroauric acid solution, heating to boiling, immediately adding 1mL of 1.5% trisodium citrate, and continuing heating for 7min. Preferably, the phosphate buffer solution of the PBST solution contains 0.26% of Na 2HPO4·12H2 O by mass, 0.044% of NaH 2PO4·2H2 O by mass, 300mM of sodium chloride and 0.05% of Triton X-100 by mass. A method for detecting a tumor marker Legumain by using the kit comprises exposing a biosensor combined with an antibody Anti-Legumian-Ab1 to a