CN113125748A - Kit for detecting heart-type fatty acid binding protein - Google Patents

Kit for detecting heart-type fatty acid binding protein Download PDF

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CN113125748A
CN113125748A CN202110340279.8A CN202110340279A CN113125748A CN 113125748 A CN113125748 A CN 113125748A CN 202110340279 A CN202110340279 A CN 202110340279A CN 113125748 A CN113125748 A CN 113125748A
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buffer solution
concentration
reagent
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kit
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王庆国
张欣
高永亮
宿明明
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Weifang 3v Biological Engineering Group Co ltd
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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials
    • 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
    • G01N33/543—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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Abstract

The invention relates to the field of in vitro diagnosis, in particular to a kit for detecting heart-type fatty acid binding protein, which comprises a reagent R1, a reagent R2 and a calibrator, wherein the reagent R1 comprises a first buffer solution, a biotin-labeled anti-human H-FABP antibody, a FITC-labeled anti-human H-FABP antibody, a first stabilizer, a first preservative and a surfactant; reagent R2, wherein the reagent R2 comprises a second buffer solution, streptavidin modified latex microspheres, anti-FITC modified latex microspheres, a salt ion compound, a second stabilizing agent, a chelating agent and a second preservative; the calibrator comprises a third buffer solution, H-FABP recombinant protein, a third stabilizer and a third preservative. The kit has the advantages of high sensitivity, high accuracy and wide linear range.

Description

Kit for detecting heart-type fatty acid binding protein
Technical Field
The invention relates to the field of in-vitro diagnosis, in particular to a kit for detecting heart-type fatty acid binding protein.
Background
Acute Coronary Syndrome (ACS) is one of the important acute events in coronary heart disease, and is classified into Acute Myocardial Infarction (AMI), Unstable Angina (UA), and Sudden Cardiac Death (SCD). The cardiac marker plays an important role in the diagnosis and prognosis processes of ACS, and the ACS has the characteristics of acute morbidity and great harm, so that an early marker with high specificity is selected, and early diagnosis and timely treatment are of great significance to ACS patients. Commonly used cardiac markers are troponin (cTn), including (cTnI and cTnT), Myoglobin (MYO) and creatine kinase isozyme (CK-MB), Heart-Type Fatty Acid Binding Protein (H-FABP), and the like. Among them, MYO is less specific and occurs earlier; CK-MB and cTnI show lower sensitivity in the early stage of ACS and appear later; the heart type fatty acid binding protein (H-FABP) has high heart specificity, when myocardial ischemic injury appears, the H-FABP is found in blood 1-3H after chest pain attack, reaches a peak value 6-8H, returns to normal within 24-30H, and then quickly returns to normal level to benefit from high renal clearance, so the H-FABP is considered to be more suitable as an early marker of myocardial injury and an ideal diagnostic marker of myocardial infarction relapse. Although the release profile of H-FABP is similar to that of myoglobin, its cardiac specificity is 15-20 times that of myoglobin, so H-FABP is a more effective marker of myocardial injury.
The existing detection method of H-FABP mainly comprises an enzyme-linked immunosorbent assay, a fluorescence immunochromatography, a chemiluminescence method, a colloidal selenium labeling method, an ortho-position connection technology, colloidal gold, a latex enhanced immunoturbidimetry and the like, wherein the enzyme-linked immunosorbent assay, the fluorescence immunochromatography, the chemiluminescence method, the colloidal selenium labeling method, the ortho-position connection technology, the colloidal gold and the like have the defects of low sensitivity, long detection time, high cost, poor stability and the like; the latex-enhanced immunoturbidimetry is a common method for measuring the concentration of H-FABP in serum, but the latex-enhanced immunoturbidimetry detection kit in the market at present has the problems of poor sensitivity, poor linearity and the like. Therefore, it is necessary to develop a kit for detecting cardiac fatty acid binding proteins.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: aiming at the defects in the prior art, the kit for detecting the heart-type fatty acid binding protein is provided, and has the advantages of high sensitivity, high accuracy and wide linear range.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a kit for detecting cardiac fatty acid binding protein, the kit comprising:
reagent R1, wherein the reagent R1 comprises a first buffer solution, a biotin-labeled anti-human H-FABP antibody, a FITC-labeled anti-human H-FABP antibody, a first stabilizing agent, a first preservative and a surfactant;
reagent R2, wherein the reagent R2 comprises a second buffer solution, streptavidin modified latex microspheres, anti-FITC modified latex microspheres, a salt ion compound, a second stabilizing agent, a chelating agent and a second preservative;
the calibrator comprises a third buffer solution, H-FABP recombinant protein, a third stabilizer and a third preservative, and the concentration gradient of the H-FABP recombinant protein is 160ng/ml, 120ng/ml, 60ng/ml, 20ng/ml, 10ng/ml, 5ng/ml and 0 ng/ml.
As an improved technical scheme, the pH of the first buffer solution is 7.0-8.0, and the concentration is 20-150 mM; the first buffer solution is phosphate buffer solution, TRIS buffer solution, MES buffer solution, Hepes buffer solution, MOPSO buffer solution or DIPSO buffer solution; the pH value of the second buffer solution is 6.5-9.5, and the concentration is 20-100 mM; the second buffer solution is phosphate buffer solution, barbital buffer solution, glycine buffer solution, TRIS buffer solution, MES buffer solution, Hepes buffer solution, MOPSO buffer solution or DIPSO buffer solution; the pH value of the third buffer solution is 6.5-9.5, and the concentration is 60-150 mM; the third buffer solution is phosphate buffer solution, barbital buffer solution, glycine buffer solution, TRIS buffer solution, MES buffer solution, Hepes buffer solution, MOPSO buffer solution or DIPSO buffer solution.
As an improved technical solution, the first stabilizer, the second stabilizer and the third stabilizer are respectively one or a combination of two or more of sorbitol, ethylene glycol, lactose, glycine, sarcosine, mannitol, PVA, PVP, sodium metavanadate, praseodymium chloride, spermidine and CHAPS; the concentration of the stabilizer is 0.1-5% w/v.
As an improved technical scheme, the surfactant is one or a combination of two or more of polyethylene glycol 6000, ethylphenyl polyethylene glycol, Tween 20, sodium dodecyl sulfate, sorbitan monostearate, polyethylene glycol octyl phenyl ether-100, dodecyl hydroxypropyl phosphate betaine, alkyl glucoside, sorbitan fatty acid and hexadecyl trimethyl ammonium bromide; the concentration of the surfactant is 0.1-5% w/v.
As an improved technical scheme, the first preservative, the second preservative and the third preservative are respectively one or a combination of two or more of sodium azide, Proclin-300, Krovin600, Kathon and gentamicin; the concentration of the first preservative, the second preservative and the third preservative is 0.05-5% w/v.
As an improved technical scheme, the chelating agent is one or the combination of two or more of EDTA, ethylenediamine, oxalic acid and sorbitol, and the concentration of the chelating agent is 1-10 mM; the salt ion compound is sodium chloride, calcium chloride or the combination of the two, and the concentration of the salt ion compound is 10-50 mM.
As an improved technical scheme, the concentration of the biotin-labeled anti-human HFABP antibody is 1-200 mu g/ml, and the concentration of the FITC-labeled anti-human H-FABP antibody is 1-200 mu g/ml.
As an improved technical scheme, the concentration of the Streptavidin (SA) -modified latex microspheres is 0.1-1mg/mL, and the particle size of the Streptavidin (SA) -modified latex microspheres is 200-400 nm; the concentration of the anti-FITC modified latex microspheres is 0.1-1mg/mL, and the sphere diameter of the anti-FITC modified latex microspheres is 50-200 nm.
After the technical scheme is adopted, the invention has the beneficial effects that:
(1) the invention introduces an SA-biotin-antibody (antigen) -antigen (antibody) -antibody (antigen) -FITC-anti-FITC multiple cascade amplification system, because SA is combined with biotin and is easily combined with protein (such as antibody and the like) by covalent bonds, avidin molecules combined with latex microspheres react with biotin molecules combined with specific antibodies, wherein one antibody can be combined with a plurality of biotin, and one biotin molecule can be combined with four streptavidin molecules, thus achieving the multi-stage amplification effect and achieving the purpose of detecting unknown antigen (or antibody) molecules. FITC can be well combined with various antibodies, the specificity of combination of the combined antibodies and antigens is not influenced, so that anti-FITC combined with latex microspheres can better react with the FITC combined with the antibodies specifically, the antigens (antibodies) react with SA-biotin-antibodies and antibodies-FITC-anti-FITC during detection, reaction complexes are further enlarged, the effect of a multi-stage amplification system is further achieved, and the sensitivity of a reagent is improved;
(2) according to the invention, small-particle-size latex microspheres are selected to be combined with FITC, the small-particle-size microspheres have good detection linearity, large-particle-size latex microspheres are combined with streptavidin, and the detection sensitivity of the large-particle-size latex microspheres is high, so that the sensitivity of the reagent can be improved to the greatest extent under the condition of good linearity.
Drawings
FIG. 1 is a standard graph of a kit of the present invention;
FIG. 2 is a graph showing the linear relationship of the kit of the present invention;
FIG. 3 is a graph of a control analysis of the results of the assay of example 2 and the inlet reagent of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
A kit for detecting heart-type fatty acid binding protein comprises a reagent R1, a reagent R2 and a calibrator, wherein the volume ratio of the reagent R1 to the reagent R2 is 2: 1;
the pH of the reagent R1 is 7.5, and the formula components are as follows:
a first buffer (MOPSO buffer 50mM), a biotin-labeled anti-human H-FABP antibody (50. mu.g/ml), a FITC-labeled anti-human H-FABP antibody (100. mu.g/ml), a first stabilizer (1% w/v PVA), a first preservative (0.9% w/v Kathon), a surfactant (0.8% w/v ethylphenylpolyethylene glycol);
the pH of the reagent R2 is 7.5, and the formula components are as follows:
a second buffer (MOPSO buffer 50mM), streptavidin-modified latex microspheres (0.8mg/mL, the particle size of the latex microspheres is 300nm), anti-FITC-modified latex microspheres (0.8mg/mL, the particle size of the latex microspheres is 150nm), a salt ion compound (sodium chloride, 10mM), a second stabilizer (1% w/v ethylene glycol), a chelating agent (EDTA 1mM), a second preservative (0.9% w/v Kathon);
the pH of the calibrator is 7.5, and the calibrator comprises the following formula components:
a third buffer (MOPSO buffer 50mM), H-FABP recombinant protein (concentration gradient of 160ng/ml, 120ng/ml, 60ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 0 ng/ml.), a third stabilizer (1% w/v mannitol), and a third preservative (0.9% w/v Kathon).
Example 2
A kit for detecting heart-type fatty acid binding protein comprises a reagent R1, a reagent R2 and a calibrator, wherein the volume ratio of the reagent R1 to the reagent R2 is 2.5: 1;
the pH of the reagent R1 is 7.5, and the formula components are as follows:
a first buffer (HEPES buffer 20mM), biotin-labeled anti-human H-FABP antibody (50. mu.g/ml), FITC-labeled anti-human H-FABP antibody (80. mu.g/ml), a first stabilizer (1% w/v sorbitol), a first preservative (0.9% w/v gentamicin), a surfactant (0.8% w/v sodium dodecyl sulfate);
FITC-labeled anti-human H-FABP antibody: taking an appropriate amount of anti-human H-FABP antibody (mouse anti-human, rabbit anti-human, sheep anti-human or cow anti-human H-FABP antibody), adding HEPES buffer solution, stirring and mixing uniformly to obtain a solution with the antibody concentration of 20 mg/ml; adding FITC into an antibody solution according to the amount of 0.01 mg added to every 1mg of antibody, stirring for 12H in the dark at 4 ℃ for primary dialysis, centrifuging for 20min at the centrifugal speed of 2500r/min (removing a small amount of precipitate), putting the supernatant into a dialysis bag, carrying out secondary dialysis with pH8.0 buffer saline at 0-4 ℃ overnight, taking the marker after overnight dialysis, passing through a sephadex G-25 or G-50 column, and separating to collect the FITC-labeled anti-human H-FABP antibody.
Biotin-labeled anti-human H-FABP antibodies: diluting anti-human H-FABP antibody to 1mg/ml with 0.1mol/L buffer solution (pH8.0), adding 120 μ L NHSB solution with concentration of 1mg/ml into 1ml antibody solution (containing antibody 1mg), stirring and maintaining temperature for 2-4H, adding 9.6 μ L1 mol/L NH4Cl (as 1. mu. lNH per 25. mu.g NHSB)4Cl amount added), stirring at room temperature for 10 minutes, then dialyzing well at 4 ℃, passing the sample through a 1ml molecular sieve column, eluting slowly with PBS, collecting 1 ml/tube, washing the protein between 1-3ml, adding the collected sample to sodium azide (final concentration 0.5g/L) and 1.0g/L BSA. Storing the combined product at 4 deg.C in dark, or adding 50% heavy steamed glycerol, and storing at-20 deg.C.
Uniformly mixing a biotin-labeled H-FABP antibody and a FITC-labeled H-FABP antibody according to the proportion of 1-3:1, and adding a corresponding amount of a stabilizer, a preservative and a surfactant to prepare a reagent R1;
the pH of the reagent R2 is 7.5, and the formula components are as follows:
a second buffer (HEPES buffer 20mM), streptavidin-modified latex microspheres (0.7mg/mL, particle size of latex microspheres 300nm), anti-FITC-modified latex microspheres (0.7mg/mL, particle size of latex microspheres 150nm), a salt ion compound (sodium chloride, 10mM), a second stabilizer (1% w/v glycine), a chelating agent (EDTA 1mM), a second preservative (0.9% w/v gentamicin);
streptavidin modified latex microspheres: diluting SA to 10mg/ml by using a reaction buffer solution (HEPES buffer solution), diluting latex microspheres to 10mg/ml by using the reaction buffer solution, adding SA into the latex microsphere solution according to the amount of adding 1ml of SA solution into each 10ml of latex microsphere solution, stirring and incubating at room temperature for 2 hours, centrifuging/ultrafiltering, removing supernate (removing unbound SA) to obtain SA modified latex microspheres, storing the SA modified latex microspheres by using the HEPES buffer solution as a storage solution, and diluting to obtain the required concentration of the SA modified latex microspheres.
Anti-FITC modified latex microspheres: diluting Anti-FITC to 10mg/ml by using a reaction buffer solution (HEPES buffer solution), diluting latex microspheres to 1% by using the reaction buffer solution, adding an FITC antibody into a latex microsphere solution, adding the Anti-FITC solution into the latex microsphere solution according to the amount of 1ml of the Anti-FITC solution added into each 10ml of the latex microsphere solution, stirring and incubating for 2 hours at room temperature, centrifuging/ultrafiltering, removing unbound FITC antibody to obtain Anti-FITC modified latex microspheres, and diluting to obtain the required concentration of the Anti-FITC modified latex microspheres.
The SA modified latex microspheres and the Anti-FITC modified latex microspheres are uniformly mixed according to the ratio of 2-3:1, and a stabilizing agent, a salt ion compound, a chelating agent and a preservative in corresponding amounts are added to prepare a reagent R2.
The pH of the calibrator is 7.5, and the calibrator comprises the following formula components:
a third buffer (MOPSO buffer 50mM), H-FABP recombinant protein (concentration gradient: 160ng/ml, 120ng/ml, 60ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 0 ng/ml.), a third stabilizer (1% w/v mannitol), and a third preservative (0.9% w/v gentamicin).
Example 3
A kit for detecting heart-type fatty acid binding protein comprises a reagent R1, a reagent R2 and a calibrator, wherein the volume ratio of the reagent R1 to the reagent R2 is 3: 1;
the pH of the reagent R1 is 7, and the formula components are as follows:
first buffer (Mes buffer 80mM), biotin-labeled anti-human H-FABP antibody (50. mu.g/ml), FITC-labeled anti-human H-FABP antibody (80. mu.g/ml), first stabilizer (1% w/v spermidine), first preservative (0.9% w/v sodium azide), surfactant (0.8% w/v sorbitan monostearate);
the pH of the reagent R2 is 6.5, and the formula components are as follows:
a second buffer (Mes buffer 50mM), streptavidin-modified latex microspheres (0.6mg/mL, particle size of latex microspheres 250nm), anti-FITC modified latex microspheres (0.6mg/mL, particle size of latex microspheres 80nm), a salt ion compound (sodium chloride, 10mM), a second stabilizer (1% w/v mannitol), a chelating agent (sorbitol 1mM), a second preservative (0.9% w/v Proclin-300);
the pH of the calibrator is 6.5, and the calibrator comprises the following formula components:
a third buffer (Mes buffer 80mM), H-FABP recombinant protein (concentration gradient: 160ng/ml, 120ng/ml, 60ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 0 ng/ml.), a third stabilizer (1% w/v mannitol), and a third preservative (0.9% w/v Proclin-300).
Example 4
A kit for detecting heart-type fatty acid binding protein comprises a reagent R1, a reagent R2 and a calibrator, wherein the volume ratio of the reagent R1 to the reagent R2 is 3.5: 1;
the pH of the reagent R1 is 8.0, and the formula components are as follows:
a first buffer (Tris buffer 20mM), a biotin-labeled anti-human H-FABP antibody (80. mu.g/ml), a FITC-labeled anti-human H-FABP antibody (80. mu.g/ml), a first stabilizer (1% w/v of pvp and 0.1% w/v of sodium metavanadate), a first preservative (0.9% w/v of sodium azide), a surfactant (0.8% w/v of sorbitan fatty acid);
the pH of the reagent R2 is 8.0, and the formula components are as follows:
a second buffer (Tris buffer 20mM), streptavidin modified latex microspheres (0.5mg/mL, the particle size of the latex microspheres is 250nm), anti-FITC modified latex microspheres (0.5mg/mL, the particle size of the latex microspheres is 150nm), a salt ion compound (sodium chloride, 10mM), a second stabilizer (1% w/v mannitol and 1% w/v chlorination spectrum), a chelating agent (EDTA 1mM), a second preservative (0.9% w/v Proclin-300);
the pH of the calibrator is 7.5, and the calibrator comprises the following formula components:
a third buffer (Tris buffer 20mM), H-FABP recombinant protein (concentration gradient: 160ng/ml, 120ng/ml, 60ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 0 ng/ml.), a third stabilizer (1% w/v mannitol), and a third preservative (0.9% w/v Proclin-300).
Example 5
A kit for detecting heart-type fatty acid binding protein comprises a reagent R1, a reagent R2 and a calibrator, wherein the volume ratio of the reagent R1 to the reagent R2 is 2.5: 1;
the pH of the reagent R1 is 8.0, and the formula components are as follows:
a first buffer (Tris buffer 80mM), a biotin-labeled anti-human H-FABP antibody (80. mu.g/ml), a FITC-labeled anti-human H-FABP antibody (100. mu.g/ml), a first stabilizer (1% w/v mannitol and 0.5% w/v glycine), a first preservative (1.5% w/v sodium azide), a surfactant (0.8% w/v ethylphenylpolyethylene glycol and 0.5% w/v Tween 20);
the pH of the reagent R2 is 8.0, and the formula components are as follows:
a second buffer (80 mM Tris buffer), streptavidin modified latex microspheres (0.50mg/mL, the particle size of the latex microspheres is 250nm), anti-FITC modified latex microspheres (0.5mg/mL, the particle size of the latex microspheres is 100nm), salt ion compounds (sodium chloride and calcium chloride, the amount of the sodium chloride and the calcium chloride is 10mM respectively), a second stabilizer (1% w/v mannitol and 0.5% w/v glycine), a chelating agent (1mM EDTA), and a second preservative (1% w/v Proclin-300);
the pH value of the calibrator is 8.0, and the calibrator comprises the following formula components:
a third buffer (MOPSO buffer 50mM), H-FABP recombinant protein (concentration gradient: 160ng/ml, 120ng/ml, 60ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 0 ng/ml.), a third stabilizer (1% w/v mannitol), and a third preservative (1% w/v Proclin-300).
Example 6
A kit for detecting heart-type fatty acid binding protein comprises a reagent R1, a reagent R2 and a calibrator, wherein the volume ratio of the reagent R1 to the reagent R2 is 4: 1;
the pH of the reagent R1 is 7, and the formula components are as follows:
first buffer (Mes buffer 100mM), biotin-labeled anti-human H-FABP antibody (50. mu.g/ml), FITC-labeled anti-human H-FABP antibody (80. mu.g/ml), first stabilizer (2.5% w/v mannitol and 1% w/v lactose), first preservative (2% w/v sodium azide), surfactant (0.8% w/v polyethylene glycol octylphenyl ether-100 and 1% w/v cetyltrimethylammonium bromide);
the pH of the reagent R2 is 7.5, and the formula components are as follows:
a second buffer (Mes buffer 100mM), streptavidin-modified latex microspheres (0.3mg/mL, particle size of latex microspheres is 150nm), anti-FITC-modified latex microspheres (0.3mg/mL), salt ion compounds (sodium chloride and calcium chloride, 10mM), a second stabilizer (1% w/v mannitol and 1% w/v sarcosine), a chelating agent (EDTA 1mM), a second preservative (2.5% w/v Proclin-300);
the pH value of the calibrator is 8.5, and the calibrator comprises the following formula components:
a third buffer (Tris buffer 100mM), H-FABP recombinant protein (concentration gradient: 160ng/ml, 120ng/ml, 60ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 0 ng/ml.), a third stabilizer (1% w/v mannitol and 1% w/v sarcosine), and a third preservative (2% w/v Proclin-300).
Example 7
A kit for detecting heart-type fatty acid binding protein comprises a reagent R1, a reagent R2 and a calibrator, wherein the volume ratio of the reagent R1 to the reagent R2 is 4: 1;
the pH of the reagent R1 is 8.5, and the formula components are as follows:
first buffer (hepes buffer 130mM), biotin-labeled anti-human H-FABP antibody (80. mu.g/ml), FITC-labeled anti-human H-FABP antibody (120. mu.g/ml), first stabilizer (2% w/v mannitol and 1% w/v chloride profile), first preservative (0.9% w/v sodium azide), saccharide compound (1% w/v maltose and 2% w/v trehalose), surfactant (1.8% w/v ethylphenylpolyethylene glycol and 0.5% w/v alkylglucoside);
the pH of the reagent R2 is 9, and the formula components are as follows:
a second buffer (Tris buffer 130mM), streptavidin modified latex microspheres (0.3mg/mL, the particle size of the latex microspheres is 300nm), anti-FITC modified latex microspheres (0.3mg/mL, the particle size of the latex microspheres is 150nm), salt ion compounds (sodium chloride and calcium chloride, 10mM respectively), a second stabilizer (4% w/v sarcosine and 2% w/v praseodymium chloride), a chelating agent (EDTA 1mM), and a second preservative (4% w/v Proclin-300);
the pH value of the calibrator is 8.5, and the calibrator comprises the following formula components:
a third buffer (Tris buffer 130mM), H-FABP recombinant protein (concentration gradient: 160ng/ml, 120ng/ml, 60ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 0 ng/ml.), a third stabilizer (1% w/v mannitol and 0.1% w/v glycine), and a third preservative (4% w/v Proclin-300).
Example 8
A kit for detecting heart-type fatty acid binding protein comprises a reagent R1, a reagent R2 and a calibrator, wherein the volume ratio of the reagent R1 to the reagent R2 is 4: 1;
the pH of the reagent R1 is 8.0, and the formula components are as follows:
first buffer (Mes buffer 150mM), biotin-labeled anti-human H-FABP antibody (100. mu.g/ml), FITC-labeled anti-human H-FABP antibody (120. mu.g/ml), first stabilizer (5% w/v VCHAPS and 4% w/v sorbitol), first preservative (5% w/v sodium azide), surfactant (5% w/v dodecyl hydroxypropyl phosphate betaine);
the pH of the reagent R2 is 9.5, and the formula components are as follows:
a second buffer (MOPSO buffer 100mM), streptavidin-modified latex microspheres (0.2mg/mL, particle size of latex microspheres 250nm), anti-FITC modified latex microspheres (0.2mg/mL, particle size of latex microspheres 100nm), a salt ion compound (sodium chloride, 50mM), a second stabilizer (5% w/v trehalose and 2% w/v CHAPS), a chelating agent (1mM EDTA and 1% w/v sorbitol), a second preservative (5% w/v Proclin-300);
the pH of the calibrator is 9.5, and the calibrator comprises the following formula components:
a third buffer (MOPSO buffer 150mM), H-FABP recombinant protein (concentration gradient: 160ng/ml, 120ng/ml, 60ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 0 ng/ml.), a third stabilizer (5% w/v mannitol), a third preservative (0.9% w/v Proclin-300 and 0.05% w/v Krovin 600).
To better demonstrate the good performance of the kit product of the invention, the following tests were performed.
1. Calibration curve of reagent
And (2) adding 320ng of the corresponding recombinant H-FABP pure product into 2ml of buffer solution of the calibrator diluent according to the required concentration of the H-FABP reference calibrator to prepare the H-FABP calibrator with the concentration of 160ng/ml, wherein when the H-FABP calibrator with the concentration of 160ng/ml is used, the H-FABP calibrator with the concentration of 160ng/ml, 120ng/ml, 60ng/ml, 20ng/ml, 10ng/ml, 5ng/ml and 0ng/ml are diluted by the buffer solution according to the proportion, and the concentrations sequentially correspond to the absorbance data 8990, 6670, 3580, 1200, 600, 250 and 0. The calibration curve is shown in figure 1.
2. Sensitivity test
The kit of example 2 was used, and a sample of 5ng/l concentration was used as a sample for testing, and the absorbance was measured at 37 ℃ and 700 nm in a full-automatic biochemical analyzer (Hitachi full-automatic biochemical analyzer 7080). Calculated with a 95% confidence limit: the blank sample is repeatedly measured for 20 times, the mean value (x)) and the Standard Deviation (SD) measured in 20 reactions are calculated, and the corresponding concentration is calculated according to the mean value (x) +2SD (sandwich method), namely the analysis sensitivity of the reagent.
TABLE 1
Figure BDA0002998946070000101
Figure BDA0002998946070000111
The test results are shown in Table 1, and it was revealed that the analytical sensitivity of this reagent was 0.12 ng/mL.
3. Linear range assay of detection reagents:
the high concentration sample near the linear range and the low concentration H-FABP sample near the lower limit of the linear range were mixed to 6 dilution gradients. And detecting each concentration, testing each sample for 3 times, calculating an average value, and judging according to the judgment basis that R is more than or equal to 0.990. The results are shown in Table 2 and FIG. 2.
TABLE 2
Sample(s) h-FABP theoretical value H-FABP measured value 1 H-FABP found value 2 H-FABP found value 3 Mean value
1 5 5.05 5.02 5.17 5.06
2 32 31.96 31.87 32.12 31.99
3 64 64.12 63.01 63.11 63.56
4 96 96.66 96.71 95.83 96.30
5 128 128.00 127.96 128.71 128.17
6 160 161.01 162.95 161.93 161.47
Performing linear regression analysis on the average value of the measured concentration and the theoretical concentration, and calculating a regression equation of which y is 1.0077x-0.3655 and the correlation coefficient is R20.9999 and R > 0.990, which shows that the kit based on the SA-biotin-antibody-antigen-antibody-FITC-Anti-FABP multiple cascade amplification system has better correlation in the linear range of 0.1ng/ml to 160.00 ng/ml.
4. Clinical sample correlation assay
Compared with the H-FABP contrast reagent (immunoturbidimetry, imported foreign reagent), the reagent provided by the invention is used for contrast determination of 40 cases of clinical specimen serum (the sample concentration covers the detection range of a standard substance), and the kit and the detection method in the embodiment 2 are the same as the foreign contrast reagent; the H-FABP contrast reagent sets related parameters according to the instruction, and a Hitachi 7080 full-automatic biochemical analyzer is used for analyzing samples, and specific results are shown in Table 3 and FIG. 3.
TABLE 3
Figure BDA0002998946070000121
Figure BDA0002998946070000131
The H-FABP concentration detected by the invention is used as an abscissa, the H-FABP concentration detected by the imported reagent is used as an ordinate, and a sample correlation curve is drawn. As shown in fig. 3 and table 3. Wherein the correlation equation of H-FABP is that y is 0.98x +0.299, and the correlation coefficient R2When the ratio is 0.991, R is more than 0.975, which completely meets the requirements of clinical trials.
The investigation proves that the kit product has high sensitivity and accuracy and wide linear range.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (8)

1. A kit for detecting cardiac fatty acid binding protein, characterized in that the kit comprises:
reagent R1, wherein the reagent R1 comprises a first buffer solution, a biotin-labeled anti-human H-FABP antibody, a FITC-labeled anti-human H-FABP antibody, a first stabilizing agent, a first preservative and a surfactant;
reagent R2, wherein the reagent R2 comprises a second buffer solution, streptavidin modified latex microspheres, anti-FITC modified latex microspheres, a salt ion compound, a second stabilizing agent, a chelating agent and a second preservative;
the calibrator comprises a third buffer solution, H-FABP recombinant protein, a third stabilizer and a third preservative, and the concentration gradient of the H-FABP recombinant protein is 160ng/ml, 120ng/ml, 60ng/ml, 20ng/ml, 10ng/ml, 5ng/ml and 0 ng/ml.
2. The kit for detecting cardiac fatty acid binding protein according to claim 1, wherein: the pH value of the first buffer solution is 7.0-8.0, and the concentration is 20-150 mM; the first buffer solution is phosphate buffer solution, TRIS buffer solution, MES buffer solution, Hepes buffer solution, MOPSO buffer solution or DIPSO buffer solution; the pH value of the second buffer solution is 6.5-9.5, and the concentration is 20-100 mM; the second buffer solution is phosphate buffer solution, barbital buffer solution, glycine buffer solution, TRIS buffer solution, MES buffer solution, Hepes buffer solution, MOPSO buffer solution or DIPSO buffer solution; the pH value of the third buffer solution is 6.5-9.5, and the concentration is 50-150 mM; the third buffer solution is phosphate buffer solution, barbital buffer solution, glycine buffer solution, TRIS buffer solution, MES buffer solution, Hepes buffer solution, MOPSO buffer solution or DIPSO buffer solution.
3. The kit for detecting cardiac fatty acid binding protein according to claim 1, wherein: the first stabilizer, the second stabilizer and the third stabilizer are respectively one or more of sorbitol, ethylene glycol, lactose, glycine, sarcosine, mannitol, PVA, PVP, sodium metavanadate, praseodymium chloride, spermidine and CHAPS; the concentration of the stabilizer is 0.1-5% w/v.
4. The kit for detecting cardiac fatty acid binding protein according to claim 1, wherein: the surfactant is one or more of polyethylene glycol 6000, ethylphenyl polyethylene glycol, tween 20, sodium dodecyl sulfate, sorbitan monostearate, polyethylene glycol octyl phenyl ether-100, dodecyl hydroxypropyl phosphate betaine, alkyl glucoside, fatty acid sorbitan, and cetyl trimethyl ammonium bromide; the concentration of the surfactant is 0.1-5% w/v.
5. The kit for detecting cardiac fatty acid binding protein according to claim 1, wherein: the first preservative, the second preservative and the third preservative are respectively one or more of sodium azide, Proclin-300, Krovin600, Kathon and gentamicin; the concentration of the first preservative, the second preservative and the third preservative is 0.05-5% w/v.
6. The kit for detecting cardiac fatty acid binding protein according to claim 1, wherein: the chelating agent is one or more of EDTA, ethylenediamine, oxalic acid and sorbitol, and the concentration of the chelating agent is 1-10 mM; the salt ion compound is sodium chloride, calcium chloride or the combination of the two, and the concentration of the salt ion compound is 10-50 mM.
7. The kit for detecting cardiac fatty acid binding protein according to claim 1, wherein: the concentration of the biotin-labeled human HFABP antibody is 1-200 mu g/ml, and the concentration of the FITC-labeled anti-human H-FABP antibody is 1-200 mu g/ml.
8. The kit for detecting cardiac fatty acid binding protein according to claim 1, wherein: the concentration of the streptavidin-modified latex microspheres is 0.1-1mg/mL, and the particle size of the streptavidin-modified latex microspheres is 200-400 nm; the concentration of the anti-FITC modified latex microspheres is 0.1-1mg/mL, and the sphere diameter of the anti-FITC modified latex microspheres is 50-200 nm.
CN202110340279.8A 2021-03-30 2021-03-30 Kit for detecting heart-type fatty acid binding protein Pending CN113125748A (en)

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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030203361A1 (en) * 1999-08-13 2003-10-30 Human Genome Sciences, Inc. 13 human colon and colon cancer associated proteins
US20050095591A1 (en) * 2000-09-12 2005-05-05 Christopherson Richard I. Diagnostic assay
CN103123319A (en) * 2012-12-20 2013-05-29 武汉生之源生物科技有限公司 Heart-type fatty acid binding protein content detection kit and preparation method thereof
CN104215772A (en) * 2014-08-14 2014-12-17 上海睿康生物科技有限公司 Heart-type fatty acid binding protein detection kit and making method thereof
CN104634980A (en) * 2015-02-10 2015-05-20 深圳市新产业生物医学工程股份有限公司 Cardiac troponin I (cTn I) hypersensitive detection kit and hypersensitive detection method
CN108445222A (en) * 2018-02-01 2018-08-24 浙江艾明德生物科技有限公司 A kind of kit and preparation method quantitatively detecting cardic fatty acid binding protein
CN110161250A (en) * 2018-02-11 2019-08-23 博阳生物科技（上海）有限公司 A kind of homogeneous human cardiac troponin I quick detection kit, system, detection method and application
CN110261621A (en) * 2019-07-16 2019-09-20 三诺生物传感股份有限公司 A kind of interleukin-6 detection kit
CN112034186A (en) * 2020-09-07 2020-12-04 南京立顶医疗科技有限公司 Glycosylated hemoglobin kit based on biotin-streptavidin amplification and preparation method thereof

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030203361A1 (en) * 1999-08-13 2003-10-30 Human Genome Sciences, Inc. 13 human colon and colon cancer associated proteins
US20050095591A1 (en) * 2000-09-12 2005-05-05 Christopherson Richard I. Diagnostic assay
CN103123319A (en) * 2012-12-20 2013-05-29 武汉生之源生物科技有限公司 Heart-type fatty acid binding protein content detection kit and preparation method thereof
CN104215772A (en) * 2014-08-14 2014-12-17 上海睿康生物科技有限公司 Heart-type fatty acid binding protein detection kit and making method thereof
CN104634980A (en) * 2015-02-10 2015-05-20 深圳市新产业生物医学工程股份有限公司 Cardiac troponin I (cTn I) hypersensitive detection kit and hypersensitive detection method
CN108445222A (en) * 2018-02-01 2018-08-24 浙江艾明德生物科技有限公司 A kind of kit and preparation method quantitatively detecting cardic fatty acid binding protein
CN110161250A (en) * 2018-02-11 2019-08-23 博阳生物科技（上海）有限公司 A kind of homogeneous human cardiac troponin I quick detection kit, system, detection method and application
CN110261621A (en) * 2019-07-16 2019-09-20 三诺生物传感股份有限公司 A kind of interleukin-6 detection kit
CN112034186A (en) * 2020-09-07 2020-12-04 南京立顶医疗科技有限公司 Glycosylated hemoglobin kit based on biotin-streptavidin amplification and preparation method thereof

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