CN117491624A - Human hemoglobin and transferrin co-detection reagent tube and preparation method and application thereof - Google Patents
Human hemoglobin and transferrin co-detection reagent tube and preparation method and application thereof Download PDFInfo
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- 102000001554 Hemoglobins Human genes 0.000 title claims abstract description 45
- 108010054147 Hemoglobins Proteins 0.000 title claims abstract description 45
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 38
- 102000004338 Transferrin Human genes 0.000 title claims abstract description 36
- 108090000901 Transferrin Proteins 0.000 title claims abstract description 36
- 239000012581 transferrin Substances 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title abstract description 18
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- 238000012360 testing method Methods 0.000 claims abstract description 37
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Classifications
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
- G01N33/726—Devices
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/79—Transferrins, e.g. lactoferrins, ovotransferrins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/795—Porphyrin- or corrin-ring-containing peptides
- G01N2333/805—Haemoglobins; Myoglobins
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Abstract
The invention discloses a human hemoglobin and transferrin co-detection reagent tube, a preparation method and application thereof, and particularly relates to the technical field of biological detection. The preparation method comprises the steps of preparing colloidal gold, preparing immune colloidal gold (colloidal gold-antibody complex), preparing immune colloidal gold pad, preparing immune nitrocellulose membrane, assembling detection test paper and the like. The test strip provided by the invention is based on the specific combination principle of the specific mouse anti-human Hb monoclonal antibody and the specific hemoglobin, and the specific combination principle of the mouse anti-human Tf monoclonal antibody and the specific transferrin, so that the test strip prepared by the monoclonal antibody has higher accuracy and sensitivity. The detection method of the detection device is simple and can be used as a household detection reagent; and the detection device is of a closed structure, so that products and an operation environment can be protected from pollution.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a human hemoglobin and transferrin co-detection reagent tube, a preparation method and application thereof.
Background
Fecal occult blood refers to small amount of hemorrhage of digestive tract, red blood cells are destroyed by digestion, the appearance of fecal is not changed abnormally, and hemorrhage can not be confirmed by naked eyes and under a microscope. Fecal occult blood examination is also known as fecal occult blood test. Is an experiment for examining hidden red blood cells or hemoglobin in feces, transferrin. This is a very useful diagnostic indicator for examining gastrointestinal bleeding.
The current method for detecting more gastrointestinal bleeding is a fecal occult blood reagent duplex detection method, which is a detection method for specifically detecting hemoglobin (Hb) and transferrin (Tf) in human feces. The combined detection Hb and Tf method has high sensitivity and strong specificity, and can play a role in complementary advantages in clinical significance of detecting gastrointestinal bleeding.
However, the existing novel method for detecting fecal occult blood by using TF/Hb duplex colloidal gold diagnostic test paper integrates an anti-human TF antibody and an anti-human Hb antibody on the same test paper belt, can synchronously detect TF and Hb without mutual interference, has complementary advantages, but has limited Hb detection (20 mug/ml can be detected and 1 mug/ml can not be detected through testing), and has further improved sensitivity and detection limit.
Chinese patent CN106680477a discloses an integrated device for fecal sampling and occult blood detection. However, the invention can only be used for placing a single test strip, has lower detection sensitivity and sample utilization rate, has lower transferrin content in blood than hemoglobin, has lower detection sensitivity on trace hemorrhage than hemoglobin, has easy denaturation of hemoglobin under the action of digestive enzymes and bacteria in gastrointestinal tracts, easily causes occult blood false negative, has strong antibacterial capability in intestinal tracts, and has stable property. The sensitivity and specificity of a single occult blood (FOB) diagnostic method are further improved.
Chinese patent CN110456048A discloses a multifunctional fecal collection and detection device. However, the invention has a plurality of test paper grooves, but the design is on the same plane, which is inconvenient for reading the result.
Chinese patent CN105467124a discloses a colloidal gold duplex test strip for detecting fecal occult blood and a preparation method thereof, and the test strip is exposed in the sampling and detecting process, which is unfavorable for home self-test.
Disclosure of Invention
Therefore, the invention provides a human hemoglobin and transferrin co-detection integrated device, which can realize one-time excrement collection, can detect two projects simultaneously, can effectively improve the clinical detection rate of fecal occult blood, and is simple and convenient to operate and beneficial to home detection. The user saves the time of going to the hospital and simultaneously knows whether the health of the user has related problems or not, and the whole device is economical and effective.
The invention is based on a double-antibody sandwich method, and utilizes a colloidal gold immunochromatography technology to rapidly detect hemoglobin and transferrin in human fecal samples.
The invention provides the test paper prepared by the monoclonal antibody based on the specific combination principle of the specific mouse anti-human Hb monoclonal antibody and the specific hemoglobin, and the mouse anti-human Tf monoclonal antibody and the specific transferrin, so that the detection reagent tube has higher accuracy and sensitivity.
In order to achieve the above object, the present invention provides the following technical solutions:
according to a first aspect of the present invention, there is provided a method for preparing a human hemoglobin and transferrin co-detection reagent tube, comprising:
preparing colloidal gold by adopting a trisodium citrate reduction method;
step two, a colloidal gold is utilized to mark the mouse anti-human Hb monoclonal antibody 1 and the mouse anti-human Tf monoclonal antibody 1, and a colloidal gold-mouse anti-human Hb monoclonal antibody 1 compound and a colloidal gold-mouse anti-human Tf monoclonal antibody 1 compound are respectively prepared;
step three, respectively coating the colloidal gold-mouse anti-human Hb monoclonal antibody 1 complex and the colloidal gold-mouse anti-human Tf monoclonal antibody 1 complex on a glass fiber membrane to prepare an immune colloidal gold pad;
preparing detection line (T line) solution by using a mouse anti-human Hb monoclonal antibody 2 and a mouse anti-human Tf monoclonal antibody 2, and preparing quality control line (C line) solution by using a sheep anti-mouse IgG;
spraying C, T lines on the nitrocellulose membrane to obtain an immune nitrocellulose membrane;
step five, respectively drying the immune colloidal gold pad and the immune nitrocellulose membrane;
sticking an immune colloidal gold pad, an immune nitrocellulose membrane, absorbent paper and a sample pad on a bottom lining to form a Hb detection test paper semi-finished product plate and a Tf detection test paper semi-finished product plate;
step six, cutting the two semi-finished plates into reagent strips, loading the reagent strips into different clamping grooves, firmly connecting a base with the clamping grooves with a detection tube, adding sample treatment liquid into the inner tube, and packaging to obtain the single Hb and Tf detection reagent tube.
Further, in the first step, the method for preparing colloidal gold includes: preparation of 0.01% HAuCl 4 Aqueous solution and 1.0% trisodium citrate aqueous solution, 0.01% HAuCl 4 Heating the aqueous solution to boiling, adding 1.0% trisodium citrate aqueous solution under stirring, and stirring while adding; and after boiling, continuously heating and boiling for 10-15 minutes until the solution is transparent purple red, thus obtaining the colloidal gold solution.
Further, in the second step, the preparation method of the colloidal gold-mouse anti-human Hb monoclonal antibody 1 complex includes: adding pH7.8 and 0.01M phosphate buffer solution into the colloidal gold obtained in the step one, and uniformly mixing; adding the anti-human Hb monoclonal antibody 1 solution under stirring, uniformly mixing, and reacting at room temperature; after the reaction is finished, adding 5% BSA as a protective agent, uniformly mixing, and reacting for 10 minutes at room temperature; centrifuging and purifying the reaction complex; adding pH7.8,0.01M phosphate buffer solution to the original volume, centrifuging, taking precipitate, and adding pH7.8,0.01M phosphate buffer solution to the final volume;
the preparation method of the colloidal gold-mouse anti-human Tf monoclonal antibody 1 complex comprises the following steps: the preparation method is exactly the same as that of the colloidal gold-mouse anti-human Hb monoclonal antibody 1 complex.
Further, in the third step, the preparation method of the immune colloidal gold pad comprises the following steps: and (3) diluting the purified colloidal gold-mouse anti-human Hb monoclonal antibody 1 complex and the colloidal gold-mouse anti-human Tf monoclonal antibody 1 complex by 10 times with a buffer solution, flatly placing a glass fiber membrane to be paved in a template, slowly and uniformly pouring a certain amount of diluted solution into the template, soaking uniformly, taking out and drying to obtain the immune colloidal gold pad.
Further, in the fourth step, the configuration of the detection line (T line) solution includes: according to the amount of the T line solution to be prepared and the concentrations of the hemoglobin monoclonal antibody 2 and the transferrin monoclonal antibody 2, the hemoglobin monoclonal antibody 2 and the transferrin monoclonal antibody 2 are calculated and measured, and the hemoglobin monoclonal antibody 2 and the transferrin monoclonal antibody 2 are diluted to the protein concentration of 1.0mg/ml by a coating buffer solution.
The configuration of the quality control line (C line) solution comprises: according to the amount of C line solution to be prepared and the concentration of goat anti-mouse IgG, the goat anti-mouse IgG stock solution is calculated and measured, and the stock solution is diluted to the protein concentration of 1.0mg/ml by the coating buffer solution.
On immunonitrocellulose membrane, T-line concentration: 1.0mg/ml; concentration of line C: 1.0mg/ml.
In the fifth step, the semi-finished plate is sequentially provided with a sample pad, an immune colloidal gold pad, an immune nitrocellulose membrane, absorbent paper, a detection line, a quality control line and a bottom lining according to the chromatography direction.
In the sixth step, the clamping groove is positioned on the outer layer of the inner tube, the inner tube is internally provided with sample treatment liquid, and the sample treatment liquid is not communicated with the clamping groove layer before the clamping groove layer is not used; the bottom of the inner tube is provided with a puncture inner wall, and the outer layer of the inner tube where the treatment liquid in the inner tube and the clamping groove are positioned is isolated.
The invention provides an application of a human hemoglobin and transferrin antibody co-detection reagent tube in preparing a fecal occult blood product.
The invention provides a co-detection device for human hemoglobin and transferrin, which is characterized by comprising a tube body (1), a sample processing cavity (2), a funnel tube section, a cover (3), a sampling rod (4), a partition piece (8), a detection reagent strip (9), a tearing strip (10), a base (11), a puncture needle (12), a receiving groove (13) and a sealing sheet (17);
the sample treatment cavity (2) is formed in the pipe body (1), the funnel pipe section is arranged in the sample treatment cavity (2), and the funnel pipe section divides the sample treatment cavity (2) into an upper liquid cavity (15) and a lower liquid cavity (16);
the lower end of the cover (3) is fixedly provided with the sampling rod (4), the sampling rod (4) is inserted into the sample processing cavity (2), and the cover (3) is used for being arranged at the upper open end of the sample processing cavity (2) in a sealing way;
the lower end of the sample processing cavity (2) is provided with the partition (8), the bottom surface of the partition (8) is provided with the sealed sealing sheet (17), the side wall of the partition (8) is provided with a plurality of detection reagent strips (9), the detection reagent strips (9) and the sample processing cavity (2) are separated from each other, the inner surface of the base (11) is upwards protruded with the needles (12), the needles (12) are arranged opposite to the sealing sheet (17) at intervals, the outer circumference side of the needles (12) is provided with the receiving grooves (13), and the lower ends of the detection reagent strips (9) are arranged in the receiving grooves (13) at the lower part of the sealing sheet (17) in a penetrating mode;
the Yi Sitiao (10) is arranged between the base (11) and the lower end outer side wall of the partition piece (8).
The invention has the following advantages:
the invention is based on a double-antibody sandwich method, and utilizes a colloidal gold immunochromatography technology to rapidly detect hemoglobin and transferrin in human fecal samples.
The detection device disclosed by the invention is integrated with quantitative collection, treatment and detection of the fecal sample, does not need to send the sample alone, is simple and convenient to operate, can be used for detecting at home at any time, is closed, does not need to worry about spilling of the sample, and is not influenced by the odor of the fecal sample in the detection process.
According to the detection process of the detection reagent tube, the lower easy-tearing strip is torn off, the sealing piece is pressed to puncture so that the sample treatment liquid enters the bottom receiving groove, the sample treatment liquid starts to contact the test strip, misoperation is not easy to occur, and in addition, the sealing piece is elliptical, so that the surface tension can be reduced, and the liquid can flow down conveniently.
The device can simultaneously place the hemoglobin and transferrin detection test strips, and simultaneously detect the hemoglobin and transferrin to play the role of complementary advantages, reduce missed detection and improve clinical detection rate of fecal occult blood.
The test strip provided by the invention is based on the specific combination principle of the specific mouse anti-human Hb monoclonal antibody and the specific hemoglobin, and the specific combination principle of the mouse anti-human Tf monoclonal antibody and the specific transferrin, so that the test strip prepared by the monoclonal antibody has higher accuracy and sensitivity.
The co-detection reagent tube can simultaneously have multiple functions of fecal sample collection, dissolution, detection of multiple colloidal gold test strips, user privacy protection and the like; according to the invention, the test paper groove capable of placing a plurality of test papers is designed, a plurality of detection test papers can be installed in the test paper groove, and various detection combinations can be realized; through the circular lumen flow direction test paper groove with easy tear strip control solution follow main part container pipe, can realize that the operation flow is more simple and convenient, avoid the detection inefficacy that the maloperation leads to.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.
The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the ambit of the technical disclosure.
Fig. 1 is a schematic structural diagram of a test strip according to embodiment 1 of the present invention; wherein, the sample pad A, the immune colloidal gold pad B, the immune nitrocellulose membrane C, the absorbent paper D, the detection line E, the quality control line F and the substrate G.
Fig. 2 is an exploded view of a closed joint detection integrated device according to some embodiments of the present invention.
Fig. 3 is an internal structure diagram of a closed joint detection integrated device according to some embodiments of the present invention.
Fig. 4 is a bottom view of a closed joint detection integrated device according to some embodiments of the present invention.
Fig. 5 is a top view of a closed type joint detection integrated device according to some embodiments of the present invention.
Fig. 6 is a cross-sectional view of A-A of a closed joint detection integrated device according to some embodiments of the present invention.
Fig. 7 is a B-B cross-sectional view of a closed joint detection integrated device according to some embodiments of the present invention.
In the figure: 1. the device comprises a tube body, 2, a sample processing cavity, 3, a cover, 4, a sampling rod, 5, a slot, 6, a splicing bulge, 7, a sampling sealing ring, 8, a separating piece, 9, a detection reagent strip, 10, an easy-to-tear strip, 11, a base, 12, a puncture needle, 13, a receiving groove, 14, a bottom sealing ring, 15, an upper liquid cavity, 16, a lower liquid cavity, 17 and a sealing sheet.
Detailed Description
Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The embodiment provides a preparation method of a human hemoglobin and transferrin co-detection reagent tube, which comprises the following steps:
1) Screening preparation process of specific mouse anti-human Hb/Tf monoclonal antibody
a. Immunization: human hemoglobin (Hb), human transferrin (Tf).
b. Preparation of murine anti-human Hb monoclonal antibody: after full emulsification, the human hemoglobin is added with Freund's complete adjuvant, and then multi-point injection is carried out in the back skin of a BALB/c mouse, after 4w interval, the purified human hemoglobin is added with Freund's incomplete adjuvant for secondary immunization, and 3d tail vein is reinforced for one needle before fusion. The method comprises the steps of fusing mouse myeloma cells (SP 2/0-Ag 14) with immune mouse spleen cells under the assistance of PEG, screening target substances of hybridoma cells of high-titer antibodies by using purified human hemoglobin as indirect ELISA, cloning the screened cells for multiple times by a limiting dilution method, expanding seeds, and preserving. And injecting the expanded hybridoma cells into a BALB/c mouse intraperitoneally, collecting ascites after 10-14 d, and centrifuging to obtain supernatant, namely the anti-human hemoglobin-McAb.
c. Preparation of murine anti-human Tf monoclonal antibody: after full emulsification, the human transferrin is added with Freund's complete adjuvant, and then multi-point injection is carried out in the back skin of a BALB/c mouse, after 4w interval, the purified human transferrin is added with Freund's incomplete adjuvant for secondary immunization, and 3d tail vein is reinforced for one needle before fusion. The method comprises the steps of fusing mouse myeloma cells (SP 2/0-Ag 14) with immune mouse spleen cells under the assistance of PEG, screening target substances of hybridoma cells of high-titer antibodies by using purified human transferrin as indirect ELISA, cloning the screened cells for multiple times by a limiting dilution method, expanding seeds, and preserving. And injecting the expanded hybridoma cells into a BALB/c mouse intraperitoneally, collecting ascites after 10-14 d, and centrifuging to obtain supernatant, namely the anti-human transferrin-McAb.
2) Anti-human Hb/Tf monoclonal antibodies are commercially available.
3) The preparation process of the colloidal gold comprises the following steps: preparing 0.01% HAuCl4 aqueous solution and 1.0% trisodium citrate aqueous solution respectively, adding 0.01% HAuCl 4 1000ml of the aqueous solution was placed in a beaker or flask and heated to boiling, and 6.5ml of 1.0% trisodium citrate aqueous solution was added with stirring. After boiling, heating and boiling are continued for 10-15 minutes until the solution is transparent and purple.
The colloidal gold particles are prepared to be 40-70 nm in size, and after the solution is cooled to room temperature, water is added to restore the original volume so as to ensure the consistency of the colloidal gold concentration.
The maximum absorption peak is found at 520-545 nm when scanned by spectrophotometry.
4) Determination of optimal pH for binding of anti-human Hb/Tf monoclonal antibody-1 to colloidal gold
Taking 8 1.5ml test tubes, adding 100 μL 3) of the prepared colloidal gold, respectively, using 0.1. 0.1mLK 2 CO 3 And 0.1M HCl to adjust pH to 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, respectively; mu.L of anti-human Hb/Tf monocgram at a concentration of 1mg/mL was added to each tubeThe hump antibody-1 is mixed and placed at room temperature for 10 minutes to 15 minutes.
The optimum pH was determined to be 7.8.
5) Preparation of colloidal gold-anti-human Hb/Tf monoclonal antibody 1 complex: taking 1000mL of colloidal gold, adding 100mL of phosphate buffer solution with pH of 7.8 and 0.01M, and uniformly mixing; under stirring, respectively adding 10mL of anti-human Hb/Tf monoclonal antibody 1 solution into the prepared colloidal gold solution, uniformly mixing, and reacting at room temperature for 10 minutes; after the reaction is finished, 5% BSA (serving as a protective agent) is added to make the final concentration of BSA in the solution be 0.01%, and the mixture is uniformly mixed and reacted for 10 minutes at room temperature; centrifuging and purifying the reaction complex at 10000 rpm; adding pH7.8,0.01M phosphate buffer to the original volume, centrifuging, collecting precipitate, and adding pH7.8,0.01M phosphate buffer to the final volume of 90ml. The optimal anti-human Hb/Tf monoclonal antibody 1 labeling amount per 1000mL of colloidal gold was 20mg.
The configuration of the detection line (T-line) solution includes: according to the amount of the T line solution to be prepared and the concentrations of the hemoglobin monoclonal antibody 2 and the transferrin monoclonal antibody 2, the hemoglobin monoclonal antibody 2 and the transferrin monoclonal antibody 2 are calculated and measured, and the hemoglobin monoclonal antibody 2 and the transferrin monoclonal antibody 2 are diluted to the protein concentration of 1.0mg/ml by a coating buffer solution.
The configuration of the quality control line (C line) solution comprises: according to the amount of C line solution to be prepared and the concentration of goat anti-mouse IgG, the goat anti-mouse IgG stock solution is calculated and measured, and the stock solution is diluted to the protein concentration of 1.0mg/ml by the coating buffer solution.
6) Preparing an immune colloidal gold pad: taking purified colloidal gold-anti-human Hb/Tf monoclonal antibody 1 complex immune colloidal gold solution, diluting by 10 times with buffer solution, flatly placing a glass fiber membrane to be laid in a template, taking a certain amount of diluted solution, slowly and uniformly pouring the diluted solution in the template, soaking the diluted solution uniformly, taking out and drying the diluted solution.
Each glass fiber membrane (300 mm. Times.220 mm gauge) requires 30mL of the immunocolloid Jin Xishi solution.
7) Preparation of immune nitrocellulose membrane: and (3) sticking a nitrocellulose membrane on a PVC plate, wherein the optimal coating concentration of the anti-human Hb/Tf monoclonal antibody 2 fixed on a detection line (T line) is 1.0mg/mL, the optimal coating concentration of the C line goat anti-mouse IgG is 1.0mg/mL, and drying the sprayed membrane.
8) As shown in fig. 1, an immune colloidal gold pad, an immune nitrocellulose membrane, water absorbing paper and a sample pad are stuck on a bottom lining sheet to form a Hb detection test paper semi-finished product plate and a Tf detection test paper semi-finished product plate;
9) The two semi-finished plates are cut into reagent strips, the reagent strips are arranged in different clamping grooves, a base with the clamping grooves is firmly connected with a detection tube, a sample treatment liquid is added into an inner tube, and the single Hb and Tf detection reagent tube is obtained after packaging.
The test strip is based on a double-antibody sandwich method, and utilizes a colloidal gold immunochromatography technology to rapidly detect hemoglobin and transferrin in a human fecal sample, and a detection line is generated in a detection line area on a membrane.
The T position of the test strip detection line is respectively coated with a mouse anti-human hemoglobin monoclonal antibody 2 and a mouse anti-human transferrin monoclonal antibody 2, and in addition, a glass fiber membrane is correspondingly coated with a mouse anti-human hemoglobin monoclonal antibody 1-colloidal gold compound and a mouse anti-human transferrin monoclonal antibody 1-colloidal gold compound. When the sample mixed solution contacts with the test strip, such as the sample contains hemoglobin or transferrin, and the content of the hemoglobin or transferrin is not lower than the minimum detection limit of the sample, the substance to be detected is combined with the colloidal gold compound coated on the glass fiber film, and a red line appears at the position of the corresponding detection line T in the chromatographic process. If the sample does not contain the corresponding substance to be detected or the content of the substance to be detected is lower than the lowest detection limit of the sample, the position of the detection line is not provided with red lines, and the negative result is indicated. The quality control line (C line) is coated with goat anti-mouse IgG polyclonal antibody, and red lines appear on the quality control line no matter what condition is detected, so as to prove that the test strip works normally.
Example 2
As shown in fig. 2-7, the present embodiment provides a device for a co-detection reagent tube of human hemoglobin and transferrin:
comprises a tube body 1, a sample processing cavity 2, a funnel tube section, a cover 3, a sampling rod 4, a partition piece 8, a detection reagent strip 9, a tearing strip 10, a base 11, a puncture needle 12, a receiving groove 13 and a sealing piece 17; a sample processing cavity 2 is arranged in the tube body 1, a funnel tube section is arranged in the sample processing cavity 2, and the funnel tube section divides the sample processing cavity 2 into an upper liquid cavity 15 and a lower liquid cavity 16; the lower end of the cover 3 is fixedly provided with a sampling rod 4, the sampling rod 4 is inserted into the sample processing cavity 2, and the cover 3 is used for sealing the upper open end of the sample processing cavity 2; the lower end of the sample processing cavity 2 is provided with a partition 8, the bottom surface of the partition 8 is provided with a closed sealing sheet 17, the side wall of the partition 8 is provided with a plurality of detection reagent strips 9, the detection reagent strips 9 and the sample processing cavity 2 are mutually separated, the inner surface of the base 11 is upwards protruded with a puncture needle 12, the puncture needles 12 are arranged opposite to the sealing sheet 17 at intervals, the outer periphery side of the puncture needles 12 is provided with a receiving groove 13, and the lower end of the detection reagent strip 9 is penetrated in the receiving groove 13 at the lower part of the sealing sheet 17; a tear-off strip 10 is arranged between the base 11 and the outer side wall of the lower end of the partition 8; the separable limiting device easy-to-tear strip 10 is positioned at the base part of the device, is independent of the sample treatment part, and can be used for preventing misoperation by tearing off the limiting device and pressing the pipe body to enable sample treatment liquid to enter the base reaction tank.
The outer surface of the pipe body 1 is an elliptic curved surface. The volume of the lower liquid chamber 16 is greater than the volume of the upper liquid chamber 15.
The device also comprises a plug-in protrusion 6 and a slot 5, wherein two oppositely arranged slots 5 are formed at the upper open end of the sample processing cavity 2, two plug-in protrusions 6 are arranged on the lower surface of the cover 3 in a protruding manner, and the plug-in protrusions 6 are in plug-in fit with the slots 5.
The device still includes sampling sealing washer 7, and sampling rod 4 is located the lateral wall of funnel pipe section hole department and is provided with sampling sealing washer 7, and the surface butt of sampling sealing washer 7 is on the hole inside wall in the funnel pipe section.
The top of the lancet 12 is provided with a pointed end for puncturing the sealing plate 17.
Example 3
Method for detecting faeces using the device of example 2:
1. the cover is connected with the sampling rod, the cover is pinched to vertically pull out the sampling rod upwards, and the free head end of the sampling rod is used for collecting the excrement, so that the threaded part of the sampling rod is fully filled with the excrement.
2. After the excrement is collected, the sampling rod is inserted back into the internal circular sample treatment tube, the cover is pressed and fastened, and the device is shaken to enable the excrement to be fully dissolved in the solution;
3. tearing the bottom easy-tearing strip, pressing the bottom, and enabling the puncture needle to puncture the sealing sheet upwards so that the sample mixed solution in the sample treatment tube enters the receiving groove of the base;
4. the receiving groove is of an inclined surface structure, so that the sample mixed solution is better contacted with the test strip, and the mixed solution starts to generate color bands on the running board under the siphoning action of the glass fiber of the test strip;
5. and the result can be judged through the observation window after waiting for 10-20 minutes, and the result is not judged after 20 minutes in order to ensure the accuracy of the result.
Positive: and a red line appears at the positions of a detection line (T line) and a quality control line (C line) of the result observation window respectively, which indicates that human hemoglobin exists in the sample.
Negative: only a red line appears at the position of the quality control line (C line) to indicate that no human hemoglobin exists in the sample.
Invalidation: the quality control line position has no red line, which indicates that the result is invalid and the retry should be performed.
The detection result judgment rule of the transferrin detection test strip is the same as that of the hemoglobin detection test strip.
While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (5)
1. A method for preparing a human hemoglobin and transferrin co-detection reagent tube, which is characterized by comprising the following steps:
preparing colloidal gold by adopting a trisodium citrate reduction method;
step two, a colloidal gold is utilized to mark the mouse anti-human Hb monoclonal antibody 1 and the mouse anti-human Tf monoclonal antibody 1, and a colloidal gold-mouse anti-human Hb monoclonal antibody 1 compound and a colloidal gold-mouse anti-human Tf monoclonal antibody 1 compound are respectively prepared;
step three, respectively coating the colloidal gold-mouse anti-human Hb monoclonal antibody 1 complex and the colloidal gold-mouse anti-human Tf monoclonal antibody 1 complex on a glass fiber membrane to prepare an immune colloidal gold pad;
preparing detection line solutions by using a mouse anti-human Hb monoclonal antibody 2 and a mouse anti-human Tf monoclonal antibody 2 respectively, and preparing quality control line solutions by using sheep anti-mouse IgG;
spraying a detection line and a quality control line on the nitrocellulose membrane to obtain an immune nitrocellulose membrane;
step five, respectively drying the immune colloidal gold pad and the immune nitrocellulose membrane;
sticking an immune colloidal gold pad, an immune nitrocellulose membrane, absorbent paper and a sample pad on a bottom lining to form a Hb detection test paper semi-finished product plate and a Tf detection test paper semi-finished product plate;
step six, cutting the two semi-finished plates into reagent strips, loading the reagent strips into different clamping grooves, firmly connecting a base with the clamping grooves with a detection tube, adding sample treatment liquid into the inner tube, and packaging to obtain the single human hemoglobin and transferrin co-detection reagent tube.
2. The method for preparing a co-detection reagent tube for human hemoglobin and transferrin according to claim 1, wherein in the fifth step, the semi-finished plate comprises a sample pad, an immune colloidal gold pad, an immune nitrocellulose membrane, a water absorbing paper, a detection line, a quality control line and a substrate in sequence according to the chromatography direction.
3. The method for preparing a tube for co-examination of human hemoglobin and transferrin according to claim 1, wherein in the sixth step, the clamping groove is positioned on the outer layer of the inner tube, the inner tube is internally provided with a sample treatment liquid, and the sample treatment liquid is not communicated with the clamping groove layer before the use; the bottom of the inner tube is provided with a puncture inner wall, and the outer layer of the inner tube where the treatment liquid in the inner tube and the clamping groove are positioned is isolated.
4. The co-detection reagent tube for human hemoglobin and transferrin is used in preparing fecal occult blood product.
5. The co-detection device for the human hemoglobin and the transferrin is characterized by comprising a tube body (1), a sample processing cavity (2), a funnel tube section, a cover (3), a sampling rod (4), a partition piece (8), a detection reagent strip (9), an easy-to-tear strip (10), a base (11), a puncture needle (12), a receiving groove (13) and a sealing sheet (17);
the sample treatment cavity (2) is formed in the pipe body (1), the funnel pipe section is arranged in the sample treatment cavity (2), and the funnel pipe section divides the sample treatment cavity (2) into an upper liquid cavity (15) and a lower liquid cavity (16);
the lower end of the cover (3) is fixedly provided with the sampling rod (4), the sampling rod (4) is inserted into the sample processing cavity (2), and the cover (3) is used for being arranged at the upper open end of the sample processing cavity (2) in a sealing way;
the lower end of the sample processing cavity (2) is provided with the partition (8), the bottom surface of the partition (8) is provided with the sealed sealing sheet (17), the side wall of the partition (8) is provided with a plurality of detection reagent strips (9), the detection reagent strips (9) and the sample processing cavity (2) are separated from each other, the inner surface of the base (11) is upwards protruded with the needles (12), the needles (12) are arranged opposite to the sealing sheet (17) at intervals, the outer circumference side of the needles (12) is provided with the receiving grooves (13), and the lower ends of the detection reagent strips (9) are arranged in the receiving grooves (13) at the lower part of the sealing sheet (17) in a penetrating mode;
the Yi Sitiao (10) is arranged between the base (11) and the lower end outer side wall of the partition piece (8).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311440653.7A CN117491624A (en) | 2023-11-01 | 2023-11-01 | Human hemoglobin and transferrin co-detection reagent tube and preparation method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311440653.7A CN117491624A (en) | 2023-11-01 | 2023-11-01 | Human hemoglobin and transferrin co-detection reagent tube and preparation method and application thereof |
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| CN117491624A true CN117491624A (en) | 2024-02-02 |
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| CN202311440653.7A Pending CN117491624A (en) | 2023-11-01 | 2023-11-01 | Human hemoglobin and transferrin co-detection reagent tube and preparation method and application thereof |
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| CN (1) | CN117491624A (en) |
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