CN110873800A - Glycosylated hemoglobin immunochromatographic test strip and preparation method and kit thereof - Google Patents
Glycosylated hemoglobin immunochromatographic test strip and preparation method and kit thereof Download PDFInfo
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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
- G01N33/723—Glycosylated 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
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- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- G01N33/577—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
Abstract
The invention relates to a glycosylated hemoglobin immunochromatographic test strip, a preparation method thereof and a kit. The test strip comprises a substrate, and a sample pad, a coating film and an absorption pad which are arranged on the substrate, wherein the sample pad, the coating film and the absorption pad are sequentially connected from one end to the other end of the substrate; the coating film is sequentially provided with a marking area, a detection area and a quality control area; the labeling area is coated with a coupling compound labeled by fluorescent latex particles, and the coupling compound is formed by coupling a first canine/feline HbA1c monoclonal antibody and a first complex; the detection area is coated with a second canine-feline HbA1c monoclonal antibody; the quality control region is coated with a second complex, and the first complex and the second complex can be specifically combined; the first canine/feline HbA1c monoclonal antibody can specifically bind to hemoglobin, and the second canine/feline HbA1c monoclonal antibody can specifically bind to glycated hemoglobin. The glycosylated hemoglobin immunochromatographic test strip provided by the invention realizes quantitative detection of canine and feline glycosylated hemoglobin, and has the characteristics of rapidness and simplicity.
Description
Technical Field
The invention relates to the technical field of immunochromatography detection, in particular to a glycosylated hemoglobin immunochromatography test strip, a preparation method thereof and a kit.
Background
Glycosylated hemoglobin (HbA1c) is a stable compound formed by glucose and a valine residue at the N-terminal end of the chain β of hemoglobin in vivo, hemoglobin is contacted with glucose to undergo non-enzymatic glycosylation, and an unstable Schiff base (HbA1c precursor) is formed at the early stage and then undergoes Amadori rearrangement to form HbA1 c.
The detection of HbA1c is the most representative means for diagnosing diabetes (the change of HbA1c of every 1 percent is approximately equal to the change of blood sugar of 2 mmol/L), can reflect the indexes of blood sugar control, treatment standard reaching and the like, and has important value in the aspects of diabetes prediction, diagnosis and judgment after cure.
The traditional methods for detecting the glycosylated hemoglobin include a latex agglutination reaction method, an ion exchange high pressure liquid chromatography method, a gold-labeled immunodiafiltration method, a chemiluminescence method and a radioimmunoassay. Wherein, the latex agglutination reaction method and the ion exchange high pressure liquid chromatography require expensive equipment and professional technicians, and can not realize field detection; the gold-labeled immunodiafiltration method and the radioimmunoassay method have low sensitivity, poor repeatability and accuracy, insufficient anti-interference capability and long detection time; the chemiluminescence method needs large instruments, the detection reagent needs to be refrigerated and stored, and the transportation cost is high. In addition, the above-mentioned detection method is mainly aimed at the detection of glycated hemoglobin in humans, and for animals with low requirements for the detection specification, the adoption of the above-mentioned detection method results in an excessively high technical cost and personnel cost, and is difficult to popularize in pet hospitals. Therefore, there is still a great limitation to the measurement of glycated hemoglobin in dogs and cats.
Disclosure of Invention
Therefore, a glycosylated hemoglobin immunochromatographic test strip, a preparation method thereof and a kit are needed to solve the problem that the detection of HbA1c of dogs and cats is limited.
A glycosylated hemoglobin immunochromatographic test strip comprises a substrate, and a sample pad, a coating film and an absorption pad which are arranged on the substrate, wherein the sample pad, the coating film and the absorption pad are sequentially connected from one end of the substrate to the other end;
the coating film is sequentially provided with a marking area, a detection area and a quality control area; the labeling area is coated with a coupling compound labeled by fluorescent latex particles, and the coupling compound is formed by coupling a first canine/feline HbA1c monoclonal antibody and a first complex; the detection area is coated with a second canine/feline HbA1c monoclonal antibody; the quality control region is coated with a second complex, and the first complex and the second complex can be specifically combined; the first canine/feline HbA1c monoclonal antibody can specifically bind to hemoglobin, and the second canine/feline HbA1c monoclonal antibody can specifically bind to glycated hemoglobin.
In one embodiment, the conjugation complex is covalently conjugated to the first complex of the first canine/feline HbA1c mab in a 1:0.1 molar ratio.
In one embodiment, the fluorescent latex particles are colloidal gold particles or magnetic bead particles.
In one embodiment, the fluorescent latex particles have a particle size of 0.1 μm to 1 μm; and/or the wavelength emitted by the fluorescent latex particles after being excited is 180 nm-800 nm.
In one embodiment, one of the first and second complexes is an antibody and the other is a secondary antibody corresponding to the antibody.
The invention provides a preparation method of the glycated hemoglobin immunochromatographic test strip, which is characterized by comprising the following steps:
applying a sample pad treatment solution to the sample pad and drying;
labeling a coupling compound of a first canine-feline HbA1c monoclonal antibody and a first complex by using fluorescent latex particles, preparing a labeling solution of the fluorescent latex particle-coupling compound, adding the labeling solution, a second canine-feline HbA1c monoclonal antibody solution and a second complex solution to a labeling area, a detection area and a quality control area of a coating film respectively, and drying;
and placing the dried sample pad, the dried coating film and the dried absorption pad on a substrate, and connecting the sample pad, the coating film and the absorption pad in sequence from one end to the other end of the substrate to obtain the glycosylated hemoglobin immunochromatography detection test strip.
In one embodiment, the method further comprises the step of preparing a labeling solution of the fluorescent latex particle-coupling complex:
preparing a coupling compound of a first dog and cat HbA1c monoclonal antibody and a first complex;
activating fluorescent latex particles, preparing an activated fluorescent latex particle solution, then uniformly mixing the coupling compound and the activated fluorescent latex particle solution, and centrifuging and then retaining a precipitate;
and re-dissolving the precipitate to obtain the labeling solution.
In one embodiment, the concentration of the activated fluorescent latex particles in the activated fluorescent latex particle solution is 800 μ g/100 μ L to 1200 μ g/100 μ L; and/or
The dosage ratio of the coupling compound to the activated fluorescent latex particle solution is (1-15) mu g:110 mu L.
In one embodiment, the labeling solution is added to the label region in an amount of 0.012. mu.L/cm2～0.024μL/cm2The concentration of the HbA1c monoclonal antibody solution of the second dog or cat is 0.5 mg/mL-1.0 mg/mL, and the adding amount in the detection area is 20 mu L/(27-35) cm2(ii) a And/or
The concentration of the second complex solution is 0.5 mg/mL-1.0 mg/mL, and the addition amount in the quality control area is 20 mu L/(27-35) cm2。
The invention also provides a kit, which comprises the glycosylated hemoglobin immunochromatographic test strip and a sample diluent, wherein the sample diluent contains a lytic agent.
The traditional methods for detecting the glycosylated hemoglobin include a latex agglutination reaction method, an ion exchange high pressure liquid chromatography method, a gold-labeled immunodiafiltration method, a chemiluminescence method and a radioimmunoassay. Wherein, the latex agglutination reaction method and the ion exchange high pressure liquid chromatography require expensive equipment and professional technicians, and can not realize field detection; the gold-labeled immunodiafiltration method and the radioimmunoassay method have low sensitivity, poor repeatability and accuracy, insufficient anti-interference capability and long detection time; the chemiluminescence method needs large instruments, the detection reagent needs to be refrigerated and stored, and the transportation cost is high. In addition, the above-mentioned detection method is mainly aimed at the detection of glycated hemoglobin in humans, and for animals with low requirements for the detection specification, the adoption of the above-mentioned detection method results in an excessively high technical cost and personnel cost, and is difficult to popularize in pet hospitals. Therefore, there is still a great limitation to the measurement of glycated hemoglobin in dogs and cats.
Based on the above, the inventor of the invention realizes immunochromatography detection by using fluorescent latex particles as a marker, and realizes quantitative detection of glycosylated hemoglobin by using a quantitative analyzer, thereby filling up the blank that no fluorescence immunochromatography quantitative detection of glycosylated hemoglobin of dogs and cats exists at present, improving the sensitivity and accuracy of detection, greatly shortening the quantitative detection time, and having the advantages of rapidness, simplicity, on-site performance, small interference and the like.
In the invention, a coupling compound is formed by firstly coupling a first canine/feline HbA1c monoclonal antibody and a first complex at a molar ratio of 1:0.1, and then the coupling compound is coupled with fluorescent latex particles to form a coupling compound labeled by the fluorescent latex particles (fluorescent coupling compound). When the fluorescent coupled compound is combined with the canine/feline HbA1c antigen in the detection sample to form a canine/feline HbA1c antigen-fluorescent coupled compound, the canine/feline HbA1c antigen-fluorescent coupled compound is captured by the coated second canine/feline HbA1c monoclonal antibody through chromatography at the detection zone of the coating film, and the more the fluorescent coupled compound is accumulated at the detection zone, the signal intensity of the fluorescent antibody reflects the amount of the captured HbA1 c. The detection area and the quality control area share one fluorescent latex particle, and the mode that two antibodies are combined in the ratio of 1:0.1 can amplify the signal quantity of a reaction system and effectively solve the problem of uneven combination of the antibodies and the latex particles, so that the sensitivity and the accuracy are greatly improved, and the linear range is widened.
And the marking area of the test strip is directly manufactured on the coating film, and the traditional marking pad is saved, so that the marking solution is directly added to the marking area of the coating film, the release speed of the fluorescent latex particles is high, the detection time is short, the detection can be realized only in 5min, and the detection sensitivity is improved. The storage temperature of the test strip is 4-30 ℃, refrigeration is not needed, transportation is convenient, and operation of a user is rapid.
In addition, the kit also comprises a sample diluent containing a cracking agent, and after the sample is diluted, red blood cells in the sample are broken, so that hemoglobin is released more sufficiently, and the detection sensitivity is further improved.
Drawings
FIG. 1 is a schematic structural diagram of a glycated hemoglobin immunochromatographic strip according to an embodiment of the present invention;
FIG. 2 is a linear regression plot of HbA1c calibrator concentration versus fitted concentration for example 1 of the present invention;
FIG. 3 is a linear regression plot of HbA1C calibrator concentration versus T/C value at different storage temperatures for example 1 of the present invention;
FIG. 4 is a linear regression plot of HbA1C calibrator concentration versus T/C value for inventive panel 1;
FIG. 5 is a linear regression plot of HbA1C calibrator concentration versus T/C value for experimental group 2 of the present invention;
FIG. 6 is a linear regression plot of HbA1C calibrator concentration versus T/C value for experimental groups 3 and 4 of the present invention.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings, which illustrate embodiments of the present invention. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present. The terms "vertical," "horizontal," "left," "right," and the like as used herein are for illustrative purposes only.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Referring to fig. 1, the present invention provides a glycated hemoglobin immunochromatographic strip 100 according to an embodiment, including a substrate 110, and a sample pad 120, a coating film 130, and an absorbent pad 140 disposed on the substrate, wherein the sample pad 120, the coating film 130, and the absorbent pad 140 are sequentially connected from one end of the substrate 110 to the other end;
the coating film 130 is sequentially provided with a marking area 131, a detection area 132 and a quality control area 133; the labeling area 131 is coated with a coupling compound labeled by fluorescent latex particles, and the coupling compound is formed by coupling a first canine/feline HbA1c monoclonal antibody and a first complex; the detection zone 132 is coated with a second canine/feline HbA1c monoclonal antibody. The quality control region 133 is coated with a second complex, and the first complex and the second complex can be specifically combined; the first canine/feline HbA1c monoclonal antibody can specifically bind to hemoglobin, and the second canine/feline HbA1c monoclonal antibody can specifically bind to glycated hemoglobin. In the invention, the canine/feline HbA1c monoclonal antibody is designated as canine/feline HbA1c monoclonal antibody.
In one embodiment, the conjugate complex is formed by covalently coupling a first canine/feline HbA1c monoclonal antibody to a first complex at a molar ratio of 1: 0.1. In the present invention, the first canine/feline HbA1c monoclonal antibody and the first complex can be covalently modified and then covalently linked using methods conventional in the art.
In one embodiment, the fluorescent latex particles may be colloidal gold particles or magnetic bead particles. The fluorescent latex microparticles have a diameter of 0.1 to 1 μm. The wavelength emitted by the fluorescent latex particles after being excited is 180 nm-800 nm.
In one embodiment, one of the first and second complexes is an antibody and the other is a secondary antibody corresponding to the antibody. If the first complex is a primary antibody and the second complex is a secondary antibody, the substitution is also possible. More specifically, in one example, the first complex is an anti-IgG antibody and the second complex is an IgG antibody. For example, the first complex is a goat anti-rabbit IgG antibody, a goat anti-mouse IgG antibody, a goat anti-chicken IgG antibody, or the like, and correspondingly, the second complex may be a rabbit IgG antibody, a mouse IgG antibody, a chicken IgG antibody, or the like.
In this embodiment, the substrate is preferably made of PVC, the sample pad may be made of glass fiber, the coating film may be made of nitrocellulose, and the absorbent pad is made of common absorbent filter paper. And (3) sequentially bonding a sample pad, a coating film and an absorption pad on the substrate in a mutual lap joint manner, and cutting into a proper width to obtain the test strip. The sample pad, the envelope, the absorbent pad and the substrate may be attached to each other by other means as long as the connection is achieved.
The storage temperature of the reagent strip is 4-30 ℃, cold storage is not needed, transportation is convenient, and operation of a user is rapid.
The invention also provides a preparation method of the glycated hemoglobin immunochromatographic test strip, which comprises the following steps:
s1, adding the sample pad treatment solution to the sample pad, and drying.
The method comprises the following specific steps: the sample pad treatment solution was sprayed onto the sample pad at a rate of 2-8. mu.L/cm, and dried overnight.
In one embodiment, the sample pad treatment solution is a sample pad diluent containing 0.5-2mg/mL mouse IgG, 0.5-1% Evans blue, and 0.1-2mg/mL RBC, and the sample pad diluent is 0.02M PB Buffer (PBS) containing 0.5-3% Tween-20 and 2-5% BSA (bovine serum albumin).
S2, labeling the coupling compound of the first canine/feline HbA1c monoclonal antibody and the first complex by using fluorescent latex particles, preparing a labeling solution of the fluorescent latex particle-coupling compound, adding the labeling solution, the second canine/feline HbA1c monoclonal antibody solution and the second complex solution to a labeling area, a detection area and a quality control area of the coating film respectively, and drying.
In one embodiment, the concentration of activated fluorescent latex particles in the activated fluorescent latex particle solution is 800 μ g/100 μ L to 1200 μ g/100 μ L. The dosage ratio of the coupling compound to the activated fluorescent latex particle solution is (1-15) mu g:110 mu L.
In one embodiment, the amount of marking solution added to the marking zone is 0.012 μ L/cm2～0.024μL/cm2The concentration of the HbA1c monoclonal antibody solution of the second dog or cat is 0.5 mg/mL-1.0 mg/mL, and the adding amount in the detection area is 20 mu L/(27-35) cm2. The preparation method of the HbA1c monoclonal antibody solution for the second dog or cat comprises the following steps: the second canine/feline HbA1c mab was diluted to a concentration of 0.5mg/mL to 1.0mg/mL with 2-5% sucrose in 0.01M PB (pH 7.4) buffer.
In one embodiment, the concentration of the second complex solution is 0.5mg/mL to 1.0mg/mL, and the addition amount in the quality control region is 20 μ L/(27 to 35) cm2. Preparing a second complex solution: the second complex was diluted to a concentration of 0.5mg/mL to 1.0mg/mL with 0.01M PB (pH 7.4) buffer containing 2-5% sucrose.
In one embodiment, the labeling solution of fluorescent latex particle-conjugate complexes is prepared as follows:
preparing a coupling compound of a first dog and cat HbA1c monoclonal antibody and a first complex;
activating the fluorescent latex particles to prepare an activated fluorescent latex particle solution, then uniformly mixing the coupling compound with the activated fluorescent latex particle solution, and centrifuging and then retaining the precipitate;
redissolving the precipitate to obtain a labeled solution.
In one embodiment, the specific steps for preparing the activated fluorescent microsphere solution are as follows: cleaning, activating and activating the fluorescent microspheres;
cleaning: absorbing the fluorescent microspheres in a centrifuge tube by using a pipette gun, centrifuging for 10-15min at 12000-15000rpm, discarding the supernatant, adding 1mL of cleaning buffer solution, and ultrasonically mixing uniformly. Further, the washing buffer was 0.01M Tris buffer.
And (3) activation: and adding an activation buffer solution into the washed fluorescent microspheres, and uniformly mixing for 20min on a rotary mixer. Further, the activation buffer was 0.01M Tris buffer containing 5-10mg/mL of N-hydroxysuccinimide and 5-10mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
And (3) stopping activation: centrifuging the activated fluorescent microspheres at 12000-15000rpm for 10-15min, discarding the supernatant, adding 1mL of stop solution, and ultrasonically mixing uniformly. Further, the stop solution was 0.1M MES buffer containing 0.1-0.5% methanol.
In one embodiment, the specific preparation steps of the labeling solution are:
adding the coupling compound into the activated fluorescent microsphere solution, uniformly mixing for 60-120min on a rotary mixer, adding the confining liquid, ultrasonically mixing uniformly, and uniformly mixing for 60min on the rotary mixer;
then centrifuging at 12000-15000rpm for 10-15min, discarding the supernatant, and retaining the precipitate;
and adding a microsphere diluent into the precipitate for redissolution, and ultrasonically mixing uniformly to obtain a labeling solution.
Further, the blocking solution is 0.01M Tris buffer solution containing 0.5-2% BSA, and the microsphere diluent is 0.02M PB buffer solution containing 0.5-3% Tween-20, 2-5% BSA and 3-10% sucrose.
The marking solution may be applied to the marking zone by spraying, spotting, uniform coating, or immersion. Similarly, the second canine/feline HbA1c mab solution and/or the second complexing agent solution can be applied to the respective zones by spraying, spotting, uniformly coating, or immersing.
S3, placing the dried sample pad, the dried coating film and the dried absorption pad on a substrate, and connecting the sample pad, the coating film and the absorption pad in sequence from one end of the substrate to the other end to obtain the glycosylated hemoglobin immunochromatography test strip.
The preparation processes of the sample pad and the coating film are not limited in sequence, and similarly, the preparation steps of the coupled compound of the first canine/feline HbA1c monoclonal antibody and the first complex and the activated fluorescent latex particle solution are not limited in sequence, and the sample pad and the coating film can be specifically manufactured according to needs, and can be simultaneously prepared if the preparation steps are simultaneously performed.
The preparation method of the glycosylated hemoglobin immunochromatographic test strip is simple in principle, simple and convenient in preparation process and convenient to popularize and use.
The invention also provides a kit, which comprises the glycosylated hemoglobin immunochromatographic test strip and a sample diluent, wherein the sample diluent contains a cracking agent. Further, the cracking agent is Sodium Dodecyl Sulfate (SDS), the sample diluent is a sample diluent containing 0.1-0.2% of SDS, 1.5-1.9% of NaCl, 0.5-1% of PVP, 0.5-1% of Tween, 0.1-0.2% of SDS, 1-2% of TX-100 and 0.02-0.05% of NaN30.01M PB buffer. After the detection sample is diluted by the diluent containing the cracking agent, red blood cells in the sample are broken, hemoglobin is released more fully, and the detection sensitivity is further improved.
The following are specific examples
Example 1
Preparation of marking solutions
1) Preparing an activated fluorescent microsphere solution:
cleaning: and (3) sucking 1mL of fluorescent microsphere solution (containing 10mg of fluorescent microspheres) into a centrifuge tube by using a pipette gun, centrifuging for 15min at 14000rpm, discarding supernatant, adding 1mL of 0.01M Tris buffer solution, and ultrasonically mixing uniformly to obtain the cleaned fluorescent microsphere solution.
And (3) activation: and adding 1mL of 0.01M Tris buffer solution containing 5mg/mL of N-hydroxysuccinimide and 5mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into the washed fluorescent microsphere solution, and uniformly mixing for 20min on a rotary mixer to obtain the activated fluorescent microsphere solution.
And (3) stopping activation: and (3) centrifuging the activated fluorescent microspheres for 15min at 14000rpm, discarding supernatant, adding 1mL of 0.1M MES buffer solution containing 0.1% methanol, and ultrasonically mixing uniformly to obtain an activated fluorescent microsphere solution.
2) Preparing a marking solution
Adding 10 mu g of a coupling compound formed by covalently coupling a first canine-feline HbA1c monoclonal antibody (labeled antibody) and goat anti-rabbit IgG in a molar ratio of 1:0.1 into the activated fluorescent microsphere solution, uniformly mixing for 120min on a rotary mixer, adding 1mL of 0.01M Tris buffer solution containing 0.5% BSA, ultrasonically mixing, and uniformly mixing for 60min on the rotary mixer.
Centrifuging at 14000rpm for 15min, discarding the supernatant, and retaining the precipitate;
and adding 1mL of 0.02M PB buffer solution containing 0.5% Tween-20, 2% BSA and 3% sucrose into the precipitate, redissolving, and ultrasonically mixing to obtain a labeling solution.
Preparation of coating film
The second canine/feline HbA1c mab (detection antibody) was diluted to 0.5mg/ml with 3% sucrose in 0.01M PB (pH 7.4) buffer as the working solution at the detection zone (T-line).
Rabbit IgG was diluted to 0.5mg/ml with 0.01M PB (pH 7.4) containing 3% sucrose as a working solution for the quality control region (C-line).
Adhering a nitrocellulose membrane on a PVC plate, scribing a marking area, a detection area and a quality control area on the membrane, wherein the scribing speed is 1.0 mu L/cm, placing at 50 ℃, and drying for 72 h.
Preparation of sample pad
Spraying the sample pad treatment solution on the glass fiber at the speed of 2 mu L/cm, and drying overnight; the sample pad treatment solution was a sample pad diluent containing 0.5mg/mL mouse IgG, 0.5% evans and 0.1mg/mL RBC, and the sample pad diluent was 0.02M PB buffer containing 0.5% tween-20 and 2% BSA.
And (3) sequentially and mutually bonding a sample pad and absorbent paper on the PVC plate, sequentially connecting the sample pad, the nitrocellulose membrane and the absorbent paper from one end to the other end of the PVC plate, and then cutting into a proper width to obtain the glycosylated hemoglobin immunochromatographic test strip.
Comparative example 1
The glycated hemoglobin fluorescence immunochromatographic test strip of comparative example 1 is similar to example 1 except that the first canine/feline HbA1c monoclonal antibody and the goat anti-rabbit IgG antibody coating are labeled with fluorescent latex microparticles in the labeling region (separately labeled), respectively.
Comparative example 2
The glycated hemoglobin fluorescence immunochromatographic test strip used in comparative example 2 is similar to that used in example 1, except that glass fibers (a label pad) are attached between the sample pad and the envelope, and a labeling solution is added to the labeling zone of the glass fibers.
The glycated hemoglobin fluorescence immunochromatographic test strip prepared in example 1 was subjected to performance evaluation
The instrument comprises the following steps: dry fluoroimmunoassay analyzer (model: AFS-1000) manufactured by Guangzhou blue Bo Biotechnology Ltd
HbA1c calibrator: is prepared from Berlol linear component quality control substance (secondary calibrator) by diluting with sample diluent containing 0.1% SDS, 1.5% NaCl, 0.5% PVP, 0.5% Tween, 0.1% SDS, 1% TX-100 and 0.02% NaN30.01M PB buffer.
1. Study of linearity
The test method comprises the following steps: the test paper prepared in example 1 was used to test HbA1c calibrants (4.4%, 6.1%, 9.7%, 14.7%, 18%) at different concentrations, the test was repeated 3 times for each calibrator, and the average value of each concentration was calculated, and the linear equation y ═ a + bx and the correlation coefficient r were calculated according to equation (1). And are provided with
And (3) performing curve fitting to obtain an equation by taking the concentration of the HbA1C calibrator as a horizontal coordinate and the T/C mean value as a vertical coordinate, obtaining a fitting concentration according to the equation, performing a linear regression curve by taking the concentration of the calibrator as the horizontal coordinate and the fitting concentration as the vertical coordinate, and calculating a correlation coefficient. The data for the linear regression curve are shown in table 1 below and the linear regression curve is shown in figure 2.
TABLE 1
The linear regression equation of fig. 2 shows that the test strip prepared by the invention has a good linear relation within the concentration range of 4% -18%, and the value of the correlation coefficient r reaches above 0.990.
2. Precision degree
The test strips prepared in example 1 were used to test the HbA1c calibrator at 4% and 14.7% concentrations, each of which was tested 20 times, and the results of the concentrations fitted are shown in table 2.
TABLE 2
The test results from table 2 show that: the test strip prepared by the invention has good batch precision, and the batch precision variation Coefficient (CV) of the two detected calibrators with the concentrations is less than 15%.
3. Accuracy of
The HbA1c calibration samples were measured at 4% and 14.7% concentrations, and the fitted concentration was obtained by measuring 3 times for each concentration, and the mean value and the relative deviation were calculated, and the results are shown in Table 3.
TABLE 3
| Concentration of |
4％ | 14.7％ |
| Item | Fitted concentration% | |
| 1 | 4.01 | 14.71 |
| 2 | 4.00 | 14.72 |
| 3 | 4.02 | 14.70 |
| Mean value | 4.01 | 14.71 |
| |
1％ | 1％ |
The results in table 3 show that: the test strip prepared by the invention has high detection accuracy, and the Bias% of the accuracy of the reference substances with two concentrations is within +/-15%.
4. Influence of temperature
The test strips prepared in example 1 were stored at 4 ℃, 25 ℃, 30 ℃ and 50 ℃, respectively, and then HbA1C calibrants at concentrations of 4%, 6%, 8% and 12% were measured, each concentration was measured 3 times for T and C values, and the average value of T/C was calculated, using the T/C value as ordinate and the concentration of HbA1C calibrant as abscissa, to establish an equation and fit a linear regression curve, the raw data of which are shown in table 4 below, the linear regression curves being shown as a, b, C and d in fig. 3, respectively.
TABLE 4
From the results shown in fig. 3 a-d: the test result of the test strip meets the requirement under the storage conditions of 4 ℃, 25 ℃, 30 ℃ and 50 ℃.
HbA1c calibrator: the sample is prepared by diluting from Berlol linear component quality control product (secondary calibrator) with sample diluent containing 0.1% SDS, 1.5% NaCl, 0.5% PVP, 0.5% Tween, 0.1% SDS, 1% TX-100 and 0.02% NaN30.01M PB buffer.
The test strips of example 1 and comparative example 1 were tested on 4 different concentrations of HbA1c calibrator (3 replicates at each concentration of 4%, 6%, 8.0%, 10%). Wherein, the detection line signal value and the quality control line signal value on the glycated hemoglobin fluorescence immunochromatographic strip provided in example 1 are respectively represented by T1 and C1, and the detection line signal value and the quality control line signal value on the glycated hemoglobin fluorescence immunochromatographic strip provided in comparative example 1 are respectively represented by T2 and C2; and (3) establishing an equation and fitting a linear regression curve by taking the ratio of the signal value of the detection line to the signal value of the quality control line as an abscissa and the concentration of the HbA1c calibrator as an ordinate, wherein the original data of the linear regression curve are shown in the following tables 5 and 6, and the linear regression curve is shown in a and b in fig. 4.
TABLE 5
TABLE 6
As shown by comparing a and b in FIG. 4, the sensitivity of the coupled labeled test strip is higher than that of the separately labeled test strip.
HbA1c calibrator: the sample diluent is prepared by diluting a Berlole linear component quality control product (a secondary calibrator) by using a sample diluent, wherein the sample diluent contains 0.1-0.2% of SDS, 1.5-1.9% of NaCl, 0.5-1% of PVP, 0.5-1% of Tween, 0.1-0.2% of SDS, 1-2% of TX-100 and 0.02-0.05% of NaN30.01M PB buffer.
The test strips of example 1 and comparative example 2 were tested on 4 different concentrations of HbA1c calibrator (3 replicates at each concentration of 4%, 6%, 8.0%, 12%). Wherein, the detection line signal value and the quality control line signal value on the glycated hemoglobin fluorescence immunochromatographic strip of example 1 are represented by T1 and C1, respectively, and the detection line signal value and the quality control line signal value on the glycated hemoglobin fluorescence immunochromatographic strip provided in comparative example 2 are represented by T2 and C2, respectively; and (3) establishing an equation and fitting a linear regression curve by taking the ratio of the signal value of the detection line to the signal value of the quality control line as an abscissa and the concentration of the HbA1c calibrator as an ordinate, wherein the original data of the linear regression curve are shown in the following tables 7 and 8, and the linear regression curve is shown in a and b in fig. 5.
TABLE 7
TABLE 8
From the detection time and comparison of a and b in FIG. 5, it was found that the detection speed was faster and the sensitivity was higher when the labeling solution was sprayed on the labeled region of the detection membrane than when the labeling solution was applied to the labeled region of the label pad.
Experimental group 3
HbA1c calibrator: the sample diluent is prepared by diluting a Berlole linear component quality control product (a secondary calibrator) with a sample diluent, wherein the sample diluent contains 0.1-0.2% of SDS, 1.5-1.9% of NaCl, 0.5-1% of PVP, 0.5-1% of Tween, 0.1-0.2% of SDS, 1-2% of TX-100 and 0.02 @0.05％NaN30.01M PB buffer.
The test strip of example 1 was tested on 4 different concentrations of HbA1c calibrator (3 replicates at each concentration of 4%, 6%, 8.0%, 11%). Wherein, the detection line signal value and the quality control line signal value on the glycated hemoglobin fluorescence immunochromatographic strip of example 1 are represented by T1 and C1, respectively; and (3) establishing an equation and fitting a linear regression curve by taking the ratio of the signal value of the detection line to the signal value of the quality control line as a horizontal coordinate and the concentration of the HbA1c calibrator as a vertical coordinate, wherein the original data of the linear regression curve is shown in the following table 9, and the linear regression curve is shown in a in fig. 6.
TABLE 9
Essentially the same as experimental group 3, except that no SDS was present in the sample dilution. And (3) establishing an equation and fitting a linear regression curve by taking the ratio of the quality control line signal value to the detection limit signal value of the detected sample as the abscissa and the concentration of the HbA1c calibrator as the ordinate, wherein the original data of the linear regression curve is shown in the following table 10, and the linear regression curve is shown in b in fig. 6.
As shown by the comparison of a and b in FIG. 6, the reaction is more complete when the sample diluent contains the cracking agent than when the sample diluent does not contain the cracking agent, and the test strip has higher detection sensitivity.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. The glycosylated hemoglobin immunochromatographic test strip is characterized by comprising a substrate, and a sample pad, a coating film and an absorption pad which are arranged on the substrate, wherein the sample pad, the coating film and the absorption pad are sequentially connected from one end to the other end of the substrate;
the coating film is sequentially provided with a marking area, a detection area and a quality control area; the labeling area is coated with a coupling compound labeled by fluorescent latex particles, and the coupling compound is formed by coupling a first canine/feline HbA1c monoclonal antibody and a first complex; the detection area is coated with a second canine/feline HbA1c monoclonal antibody; the quality control region is coated with a second complex, and the first complex and the second complex can be specifically combined; the first canine/feline HbA1c monoclonal antibody can specifically bind to hemoglobin, and the second canine/feline HbA1c monoclonal antibody can specifically bind to glycated hemoglobin.
2. The glycated hemoglobin immunochromatographic strip of claim 1, wherein said conjugated complex is covalently coupled to said first complex by a molar ratio of 1:0.1 of said first canine/feline HbA1c monoclonal antibody.
3. The glycated hemoglobin immunochromatographic strip according to claim 1, wherein the fluorescent latex particles are colloidal gold particles or magnetic bead particles.
4. The glycated hemoglobin immunochromatographic strip according to claim 1, wherein the fluorescent latex microparticles have a particle size of 0.1 to 1 μm; and/or the wavelength emitted by the fluorescent latex particles after being excited is 180 nm-800 nm.
5. The glycated hemoglobin immunochromatographic strip according to claims 1 to 4, wherein one of the first complex and the second complex is an antibody, and the other is a secondary antibody corresponding to the antibody.
6. A method for preparing a glycated hemoglobin immunochromatographic strip according to any one of claims 1 to 5, comprising the steps of:
applying a sample pad treatment solution to the sample pad and drying;
labeling a coupling compound of a first canine-feline HbA1c monoclonal antibody and a first complex by using fluorescent latex particles, preparing a labeling solution of the fluorescent latex particle-coupling compound, adding the labeling solution, a second canine-feline HbA1c monoclonal antibody solution and a second complex solution to a labeling area, a detection area and a quality control area of a coating film respectively, and drying;
and placing the dried sample pad, the dried coating film and the dried absorption pad on a substrate, and connecting the sample pad, the coating film and the absorption pad in sequence from one end to the other end of the substrate to obtain the glycosylated hemoglobin immunochromatography detection test strip.
7. The method for preparing a glycated hemoglobin immunochromatographic strip according to claim 6, further comprising a step of preparing a labeling solution of a fluorescent latex microparticle-conjugate complex:
preparing a coupling compound of a first dog and cat HbA1c monoclonal antibody and a first complex;
activating fluorescent latex particles, preparing an activated fluorescent latex particle solution, then uniformly mixing the coupling compound and the activated fluorescent latex particle solution, and centrifuging and then retaining a precipitate;
and re-dissolving the precipitate to obtain the labeling solution.
8. The method for preparing a glycated hemoglobin immunochromatographic strip according to claim 7, wherein the concentration of the activated fluorescent latex microparticles in the activated fluorescent latex microparticle solution is 800 μ g/100 μ L to 1200 μ g/100 μ L; and/or
The dosage ratio of the coupling compound to the activated fluorescent latex particle solution is (1-15) mu g:110 mu L.
9. The method for preparing a glycated hemoglobin immunochromatographic strip according to any one of claims 6 to 8, wherein the amount of the labeling solution added to the labeling region is 0.012 μ L/cm2～0.024μL/cm2The concentration of the HbA1c monoclonal antibody solution of the second dog or cat is 0.5 mg/mL-1.0 mg/mL, and the adding amount in the detection area is 20 mu L/(27-35) cm2(ii) a And/or
The concentration of the second complex solution is 0.5 mg/mL-1.0 mg/mL, and the addition amount in the quality control area is 20 mu L/(27-35) cm2。
10. A kit comprising the glycated hemoglobin immunochromatographic strip according to any one of claims 1 to 4 and a sample diluent containing a lysing agent.
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