Preparation method of reagent for detecting FOB (fecal occult blood) content in whole blood

Abstract

The invention relates to a preparation method of a reagent for detecting the content of fecal occult blood (FOB) in whole blood, which comprises the following steps: preparation of an anti-reagent: an alkaline phosphatase labeled fecal occult blood (FOB) antibody is formed by connecting alkaline phosphatase and a fecal occult blood (FOB) antibody through a cross-linking agent suberic acid succinimide ester, the solution concentration of the alkaline phosphatase labeled fecal occult blood (FOB) antibody is 0.8-1.5 mu g / mL, the pH value is 7-9, and the pH value is 7-9; the concentration of the magnetic particle reagent is 1-2 mg / mL; and preparation of a magnetic particle reagent: fully and uniformly mixing the magnetic bead concentrated solution, placing the magnetic bead concentrated solution in a magnetic field for 15 minutes after the magnetic bead concentrated solution is fully and uniformly mixed, absorbing a supernatant after carboxyl magnetic beads are completely settled, adding a phosphate buffer solution of which the volume is 5 times that of the carboxyl magnetic beads, mixing, placing the magnetic bead concentrated solution in the magnetic field, absorbing the supernatant after the magnetic beads are completely settled, and fixing the volume of the carboxyl magnetic bead solution to 10-50 mg / mL. In order to solve the problem of low detection sensitivity of the existing colloidal gold method, a magnetic particle chemiluminescence detection mode is adopted, so that the detection sensitivity can be greatly improved from 0.2 ug / ml of the colloidal gold method to 0.05 ng / ml of the detection method, and the detection sensitivity is improved by about 4,000 times.

Description

Technical Field

[0001] The present invention relates to the technical field of magnetic particle chemiluminescence detection, and specifically relates to a method for preparing a reagent used for detecting the content of fecal occult blood (FOB) in whole blood. Background Art

[0002] Fecal occult blood (FOB) (also known as fecal hidden blood) refers to a small amount of bleeding in the digestive tract. Red blood cells are digested and destroyed, and the appearance of feces has no abnormal changes, which cannot be confirmed by the naked eye or under a microscope. In the early stage of clinical digestive tract malignancies, 20% of patients may show a positive occult blood test. The positive rate of occult blood in advanced patients can reach more than 90%, and it can be continuously positive. For patients with digestive tract ulcers, the fecal occult blood (FOB) test is mostly intermittently positive. Dysentery, rectal polyps, hemorrhoids bleeding, etc. can also cause more red blood cells in the feces, resulting in a positive occult blood test. Therefore, the fecal occult blood (FOB) test can be used as an important test for detecting digestive tract bleeding caused by various reasons and early screening of digestive tract malignancies, and it is an effective method for detecting fecal occult blood.

[0003] Currently, the main method for clinically detecting fecal occult blood is the colloidal gold method.

[0004] The colloidal gold method uses colloidal gold as an indicator method and applies the principle of "double antibody sandwich method" to detect human hemoglobin in feces.

[0005] The lowest detection limit of the FOB colloidal gold method is 0.2 μg / ml, and the analytical sensitivity is poor. The detection rate of early small amounts of bleeding in the digestive tract of patients with digestive tract malignancies is significantly insufficient, which limits the early detection of digestive tract tumor patients. Summary of the Invention

[0006] The object of the present invention is to overcome the deficiencies of the existing technology and provide a method for preparing a reagent used for detecting the content of fecal occult blood (FOB) in whole blood. The preparation method of the anti-reagent is as follows: The fecal occult blood (FOB) antibody labeled with alkaline phosphatase is formed by connecting alkaline phosphatase and the fecal occult blood (FOB) antibody through a cross-linking agent, disuccinimidyl suberate. The solution concentration of the fecal occult blood (FOB) antibody labeled with alkaline phosphatase is 0.8 - 1.5 μg / mL, pH 7 - 9, and the concentration of the magnetic particle reagent (7) is 1 - 2 mg / mL. The preparation method of the magnetic particle reagent is as follows: fully mix the magnetic bead concentrate, place it in a magnetic field for 15 min after full mixing, aspirate the supernatant after all the carboxyl magnetic beads have settled, add a phosphate buffer solution 5 times the volume of the carboxyl magnetic beads, mix for 20 - 30 min, place it in a magnetic field, aspirate the supernatant after all the magnetic beads have settled, repeat the washing step three times, and make the volume of the carboxyl magnetic bead solution constant at 10 - 50 mg / mL; according to the mass ratio of magnetic beads: antibody = 100:1, add the treated antibody to the magnetic beads, react at 2 - 8 °C for 18 hours while maintaining a mixed state, wash three times with a phosphate buffer solution, make the volume constant at 1 mg / mL, and store at 2 - 8 °C for use, thus obtaining the magnetic particle reagent; The preparation method of the calibrator and the quality control product is as follows: the buffer solution of the calibrator and the quality control product is prepared by adding 50 - 70 g of bovine serum albumin, 1 - 3 mL of PC - 300, and 10 - 15 g of HEPES to 1 L of purified water. The calibrator and the quality control product have the same composition and are obtained by diluting the buffer solution of the fecal occult blood (FOB) antigen with the buffer solution containing bovine serum albumin; The preparation method of the luminescent substrate is as follows: the luminescent substrate is prepared by diluting the ALPS luminescent substrate in a ratio of 1:4 - 1:10 to a buffer solution formed by 0.1 - 1 M Tris, 0.1% sodium sulfite, 1% SDS, 0.3% lucigenin, 0.15% bovine serum albumin, and with a pH of 9.5; The preparation method of the washing solution is as follows: the washing solution is a Tris buffer solution containing about 1% Tween 20, with a pH of 8.0 ± 0.1.

[0007] A method for detecting the content of fecal occult blood (FOB) in whole blood, step one: lysis of the sample: add 200 μL of lysis agent to the sample to be tested for lysis; Step two: water bath: on the basis of step one, add 80 μL of anti - reagent and 80 μL of magnetic particle reagent, and then perform a water bath at 37 °C for 15 minutes; Step three: magnetic sleeve washing: on the basis of step two, add 300 μL of washing solution and repeat three times for magnetic sleeve elution; Step four: detection by chemiluminescence analyzer: on the basis of step three, add 180 μL of luminescent substrate and mix well, and then detect the luminescence intensity with a chemiluminescence analyzer.

[0008] A kit formed by a method for detecting the content of fecal occult blood (FOB) in whole blood, including the kit. The kit includes a calibrator, a quality control product, an anti - reagent, a magnetic particle reagent, a luminescent substrate, and a washing solution. The anti - reagent is an alkaline phosphatase - labeled fecal occult blood (FOB) antibody. The magnetic particle reagent is a non - fecal occult blood (FOB) antibody and carboxyl magnetic beads. The washing solution includes a buffer solution and a surfactant; the calibrator, the quality control product, the anti - reagent, the magnetic particle reagent, the luminescent substrate, and the washing solution are independently packaged.

[0009] The beneficial effects of the present invention are as follows: In view of the problem of low detection sensitivity of the existing colloidal gold method, the present invention adopts a magnetic particle chemiluminescence detection method, which can detect extremely low amounts of human hemoglobin in feces, greatly improving the detection sensitivity. It has been increased from 0.2 μg / ml of the colloidal gold method to 0.05 ng / ml of this detection method, an increase of nearly 4,000 times. The detection results are more accurate and worthy of being vigorously promoted and applied. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 It is a schematic structural diagram of the kit of the present invention.

[0011] In the figure: 1. Kit, 2. Luminescent substrate, 3. Cleaning solution, 4. Calibrator, 5. Quality control product, 6. Anti-reagent, 7. Magnetic particle reagent. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0012] The drawings are only for illustrative purposes and should not be construed as a limitation of this patent; for better illustration of this embodiment, some components in the drawings may be omitted, enlarged or reduced, which do not represent the dimensions of the actual product; for those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted.

[0013] Example 1, A method for detecting the content of fecal occult blood (FOB) in whole blood: Step 1: Lysis of the sample: Add 200 μL of lysis agent to the sample to be tested for lysis; Step 2: Water bath: On the basis of Step 1, add 80 μL of anti-reagent 6 and 80 μL of magnetic particle reagent 7, and then perform a water bath at 37 °C for 15 minutes; Step 3: Magnetic sleeve washing: On the basis of Step 2, add 300 μL of cleaning solution 3 and repeat three times for magnetic sleeve elution; Step 4: Detection by chemiluminescence analyzer: On the basis of Step 3, add 180 μL of luminescent substrate 2 and mix well, and then detect the luminescence intensity with a chemiluminescence analyzer.

[0014] Example 2, A method for preparing a reagent (I) for detecting the content of fecal occult blood (FOB) in whole blood: The preparation method of the anti-reagent 6 is as follows: The alkaline phosphatase-labeled fecal occult blood (FOB) antibody is formed by connecting alkaline phosphatase and fecal occult blood (FOB) antibody through a cross-linking agent disuccinimidyl suberate, and the solution concentration of the alkaline phosphatase-labeled fecal occult blood (FOB) antibody is 0.8 μg / mL, pH 7-9, and the concentration of the magnetic particle reagent 7 is 1 mg / mL; The preparation method of magnetic particle reagent 7 is as follows: fully mix the magnetic bead concentrate, place it in a magnetic field for 15 min after full mixing, aspirate the supernatant after all carboxyl magnetic beads have settled, add a phosphate buffer solution 5 times the volume of the carboxyl magnetic beads, mix for 20 - 30 min, place it in a magnetic field, aspirate the supernatant after all magnetic beads have settled, repeat the washing step three times, and make the volume of the carboxyl magnetic bead solution up to 10 mg / mL; according to the mass ratio of magnetic beads: antibody = 100:1, add the treated antibody to the magnetic beads, react at 2 - 8 °C for 18 hours while maintaining a mixed state, wash three times with phosphate buffer solution, make the volume up to 1 mg / mL, and store at 2 - 8 °C for later use to obtain the magnetic particle reagent; The preparation methods of calibrator 4 and quality control product 5 are as follows: the buffer solutions of the calibrator 4 and quality control product 5 are prepared by adding 50 g of bovine serum albumin, 1 mL of PC - 300, and 10 g of HEPES to 1 L of purified water. The calibrator 4 and quality control product 5 have the same composition and are obtained by diluting the buffer solution of fecal occult blood (FOB) antigen with the buffer solution containing bovine serum albumin; The preparation method of luminescent substrate 2 is as follows: the luminescent substrate 2 is prepared by diluting the ALPS luminescent substrate 2 at a ratio of 1:4 into a buffer solution formed by 0.1 M Tris, 0.1% sodium sulfite, 1% SDS, 0.3% lucigenin, 0.15% bovine serum albumin, and with a pH of 9.5; The preparation method of washing solution 3 is as follows: the washing solution 3 is a Tris buffer solution containing about 1% Tween 20, with a pH of 8.0 ± 0.1.

[0015] Example 3, a preparation method (two) of a reagent for detecting the content of fecal occult blood (FOB) in whole blood: The preparation method of anti - reagent 6 is as follows: the alkaline phosphatase - labeled fecal occult blood (FOB) antibody is formed by connecting alkaline phosphatase and fecal occult blood (FOB) antibody through a cross - linker disuccinimidyl suberate. The solution concentration of the alkaline phosphatase - labeled fecal occult blood (FOB) antibody is 1.5 μg / mL, with a pH of 7 - 9, and the concentration of the magnetic particle reagent 7 is 2 mg / mL; The preparation method of magnetic particle reagent 7 is as follows: fully mix the magnetic bead concentrate, place it in a magnetic field for 15 min after full mixing, aspirate the supernatant after all carboxyl magnetic beads have settled, add a phosphate buffer solution 5 times the volume of the carboxyl magnetic beads, mix for 20 - 30 min, place it in a magnetic field, aspirate the supernatant after all magnetic beads have settled, repeat the washing step three times, and make the volume of the carboxyl magnetic bead solution up to 50 mg / mL; according to the mass ratio of magnetic beads: antibody = 100:1, add the treated antibody to the magnetic beads, react at 2 - 8 °C for 18 hours while maintaining a mixed state, wash three times with phosphate buffer solution, make the volume up to 1 mg / mL, and store at 2 - 8 °C for later use to obtain the magnetic particle reagent; The preparation methods of calibrator 4 and quality control product 5 are as follows: The buffer solution of calibrator 4 and quality control product 5 is prepared by adding 70 g of bovine serum albumin, 3 mL of PC-300, and 15 g of HEPES to 1 L of purified water. The components of calibrator 4 and quality control product 5 are the same and are obtained by diluting the buffer solution of fecal occult blood (FOB) antigen with a buffer solution containing bovine serum albumin. The preparation method of luminescent substrate 2 is as follows: Luminescent substrate 2 is prepared by diluting the ALPS luminescent substrate 2 at a ratio of 1:10 into a buffer solution formed by Tris 1 M, sodium sulfite 0.1%, SDS 1%, lucigenin 0.3%, bovine serum albumin 0.15%, and with a pH of 9.5. The preparation method of washing solution 3 is as follows: Washing solution 3 is a Tris buffer solution containing approximately 1% Tween 20, with a pH of 8.0 ± 0.1 Example 4, a kit 1 formed by a method for detecting the content of fecal occult blood (FOB) in whole blood: The kit 1 includes calibrator 4, quality control product 5, anti-reagent 6, magnetic particle reagent 7, luminescent substrate 2, and washing solution 3. The anti-reagent 6 is an alkaline phosphatase-labeled fecal occult blood (FOB) antibody. The magnetic particle reagent 7 is a non-fecal occult blood (FOB) antibody and carboxyl magnetic beads. The washing solution 3 includes a buffer solution and a surfactant. Calibrator 4, quality control product 5, anti-reagent 6, magnetic particle reagent 7, luminescent substrate 2, and washing solution 3 are independently packaged.

[0016] Aiming at the problem of low detection sensitivity of the existing colloidal gold method, the present invention adopts a magnetic particle chemiluminescence detection method, which can greatly improve the detection sensitivity, from 0.2 μg / ml of the colloidal gold method to 0.05 ng / ml of this detection method, an increase of nearly 4,000 times.

[0017] Finally, it should be noted that the above embodiments are selected and described in detail to better illustrate the technical solutions of the present invention, rather than being intended to be limited to the shown details. Those skilled in the art who modify or make equivalent substitutions to the technical solutions of the present invention without departing from the purpose and scope of the technical solutions of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a reagent for detecting the content of fecal occult blood (FOB) in whole blood, characterized in that: The preparation method of the anti-reagent (6) is as follows: the alkaline phosphatase-labeled fecal occult blood (FOB) antibody is formed by linking alkaline phosphatase and the fecal occult blood (FOB) antibody through a cross-linking agent, disuccinimidyl suberate, and the concentration of the alkaline phosphatase-labeled fecal occult blood (FOB) antibody solution is 0.8-1.5 μg / mL, pH 7-9, and the concentration of the magnetic particle reagent (7) is 1-2 mg / mL; The preparation method of magnetic particle reagent (7) is as follows: the magnetic bead concentrate is fully mixed, and after being fully mixed, it is placed in a magnetic field for 15 minutes, and after the carboxyl magnetic beads are completely precipitated, the supernatant is removed, and a phosphate buffer solution of 5 times the volume of the carboxyl magnetic beads is added, and after mixing for 20-30 minutes, it is placed in a magnetic field and after the magnetic beads are completely precipitated, the supernatant is removed, and the washing step is repeated three times, and the carboxyl magnetic bead solution is fixed to 10-50 mg / mL; according to the mass ratio of magnetic beads: antibody = 100:1, the treated antibody is added to the magnetic beads, and the reaction is maintained at 2-8°C for 18 hours in a mixed state, and the phosphate buffer is used for washing three times, and the volume is fixed to 1 mg / mL, and stored at 2-8°C for use, thereby preparing the magnetic particle reagent; The preparation method of the calibration product (4) and the quality control product (5) is as follows: the buffer solution of the calibration product (4) and the quality control product (5) is prepared by adding 50-70g of bovine serum albumin, 1-3mL of PC-300 and 10-15g of HEPES to 1L of purified water, and the components of the calibration product (4) and the quality control product (5) are the same, and are obtained by diluting a buffer solution of fecal occult blood (FOB) antigen with a buffer solution containing bovine serum albumin; The preparation method of the luminescent substrate (2) is as follows: the luminescent substrate (2) is prepared by diluting the ALPS luminescent substrate (2) at a ratio of 1:4-1:10 into a buffer solution formed by Tris 0.1-1M, sodium sulfite 0.1%, SDS 1%, lucigenin 0.3%, bovine serum albumin 0.15%, and pH 9.5; The preparation method of the cleaning solution (3) is as follows: the cleaning solution (3) is a Tris buffer containing about 1% Tween 20, with a pH of 8.0±0.

1.

2. A method for detecting the content of fecal occult blood (FOB) in whole blood, characterized in that: Step 1: Sample lysis: Add 200 μL of lysis agent to lyse the sample to be tested; Step 2: Water bath: Based on step 1, add 80 μL of anti-reagent (6) and 80 μL of magnetic particle reagent (7), and then bathe in 37°C water for 15 minutes; Step 3: Magnetic elution: Based on step 2, add 300 μL of cleaning solution (3) and repeat the magnetic elution three times; Step 4: Chemiluminescence detection: Based on step 3, add 180 μL of luminescent substrate (2) and mix well, then use a chemiluminescence instrument to detect the luminescence intensity.

3. A kit for detecting the content of fecal occult blood (FOB) in whole blood, comprising a kit (1), characterized in that: The kit (1) comprises a calibrator (4), a quality control product (5), an anti-reagent (6), a magnetic particle reagent (7), a luminescent substrate (2) and a cleaning solution (3). The anti-reagent (6) is an alkaline phosphatase-labeled fecal occult blood (FOB) antibody, the magnetic particle reagent (7) is a non-fecal occult blood (FOB) antibody and carboxyl magnetic beads, and the cleaning solution (3) comprises a buffer solution and a surfactant; the calibrator (4), the quality control product (5), the anti-reagent (6), the magnetic particle reagent (7), the luminescent substrate (2) and the cleaning solution (3) are packaged separately.

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