Kit for detecting tumor marker Legumain and detection method

Abstract

The invention provides a kit for detecting a tumor marker Legumain. The kit comprises a biosensor and a Legumain antibody solution, wherein the biosensor is combined with an antibody Anti-Legumian-Ab1, and the Legumain antibody solution is marked by colloidal gold. The invention also provides a method for detecting the tumor marker Legumain by using the kit, and the method comprises the following steps: exposing the biosensor combined with the antibody Anti-Legumian-Ab1 to a human serum sample, transferring the biosensor combined with the target molecule to a colloidal gold labeled Legumain antibody solution, and detecting the tumor marker Legumain by using the kit. Determining the amount of an Anti-Legumain-Ab2 antibody labeled by colloidal gold deposited on the biosensor by using a biological membrane interference method; and according to the deposition amount of the Anti-Legumain-Ab2 antibody labeled by colloidal gold, carrying out quantitative analysis on the antibody. The method has high detection sensitivity.

Description

Technical Field

[0001] This invention relates to the field of biological detection, specifically to a method and kit for detecting the tumor marker Legumain. Background Technology

[0002] Ovarian cancer is a common gynecological malignancy. Currently, there are no specific and effective diagnostic markers, and its mortality rate is the highest among all gynecological malignancies, posing a significant threat to women's health. Because early-stage ovarian cancer often presents with no obvious symptoms and lacks simple and effective early diagnostic methods, approximately 70% of patients are already in advanced stages when symptoms appear. The 5-year survival rate for advanced-stage patients is 20%-30%, while the 5-year survival rate for early-stage patients is 70%-90%. Therefore, early detection of ovarian cancer plays a crucial role in the selection of treatment options and improving patient prognosis.

[0003] Currently, the commonly used methods for early detection of ovarian cancer in clinical practice mainly include gynecological examination, imaging examination, and tumor marker detection. Although these methods, when used in combination, can accurately diagnose ovarian cancer, they require sophisticated equipment and skilled operators, increasing detection costs and hindering the widespread adoption and promotion of early ovarian cancer detection.

[0004] Tumor marker detection is widely used for early cancer screening. Carbohydrate antigen 125 (CA125) is a commonly used tumor marker in the diagnosis of ovarian cancer. However, its specificity in the diagnosis of ovarian cancer is low and it is prone to false positives. Therefore, it is necessary to find new tumor markers to replace it or complement it. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of low specificity and false positives in existing methods for early screening of ovarian cancer tumor markers, and to provide a kit and detection method for detecting the tumor marker Legumain, so as to improve the detection sensitivity.

[0006] To achieve the above objectives, the present invention provides a kit for detecting the tumor marker Legumian, the kit comprising: a biosensor incorporating the antibody Anti-Legumian-Ab1, wherein the biosensor is a suspension prepared by immersing a surface-silanized fiber optic biosensor in a mixed solution of Anti-Legumian-Ab1 antibody, EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), and incubating thereon; a colloidal gold-labeled Legumian antibody solution, wherein the antibody is a suspension prepared by labeling Anti-Legumian-Ab2 with colloidal gold and then blocking it with bovine serum albumin; and a PBST (phosphate buffer) solution, wherein the concentration of the PBST solution is 0.01 mol / L and the pH is 7.2.

[0007] Preferably, the fiber optic biosensor is prepared by the following method: using a multimode fiber, with the far end of the fiber as the biolayer mounting surface, coating a high refractive index layer on the biolayer mounting surface, and then coating a transparent silica film layer on the high refractive index layer using physical vapor deposition; the other end of the fiber optic biosensor is coupled to a light source and an analysis device.

[0008] Preferably, the multi-film optical fiber has a length of 10mm to 30mm and a diameter of 200μm to 1000μm.

[0009] 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 nm–800 nm.

[0010] Preferably, the surface-silanized fiber optic biosensor is prepared by immersing the fiber optic biosensor in a 2% APTES (aminopropyltriethoxysilane) ethanol solution after plasma surface activation.

[0011] Preferably, the method for preparing the mixed solution of Anti-Legumian-Ab1 antibody with EDC and NHS is as follows: 10mM EDC and 10mM Sulfo-NHS (N-hydroxythiosuccinimide) are added to the activation buffer and mixed in equal volumes to form a 5mM / 5mM EDC / sulfo-NHS mixture. Anti-Legumian-Ab1 antibody is then added to achieve a final concentration of 4ug / ml.

[0012] Preferably, the incubation step lasts for 2 hours.

[0013] Preferably, the colloidal gold is prepared by the following method: placing a rotor in ultrapure water and heating it on a heatable magnetic stirrer until bubbles emerge evenly; then quickly adding 1 mL of 1% chloroauric acid solution; heating to boiling; and immediately adding 1 mL of 1.5% trisodium citrate; and continuing to heat for 7 minutes.

[0014] Preferably, the PBST solution contains 0.26% Na2HPO4·12H2O and 0.044% NaH2PO4·2H2O by mass in the phosphate buffer solution; the sodium chloride concentration is 300 mM; and the Triton X-100 concentration is 0.05%.

[0015] A method for detecting the tumor marker Leguminian using the kit includes: exposing a biosensor bound to the antibody Anti-Legumian-Ab1 to a human serum sample to allow the biosensor to bind to the target molecule; transferring the biosensor bound to the target molecule to a colloidal gold-labeled Leguminian antibody solution to allow the biosensor bound to the target molecule to bind to the Anti-Legumian-Ab2 antibody; using a biosensor bound to the antibody Anti-Legumian-Ab1 equilibrated in PBST solution as a control, determining the amount of colloidal gold-labeled Anti-Legumian-Ab2 antibody deposited on the biosensor using a biomembrane interferometry method; and determining the amount of the target molecule on the biosensor based on the amount of colloidal gold-labeled Anti-Legumian-Ab2 antibody deposited. Attached Figure Description

[0016] Figure 1 The image shows the results of detecting Legumain using the biomembrane interferometry method in Example 1. Detailed Implementation

[0017] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0018] This invention provides a kit for detecting the tumor marker Legumian, comprising: a biosensor incorporating the antibody Anti-Legumian-Ab1, wherein the biosensor is a suspension prepared by immersing a surface-silanized fiber optic biosensor in a mixed solution of Anti-Legumian-Ab1 antibody, EDC, and NHS, followed by incubation; a colloidal gold-labeled Legumian antibody solution, wherein the antibody is a suspension prepared by labeling Anti-Legumian-Ab2 with colloidal gold and then blocking it with bovine serum albumin; and a PBST solution with a concentration of 0.01 mol / L and a pH of 7.2.

[0019] Preferably, the fiber optic biosensor is prepared by the following method: using a multimode fiber, with the far end of the fiber as the biolayer mounting surface, coating a high refractive index layer on the biolayer mounting surface, and then coating a transparent silica film layer on the high refractive index layer using physical vapor deposition; the other end of the fiber optic biosensor is coupled to a light source and an analysis device.

[0020] When the transparent silica film layer combines with the biomolecular layer, the reflected incident light will primarily be reflected from the interface between the biomolecular layer and the buffer solution, rather than from the interface between the transparent film layer and the biomolecular layer. When detecting a sample, the distal end of the optical fiber is immersed in the sample solution. If ligand binding to the analyte occurs at this time, an interference pattern will be generated between the first and second reflected beams. By analyzing the displacement of the second reflected beam in the interference pattern, changes in the biomolecular layer can be determined.

[0021] This invention provides a method for detecting the tumor marker Legumian using the aforementioned kit. The method includes: exposing a biosensor bound to the antibody Anti-Legumian-Ab1 to a human serum sample, allowing the biosensor to bind to the target molecule; transferring the biosensor bound to the target molecule to a colloidal gold-labeled Legumian antibody solution, allowing the biosensor bound to the target molecule to bind to the Anti-Legumian-Ab2 antibody; using a biosensor bound to the antibody Anti-Legumian-Ab1 equilibrated in PBST solution as a control, determining the amount of colloidal gold-labeled Anti-Legumian-Ab2 antibody deposited on the biosensor using a biomembrane interferometry method; and determining the amount of the target molecule on the biosensor based on the amount of colloidal gold-labeled Anti-Legumian-Ab2 antibody deposited.

[0022] The present invention will be described in detail below through examples. Unless otherwise specified, all raw materials used in the following examples and comparative examples are commercially available.

[0023] All the following embodiments and comparative examples,

[0024] Anti-Legumain-Ab1 and Anti-Legumain-Ab2 used were purchased from Abcom, with product numbers ab183028 and ab232870 respectively.

[0025] Sensitivity was determined by the following method: 1 mg / mL of Legumain was diluted with negative human serum to prepare standard solutions with concentrations of 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL, and 1 pg / mL, respectively. Negative serum was defined as serum without Legumain. 0.1 nm was used as the positive criterion. The serum samples of each concentration were tested to determine the concentration that could be effectively detected.

[0026] Example 1

[0027] 1. Fabrication of fiber optic biosensors

[0028] Multimode optical fibers with a length of 10mm to 30mm are selected, and the diameter of the selected multimode optical fibers is required to be in the range of 200μm to 1000μm. The distal end of the multimode optical fiber is used as the mounting surface for the biolayer. First, a high-refractive-index layer with a thickness between 10 and 50 nm is coated on the mounting surface. Next, a transparent silica film with a thickness between 500 nm and 800 nm is coated on the high-refractive-index layer using physical vapor deposition. Finally, a light source and analytical equipment are coupled to the other end of the fiber optic biosensor.

[0029] 2. Preparation of a biosensor incorporating the antibody Anti-Legumian-Ab1

[0030] 1) Silanization of sensor surface

[0031] First, the bare fiber optic biosensor prepared in step 1 was subjected to plasma surface activation (Shanghai Pluto-T, power 750w, 13.6MHZ, dry air, bombardment for 1min); after activation, the probe end face was immersed in 2% APTES ethanol solution and placed at 68℃ for 1h; then it was washed three times with deionized water and dried and stored in nitrogen.

[0032] 2) Binding antibody Anti-Legumian-Ab1

[0033] Preparation of a mixed solution of Anti-Legumian-Ab1 antibody with EDC and NHS: Add 10mM EDC and 10mM Sulfo-NHS to activation buffer (0.1M MES, 0.5M NaCl, pH 6.0) and mix in equal volumes to form a 5mM / 5mM EDC / sulfo-NHS mixture. Add Anti-Legumian-Ab1 antibody to make a final concentration of 4ug / ml. After mixing, let stand at room temperature for 15min.

[0034] The silanized fiber optic biosensor was then immersed in a mixed solution of EDC / sulfo-NHS-rAb1 and incubated at room temperature for 2 hours. It was then washed three times with deionized water and dried in nitrogen for storage.

[0035] 3. Preparation of colloidal gold-labeled Legumain antibody solution

[0036] 1) Preparation of colloidal gold

[0037] Take 80 mL of ultrapure water into a clean conical flask and place a clean rotor inside. Heat the flask on a heatable magnetic stirrer until bubbles emerge evenly. Quickly add 1 mL of 1% chloroauric acid solution and heat to boiling. Immediately add 1 mL of 1.5% trisodium citrate. At this point, the color of the solution in the conical flask changes from grayish-black to wine-red. Continue heating for 7 minutes. Remove the conical flask and allow it to cool naturally at room temperature. Then, dilute to 100 mL using a volumetric flask and seal for later use.

[0038] 2) Antibody labeling

[0039] Take 1 mL of the colloidal gold prepared in step 1), add a 0.1 mol / L potassium carbonate solution, and adjust the pH of the solution to 7.5; then add 3 μL of 2 mg / mL Anti-Legumian-Ab2, mix at room temperature for 30 min, so that Anti-Legumain-Ab2 is fully bound to the colloidal gold particles, thus completing the labeling of Anti-Legumain-Ab2.

[0040] 3) Enclosed

[0041] Add bovine serum albumin solution to the colloidal gold-labeled Anti-Legumain-Ab2 prepared in step 2), control the final mass concentration of bovine serum albumin to 1.5%, and mix at room temperature for 30 min to block the exposed sites on the colloidal gold particles.

[0042] 4) Heavy suspension

[0043] The blocked colloidal gold-labeled antibody was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the precipitate was resuspended in 120 μL of resuspension buffer. The resuspension buffer consisted of phosphate buffer, bovine serum albumin, sucrose, trehalose, and Tween. The phosphate buffer concentration was 0.01 mol / L, the pH was 7.2, the final concentration of bovine serum albumin was 1.5%, the final concentration of sucrose was 3%, the final concentration of trehalose was 3%, and the final concentration of Tween was 2%. The phosphate buffer contained 0.26% Na2HPO4·12H2O and 0.044% NaH2PO4·2H2O.

[0044] Preparation method for 1L resuspension:

[0045] Take 600 ml of distilled water, and accurately weigh each component according to the following parameters: phosphate buffer concentration of 0.01 mol / L, pH of 7.2, final concentration of bovine serum albumin of 1.5%, final concentration of sucrose of 3%, final concentration of trehalose of 3%, and final concentration of Tween of 2%. Add the components to the distilled water and stir well. Pour the mixture into a 1 L volumetric flask and dilute to volume with distilled water. Set aside for later use.

[0046] 4. Detection of the tumor marker Legumain

[0047] A capture biosensor bound to Anti-Legumain-Ab1 was exposed to human serum samples to allow the biosensor to bind to the target molecule. This biosensor was then transferred to the wells of a sample plate as a detection sample. The capture biosensor bound to Anti-Legumain-Ab1 was equilibrated in PBST solution for 5 min and then transferred to the wells of the sample plate as a control sample. The sample plate containing both the detection and control samples was then oscillated at 1000 rpm at room temperature for 2 min, followed by a PBST wash at 1000 rpm for 20 s. The treated sample plate was then transferred to a colloidal gold-labeled Anti-Legumain-Ab2 solution and maintained for 2 min. The amount of colloidal gold-labeled Anti-Legumain-Ab2 deposited in each well was detected using biofilm interferometry. All biofilm detection data were read at 30°C and 1000 rpm.

[0048] The detection sensitivity of this embodiment reaches 10 pg / mL.

[0049] Comparative Example 1

[0050] The method described in Example 1 was followed, except that Anti-Legumian-Ab2 was not labeled with colloidal gold. The detection sensitivity of this comparative example reached 1 ng / μL.

[0051] Comparative Example 2

[0052] The comparison example uses a commonly used method in the prior art for detecting Legumain tumor markers. The method is as follows:

[0053] (1) Anti-Legumain-Ab2 antibody was labeled with colloidal gold.

[0054] (2) Preparation of sample pad

[0055] After the glass fiber membrane is cut, it is soaked in the sample pad treatment solution and dried to obtain the sample pad. The sample pad treatment solution consists of phosphate buffer, sucrose, sodium chloride, bovine serum albumin and Tween. The concentration of phosphate buffer is 0.01 mol / L, the pH is 7.2, the final mass concentration of sucrose is 3%, the concentration of sodium chloride is 300 mM, the final volume concentration of bovine serum albumin is 2%, the final volume concentration of Tween is 0.05%, the mass content of Na2HPO4·12H2O in phosphate buffer is 0.26%, the mass content of NaH2PO4·2H2O is 0.044%, and the balance is water.

[0056] (3) Preparation of binding pad

[0057] The colloidal gold-labeled Anti-Legumain-Ab1 from step (2) is evenly spread on a glass fiber membrane and dried to solidify the labeled antibody colloidal gold particles on the glass fiber membrane, thus obtaining the binding pad.

[0058] (4) Preparation of reaction pad

[0059] Nitrocellulose membranes were attached to PVC boards, and the above-mentioned colloidal gold-labeled Anti-Legumain-Ab2 was used as the detection line coating agent; goat anti-mouse IgG was used as the control line coating agent; the membrane was scratched with a gold spraying membrane at 1 μL / cm, and dried to obtain the reaction pad.

[0060] (5) Detection

[0061] The sample pad, conjugate pad, and reaction pad are assembled into a test strip, which is then used to test human serum samples.

[0062] The detection sensitivity of this comparative example reached 100 ng / mL.

[0063] The test results above show that the method of the present invention can effectively improve the detection sensitivity, reaching 10 pg / mL.

[0064] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A kit for detecting the tumor marker Legumain, characterized in that, The kit includes: a biosensor incorporating the antibody Anti-Legumian-Ab1, wherein the biosensor is a suspension prepared by immersing a surface-silanized fiber optic biosensor in a mixed solution of Anti-Legumian-Ab1 antibody, EDC, and NHS, followed by incubation; a colloidal gold-labeled Legumin antibody solution, wherein the antibody is a suspension prepared by labeling Anti-Legumian-Ab2 with colloidal gold and then blocking it with bovine serum albumin; and a PBST solution with a concentration of 0.01 mol / L and a pH of 7.

2.

2. The kit for detecting the tumor marker Legumain according to claim 1, characterized in that, The fiber optic biosensor is fabricated by the following method: using a multimode fiber, the far end of the fiber is used as the biolayer mounting surface, a high refractive index layer is coated on the biolayer mounting surface, and then a transparent silica film layer is coated on the high refractive index layer using physical vapor deposition; the other end of the fiber optic biosensor is coupled to a light source and an analysis device.

3. The kit for detecting the tumor marker Legumain according to claim 2, characterized in that, The multi-film optical fiber has a length of 10mm to 30mm and a diameter of 200μm to 1000μm.

4. The kit for detecting the tumor marker Legumain according to claim 2, characterized in that, 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 nm–800 nm.

5. The kit for detecting the tumor marker Legumain according to claim 1, characterized in that, The surface-silanized fiber optic biosensor is prepared by immersing the fiber optic biosensor in a 2% APTES ethanol solution after plasma surface activation.

6. The kit for detecting the tumor marker Legumain according to claim 1, characterized in that, The method for preparing the mixed solution of Anti-Legumian-Ab1 antibody with EDC and NHS is as follows: 10mM EDC and 10mM Sulfo-NHS are added to the activation buffer and mixed in equal volumes to form a 5mM / 5mM EDC / sulfo-NHS mixture. Anti-Legumian-Ab1 antibody is then added to achieve a final concentration of 4ug / ml.

7. The kit for detecting the tumor marker Legumain according to claim 1, characterized in that, The incubation step lasts for 2 hours.

8. The kit for detecting the tumor marker Legumain according to claim 1, characterized in that, The colloidal gold was prepared by placing a rotor in ultrapure water and heating it on a heatable magnetic stirrer until bubbles emerged uniformly. Then, 1 mL of 1% chloroauric acid solution was quickly added, and after heating to boiling, 1 mL of 1.5% trisodium citrate was immediately added, and heating continued for 7 minutes.

9. The kit for detecting the tumor marker Legumain according to claim 1, characterized in that, The PBST solution contained 0.26% Na2HPO4·12H2O and 0.044% NaH2PO4·2H2O by mass; the sodium chloride concentration was 300 mM; and the Triton X-100 concentration was 0.05%.

10. A method for detecting the tumor marker Legumain using the kit according to claim 1, characterized in that, The method includes: exposing a biosensor bound to the antibody Anti-Legumian-Ab1 to a human serum sample, allowing the biosensor to bind to a target molecule; transferring the biosensor bound to the target molecule to a colloidal gold-labeled Legumain antibody solution, allowing the biosensor bound to the target molecule to bind to the Anti-Legumian-Ab2 antibody; using a biosensor bound to the antibody Anti-Legumian-Ab1 after equilibration in PBST solution as a control, determining the amount of colloidal gold-labeled Anti-Legumian-Ab2 antibody deposited on the biosensor using a biomembrane interferometry method; and determining the amount of the target molecule on the biosensor based on the amount of colloidal gold-labeled Anti-Legumian-Ab2 antibody deposited.

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