CN107656072A - Liver fatty acid binding protein detection kit - Google Patents
Liver fatty acid binding protein detection kit Download PDFInfo
- Publication number
- CN107656072A CN107656072A CN201711147897.0A CN201711147897A CN107656072A CN 107656072 A CN107656072 A CN 107656072A CN 201711147897 A CN201711147897 A CN 201711147897A CN 107656072 A CN107656072 A CN 107656072A
- Authority
- CN
- China
- Prior art keywords
- fabp
- magnetic bead
- solution
- fatty acid
- binding protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Plasma & Fusion (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of liver fatty acid binding protein detection kit, including seven storage assemblies, it is respectively used to store L FABP calibration objects, L FABP quality-control products, enzyme conjugates working solution, magnetic bead working solution, cleaning fluid, substrate solution and pre-treatment reagent；Magnetic bead working solution includes the carboxyl magnetic bead for being marked with L FABP antibody, and enzyme conjugates working solution includes the anti-L FABP antibody of alkali phosphatase enzyme mark.The invention also discloses the detection method of detection liver fatty acid binding protein.Detection kit disclosed by the invention, lowest detection are limited to 0.5ng/ml, and the range of linearity is 0.5 500ng/ml, and detection sensitivity is high, and the range of linearity is wide, and detection duration foreshortens to 15 minutes, and simplifies detecting step.
Description
Technical field
The present invention relates to field of biological detection, and in particular to a kind of liver fatty acid binding protein detection kit.
Background technology
Acute injury of kidney (Acutekidneyinjury, AKI) is as caused by many reasons, can be occurred in various clinics
In the case of a kind of complicated renal hypofunction syndrome.It can show as the horizontal slight rise of serum creatinine, can also table
It is now acute renal failure.Research finds that AKI generation and the increase of case fatality rate are closely related.This requires doctor in clinical work
It can early diagnose in work, preferably be begun to decline in glomerular filtration rate(GFR, or only tissue damage and glomerular filtration rate(GFR be still just
The normal stage finds and intervened in time, to improve prognosis, reduce case fatality rate.Serum creatinine and urine volume are current AKI diagnosis indexs,
But influenceed by several factors, Sensitivity and Specificity is not high.Lagged because AKI is diagnosed, often affect the best period for the treatment of adversely.Seek
Ask and have become one of current research focus with hypersensitivity, specificity, the new biomarker that easily detects.
Liver fatty acid binding protein (Liver-typefattyacidbindingprotein, L-FABP) is one group
The high conservative cytoplasmic protein that can combine long chain fatty acids of low molecule amount (14~15kD or so) is a kind of expression in the mankind
The intracellular small protein of renal proximal tubules, it is as participation free fatty (FFAs) in the pass of renal tubule intracellular metabolite
Key albumen, in AKI in addition to renal protection, moreover it is possible to the early sign thing as reflection injury of kidney.In human kidney
There is two kinds of fatty acid binding protein (fattyacidbindingprotein, FABP), liver type (L-FABP, is expressed in
Proximal tubular) and heart-type (H-FABP, being expressed in distal renal tubular).In proximal tubular free fatty and epithelial cell
Kytoplasm FABPs is combined, and then is transported to mitochondria or peroxisome progress beta oxidation metabolism.In CKD, diabetes
Have been found that urine L-FABP increases under a variety of pathologic conditions such as nephrosis, IgA nephrosis and radiographic contrast nephropathy.In protein-bearing nephrosis and list
Side ureteral obstruction animal model also observes that L-FABP expression is increased, urine L-FABP discharge increases, and transgenic mice experiments
Prompting L-FABP may have protective effect to renal tubule.Recently report that 40 cardiopulmonary bypass children arts are determined using ELISA method
L-FABP is urinated, increases 94 times and 45 times respectively AKI person (Scr is compared with basic value increase by 50%) postoperative 4h, 12h occurs, changes compared with Scr
Become and do sth. in advance l~3d, increase by 24 times to urinate L-FABP as point of contact, it predicts that AKI sensitiveness is 71.4%.Specificity 68.4%,
Display urine L-FABP is to predict the sensitive, special of AKI, early stage and independent biological markers after openheart surgery.
Chinese patent CN104880562A discloses a kind of L-FABP quick detection reagents and preparation method thereof, including substrate
With the cover plate being covered on substrate, sample application zone, reaction zone, detection zone and waste are machined with successively on substrate；Sample application zone, reaction
Area, detection zone and waste have microchannel composition；The polyester film for filtering biological sample is placed with sample application zone, is reacted
Area includes detection line and control line, and detection line is coated with dry fluorescent microsphere label L-FABP polyclonal antibodies, control line bag
There is fluorescent microsphere to mark sheep anti-mouse igg antibody.After sample-adding, fluorescent microsphere mark is combined with the L-FABP and detection line in sample
Remember that the L-FABP of antibody is combined, form the fluorescence signal of detection line, L-FABP content in the fluorescence signal and sample of detection line
It is proportional.It is not high using this method detection L-FABP sensitivity.
The content of the invention
Therefore, the technical problem to be solved in the present invention is detection liver fatty acid binding protein sensitivity in the prior art
The defects of not high.And then provide a kind of detection reagent for detecting quick and high sensitivity detection liver fatty acid binding protein
Box.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
A kind of liver fatty acid binding protein detection kit, including
First liquid storage component, for storing L-FABP calibration objects；
Second liquid storage component, for storing enzyme conjugates working solution；
3rd liquid storage component, for storing magnetic bead working solution；
4th liquid storage component, for storing cleaning fluid；
5th liquid storage component, for storing substrate solution；
6th liquid storage component, for storing pre-treatment reagent；
7th liquid storage component, for storing L-FABP quality-control products.
Optionally, the magnetic bead working solution includes the carboxyl magnetic bead for being marked with L-FABP antibody；The particle diameter of the magnetic bead is
1-5μm。
Optionally, it is 0.1~1mg/ml that the mark, which has the concentration of the carboxyl magnetic bead of antibody,.
Optionally, the enzyme conjugates working solution includes the anti-L-FABP antibody of alkali phosphatase enzyme mark.
Optionally, the concentration of the anti-L-FABP antibody of the alkali phosphatase enzyme mark is 0.5~2 μ g/ml.
Optionally, the L-FABP calibration objects and the L-FABP quality-control products are the Tris bufferings containing L-FABP antigens
Liquid.
Optionally, the substrate solution is enzyme-catalyzed chemical luminescence substrate solution.
Optionally, the pre-treatment reagent is the buffer solution for including NaCl, sucrose, glycerine, polysorbas20 and preservative.
The present invention provides a kind of method for detecting liver fatty acid binding protein, uses described liver fatty acid knot
Hop protein detection kit detects, and comprises the following steps：
(1) sample to be tested, L-FABP calibration objects and L-FABP quality-control products are added separately in pre-treatment reagent, mixed
Magnetic bead working solution, enzyme conjugates working solution are added afterwards, and 37 DEG C of -42 DEG C of reaction 5min-10min, obtain the first reaction solution after mixing；
(2) first reaction solution is subjected to Magneto separate, collects magnetic bead；The magnetic bead is cleaned by cleaning fluid；
(3) substrate solution is added in the magnetic bead after cleaning, obtains mixed liquor；Detect the luminous strong of the mixed liquor
Degree.
Optionally, the sample to be tested, the L-FABP calibration objects or the L-FABP quality-control products try with the pre-treatment
The ratio of agent is 1:1.
Optionally, the ratio of the magnetic bead working solution, the enzyme conjugates working solution and the substrate solution is 1:1:2.
The above-mentioned technical proposal of the present invention has advantages below compared with prior art：
1. detection kit provided in an embodiment of the present invention is combined detection liver type with magnetic bead using chemiluminescence law technology
Fatty acid binding protein, by pre-treatment reagent, the cooperation of enzyme conjugates working solution, magnetic bead working solution and substrate solution, make
The lowest detection that liver fatty acid binding protein must be detected is limited to 0.5ng/ml；The range of linearity is 0.5-500ng/ml, linearly
Coefficient correlation is r>0.99；；High sensitivity, the range of linearity are wide.Compared with prior art, detection time only has 15min, contracts significantly
Short detection duration.
2. detection kit provided in an embodiment of the present invention includes pre-treatment reagent, enzyme conjugates working solution and magnetic bead work
Liquid, without the processing such as being filtered, being centrifuged to urine specimen, add pre-treatment reagent, be directly added into enzyme conjugates working solution and
Magnetic bead working solution, simplifies detecting step, improves detection efficiency.
3. detection kit provided in an embodiment of the present invention provides L-FABP calibration objects and L-FABP quality-control products, ensure that
The accuracy of testing result.
4. the method for detection liver fatty acid binding protein provided in an embodiment of the present invention, simple to operate, detection time
Short, high sensitivity, the range of linearity is wide.
Embodiment
In order that the object, technical solutions and advantages of the present invention are clearer, illustrate this below by way of specific embodiment
The embodiment of invention, unless otherwise indicated, disclosed in this invention experimental method use the art routine techniques.
Embodiment 1
The present embodiment provides a kind of method for preparing enzyme conjugates, comprises the following steps：
1. it is 2mg/mL by L-FABP labelled antibodies preservation concentration；0.5M TCEP solution is added into the solution makes its end
Concentration is 1.25mM, is mixed at once, and 25 DEG C stand reaction 60 minutes.
2. weighing appropriate Sulfo-SMCC reagents, it is 17.5mg/mL solution to be made into concentration with dimethylformamide DMF.
It is molten that 3. the Sulfo-SMCC that step 2 is prepared is added into alkaline phosphatase enzyme solutions (AP solution, concentration 20mg/ml)
Liquid, alkaline phosphatase and Sulfo-SMCC mol ratios 1：10, mix at once, 25 DEG C stand reaction 15 minutes.
4. 1M glycine solution, glycine and Sulfo- are added in the AP solution for terminating to obtain to step 3 priming reaction
The mol ratio of SMCC reagents is 1:10, mix at once, 25 DEG C stand reaction 10 minutes.
5. step 1 to be reacted to the antibody-solutions desalination at once of end, (100mM triethanolamines are replaced in cross-linking buffer
Buffer solution, pH7.3), ultraviolet spectrophotometry measures antibody concentration；
6. step 4 to be reacted to the AP solution desalination at once of end, replace in cross-linking buffer that (100mM triethanolamines delay
Fliud flushing, pH7.3), ultraviolet spectrophotometry measures AP concentration；
7. the AP solution that step 6 has activated is added immediately in the antibody-solutions activated to step 5, the L-FABP marks of activation
The mol ratio for remembering antibody and the alkaline phosphatase of activation is 1：2, fully mix.2~8 DEG C stand reaction 18 hours.
8. weighing appropriate NEM reagents, it is made into antibody activation buffer solution (100mM Triethanolamine buffers, pH7.3)
12.5mg/mL solution.
9. the NEM solution that 1 percent debulking steps 10 are prepared is added in the solution terminated to step 7 cross-linking reaction, fully
Mix, be stored at room temperature reaction 30 minutes.Close unnecessary sulfydryl.
10. the cross-linking agent solution that step 9 terminating reaction terminates is purified by modes such as dialysis, desalination or ultrafiltration concentrations.
11. concentration will be determined enzyme conjugates concentrate after purification, isometric glycerine is added, is fully mixed, -20 DEG C of preservations are standby
With.
Embodiment 2
The present embodiment provides a kind of method for preparing enzyme conjugates, comprises the following steps：
1. it is 2mg/mL by L-FABP labelled antibodies preservation concentration；0.5M TCEP solution is added into the solution makes its end
Concentration is 1.25mM, is mixed at once, and 25 DEG C stand reaction 60 minutes.
2. weighing appropriate Sulfo-SMCC reagents, it is 17.5mg/mL solution to be made into concentration with dimethylformamide DMF.
It is molten that 3. the Sulfo-SMCC that step 2 is prepared is added into alkaline phosphatase enzyme solutions (AP solution, concentration 20mg/ml)
Liquid, alkaline phosphatase and Sulfo-SMCC mol ratios 1：15, mix at once, 25 DEG C stand reaction 15 minutes.
4. 1M glycine solution, glycine and Sulfo- are added in the AP solution for terminating to obtain to step 3 priming reaction
The mol ratio of SMCC reagents is 1:20, mix at once, 25 DEG C stand reaction 10 minutes.
5. step 1 to be reacted to the antibody-solutions desalination at once of end, (100mM triethanolamines are replaced in cross-linking buffer
Buffer solution, pH7.3), ultraviolet spectrophotometry measures antibody concentration；
6. step 4 to be reacted to the AP solution desalination at once of end, replace in cross-linking buffer that (100mM triethanolamines delay
Fliud flushing, pH7.3), ultraviolet spectrophotometry measures AP concentration；
7. the AP solution that step 6 has activated is added immediately in the antibody-solutions activated to step 5, the L-FABP marks of activation
The mol ratio for remembering antibody and the alkaline phosphatase of activation is 1：2, fully mix.2~8 DEG C stand reaction 24 hours.
8. weighing appropriate NEM reagents, it is made into antibody activation buffer solution (100mM Triethanolamine buffers, pH7.3)
12.5mg/mL solution.
9. the NEM solution that 1 percent debulking steps 10 are prepared is added in the solution terminated to step 7 cross-linking reaction, fully
Mix, be stored at room temperature reaction 30 minutes.Close unnecessary sulfydryl.
10. the cross-linking agent solution that step 9 terminating reaction terminates is purified by modes such as dialysis, desalination or ultrafiltration concentrations.
11. concentration will be determined enzyme conjugates concentrate after purification, isometric glycerine is added, is fully mixed, -20 DEG C of preservations are standby
With.
Embodiment 3
The present embodiment provides a kind of coated method of magnetic bead, comprises the following steps：
1. L-FABP coated antibodies are stored in pH5.0 100mM MES buffer solutions, concentration 1mg/mL；
2. taking the carboxyl magnetic bead solution of 100 times of antibody weight, carboxyl magnetic bead particle diameter is 3 μm, and Magneto separate abandons supernatant；
3. washing：The magnetic bead that above-mentioned steps obtain is dissolved again with pH5.0 100mM MES buffer solutions, and Magneto separate is abandoned
Clearly.
4. repeat step 3 is once
5. add the magnetic bead that appropriate 100mM MES buffer solution steps 4 obtain.
6. appropriate NHS reagents are weighed, the solution for being 10mg/mL into concentration with pH5.0 100mM MES buffer solutions；
7. appropriate EDC reagents are weighed, the solution for being 10mg/mL into concentration with pH5.0 100mM MES buffer solutions；
8. the magnetic bead solution obtained to step 5 adds the NHS solution of the step 6 of 0.23 times of magnetic bead weight and enters 0.1 times of magnetic
The EDC solution of the step 7 of pearl weight, reacted at room temperature 30 minutes on blending instrument.
9. reacting the magnetic bead solution Magneto separate after terminating, supernatant is abandoned.
10. add the magnetic bead that appropriate 100mM MES buffer solution steps 9 obtain.
11. the L-FABP coated antibodies of step 1 are added in the magnetic bead that step 10 obtains, L-FABP coated antibodies and carboxylic
Base magnetic bead solution quality ratio is 1:100, room temperature cross-linking reaction 16 hours on blending instrument.
After 12. crosslinking terminates, Magneto separate, supernatant, which is collected, surveys crosslinking rate.Repeat step 3 is twice.
13. the magnetic bead obtained to step 12 adds magnetic bead confining liquid CE210, reacted at room temperature 16 hours on blending instrument.
14. above-mentioned reaction terminate after magnetic bead solution Magneto separate, abandon supernatant.
15. wash magnetic bead 3 times with magnetic bead cleaning fluid.
16. the magnetic bead after washing is resuspended in magnetic bead and preserved in liquid, 2~8 DEG C save backup.
Embodiment 4
The present embodiment provides a kind of coated method of magnetic bead, comprises the following steps：
1. L-FABP coated antibodies are stored in pH5.0 100mM MES buffer solutions, concentration 5mg/mL；
2. taking the carboxyl magnetic bead solution of 200 times of antibody weight, carboxyl magnetic bead particle diameter is 1.5 μm, and Magneto separate abandons supernatant；
3. washing：The magnetic bead that above-mentioned steps obtain is dissolved again with pH5.0 100mM MES buffer solutions, and Magneto separate is abandoned
Clearly.
4. repeat step 3 is once
5. add the magnetic bead that appropriate 100mM MES buffer solution steps 4 obtain.
6. appropriate NHS reagents are weighed, the solution for being 10mg/mL into concentration with pH5.0 100mM MES buffer solutions；
7. appropriate EDC reagents are weighed, the solution for being 10mg/mL into concentration with pH5.0 100mM MES buffer solutions；
8. the magnetic bead solution obtained to step 5 adds the NHS solution of the step 6 of 0.23 times of magnetic bead weight and enters 0.1 times of magnetic
The EDC solution of the step 7 of pearl weight, reacted at room temperature 30 minutes on blending instrument.
9. reacting the magnetic bead solution Magneto separate after terminating, supernatant is abandoned.
10. add the magnetic bead that appropriate 100mM MES buffer solution steps 9 obtain.
11. the L-FABP coated antibodies of step 1 are added in the magnetic bead that step 10 obtains, L-FABP coated antibodies and carboxylic
Base magnetic bead solution quality ratio is 1:200, room temperature cross-linking reaction 24 hours on blending instrument.
After 12. crosslinking terminates, Magneto separate, supernatant, which is collected, surveys crosslinking rate.Repeat step 3 is twice.
13. the magnetic bead obtained to step 12 adds magnetic bead confining liquid CE210, reacted at room temperature 16 hours on blending instrument.
14. above-mentioned reaction terminate after magnetic bead solution Magneto separate, abandon supernatant.
15. wash magnetic bead 3 times with magnetic bead cleaning fluid.
16. the magnetic bead after washing is resuspended in magnetic bead and preserved in liquid, 2~8 DEG C save backup.
Embodiment 5
The present embodiment provides a kind of detection kit, including the first liquid storage component, the second liquid storage component, the 3rd liquid storage group
Part, the 4th liquid storage component, the 5th liquid storage component, the 6th liquid storage component, the 7th liquid storage component.Wherein, the first liquid storage component is used for
L-FABP calibration objects are stored, the second liquid storage component is used to store enzyme conjugates working solution, and the 3rd liquid storage component is used to store magnetic bead
Working solution, the 4th liquid storage component are used to store cleaning fluid, and the 5th liquid storage component is used to store substrate solution, and the 6th liquid storage component is used
In storage pre-treatment reagent, the 7th liquid storage component is used to store L-FABP quality-control products.
As one embodiment of the present of invention, magnetic bead working solution includes the carboxyl magnetic bead for being marked with L-FABP antibody；Magnetic bead
Particle diameter be 3 μm, the concentration for being marked with the carboxyl magnetic bead of L-FABP antibody is 0.5mg/ml；Enzyme conjugates working solution includes alkalescence
The anti-L-FABP antibody of phosphatase enzyme mark, the concentration of the anti-L-FABP antibody of alkali phosphatase enzyme mark is 2 μ g/ml；L-FABP schools
Quasi- product and L-FABP quality-control product is the Tris solution containing L-FABP antigens；Substrate solution is enzyme-catalyzed chemical luminescence substrate solution；
Pre-treatment reagent is the buffer solution for including NaCl, sucrose, glycerine, polysorbas20 and preservative.
The present embodiment provides a kind of liver fatty acid binding protein detection kit component collocation method, specific as follows：
1. calibration object and quality-control product configuration
L-FABP antigens are determined into concentration by tracing to the source, L-FABP calibration objects and L- are prepared with calibration object diluted
FABP quality-control products.Such as the concentration of calibration object is respectively：5,10,20,40,80,160ng/ml, quality-control product concentration is 100ng/ml
The specific composition of calibration object dilution is：50mM Tris, 250mmol/L NaCl, 1% horse serum, 1% excipient
With 1% preservative, pH 8.0；Excipient is preferably mannitol；Preservative is preferably Proclin-300.
2. pre-treatment reagent
Pre-treatment reagent is buffer solution, contains 100mM Tris；150mM NaCl；5% sucrose；5% glycerine；0.1%BSA
With 0.15% preservative；Preservative is preferably Proclin-300.
3. enzyme conjugates working solution
Enzyme conjugates concentrate in embodiment 1 or embodiment 2 is diluted with enzyme combination diluent, is configured to suitable dense
The kit of degree enzyme conjugates working solution, enzyme conjugates working solution concentration are 2 μ g/ml.The specific composition of enzyme combination diluent
For：50mM MOPS, 250mmol/L NaCl, 1%BSA, 5% sucrose, 5% glycerine, 0.1% polysorbas20,0.15mmol/L
ZnCl2、0.15mmol/L MgCl2With 0.15% preservative, pH pH6.5；Preservative is preferably Proclin-300.
4. magnetic bead working solution (M)
Magnetic bead in embodiment 3 or embodiment 4 is preserved into liquid magnetic bead diluted, is configured to the reagent of suitable concentration
Box magnetic bead working solution, magnetic bead working solution concentration are 0.5mg/ml.The specific composition of magnetic bead dilution is：50mM HEPES,
250mmol/LNaCl, 1%BSA, 1% sucrose, 1% gelatin, 0.1% polysorbas20,1%PVP360 and 3% preservative, pH are
8.0；Preservative is preferably Proclin-300.
5. substrate solution
Accurately weigh 3- (2- spirals adamantane) -4- methoxyl groups -4- (3- phosphorus oxygens acyl)-phenyl -1,2- dioxanes two
Sodium salt (AMPPD) 0.5g, trishydroxymethylaminomethane 18g, NaCl 100g, hexadecyltrimethylammonium chloride 0.02g, water are fixed
It is 9.4 ± 0.05 to hold to 1000mL, adjustment chemical luminous substrate pH value of solution；This chemical luminous substrate is for alkaline phosphatase
Epidioxy ethane substrate, it is necessary to be kept in dark place under the conditions of 2~8 DEG C, the used time slowly mixes again.
6. cleaning fluid
Trishydroxymethylaminomethane 1211.4mg, NaCl 9g, polysorbas20 1g, water 900mL are added into container successively, is mixed
It is even to be dissolved to each component, pH value of solution is adjusted to 8.0 ± 0.5 with sodium hydroxide solution, is added water and is settled to 1000mL, mixes 30 points
Clock, 0.22 μm of filtering obtain cleaning fluid.
Embodiment 6
The present embodiment provides a kind of detection kit, including the first liquid storage component, the second liquid storage component, the 3rd liquid storage group
Part, the 4th liquid storage component, the 5th liquid storage component, the 6th liquid storage component, the 7th liquid storage component.Wherein, the first liquid storage component is used for
L-FABP calibration objects are stored, the second liquid storage component is used to store enzyme conjugates working solution, and the 3rd liquid storage component is used to store magnetic bead
Working solution, the 4th liquid storage component are used to store cleaning fluid, and the 5th liquid storage component is used to store substrate solution, and the 6th liquid storage component is used
In storage pre-treatment reagent, the 7th liquid storage component is used to store L-FABP quality-control products.
As one embodiment of the present of invention, magnetic bead working solution includes the carboxyl magnetic bead for being marked with L-FABP antibody；Magnetic bead
Particle diameter be 3 μm, the concentration for being marked with the carboxyl magnetic bead of L-FABP antibody is 0.1/ml；Enzyme conjugates working solution includes alkaline phosphorus
The anti-L-FABP antibody of sour enzyme mark, the concentration of the anti-L-FABP antibody of alkali phosphatase enzyme mark is 0.5 μ g/ml；L-FABP schools
Quasi- product and L-FABP quality-control product is the Tris solution containing L-FABP antigens；Substrate solution is enzyme-catalyzed chemical luminescence substrate solution；
Pre-treatment reagent is the buffer solution for including NaCl, sucrose, glycerine, polysorbas20 and preservative.
The present embodiment provides a kind of liver fatty acid binding protein detection kit component collocation method, specific as follows：
1. calibration object and quality-control product configuration
L-FABP antigens are determined into concentration by tracing to the source, L-FABP calibration objects and L- are prepared with calibration object diluted
FABP quality-control products.Such as the concentration of calibration object is respectively：5,10,20,40,80,160ng/ml, quality-control product concentration is 100ng/ml
The specific composition of calibration object dilution is：50mM Tris, 250mmol/L NaCl, 1% horse serum, 1% excipient
With 1% preservative, pH 8.0；Excipient is preferably mannitol；Preservative is preferably Proclin-300.
2. pre-treatment reagent
Pre-treatment reagent is buffer solution, contains 100mM Tris；150mM NaCl；5% sucrose；5% glycerine；0.1%BSA
With 0.15% preservative；Preservative is preferably Proclin-300.
3. enzyme conjugates working solution
Enzyme conjugates concentrate in embodiment 1 or embodiment 2 is diluted with enzyme combination diluent, is configured to suitable dense
The kit of degree enzyme conjugates working solution, enzyme conjugates working solution concentration are 0.5 μ g/ml.Enzyme combination diluent specifically into
It is divided into：20mM MOPS, 250mmol/L NaCl, 1%BSA, 5% sucrose, 5% glycerine, 0.1% polysorbas20,0.15mmol/
LZnCl2、0.15mmol/L MgCl2With 0.15% preservative, pH pH6.5；Preservative is preferably Proclin-300.
4. magnetic bead working solution (M)
Magnetic bead in embodiment 3 or embodiment 4 is preserved into liquid magnetic bead diluted, is configured to the reagent of suitable concentration
Box magnetic bead working solution, magnetic bead working solution concentration are 0.1mg/ml.The specific composition of magnetic bead dilution is：20mM HEPES,
250mmol/LNaCl, 1%BSA, 1% sucrose, 1% gelatin, 0.1% polysorbas20,1%PVP360 and 3% preservative, pH are
8.0；Preservative is preferably Proclin-300.
5. substrate solution
Accurately weigh 3- (2- spirals adamantane) -4- methoxyl groups -4- (3- phosphorus oxygens acyl)-phenyl -1,2- dioxanes two
Sodium salt (AMPPD) 0.5g, trishydroxymethylaminomethane 18g, NaCl 100g, hexadecyltrimethylammonium chloride 0.02g, water are fixed
It is 9.4 ± 0.05 to hold to 1000mL, adjustment chemical luminous substrate pH value of solution；This chemical luminous substrate is for alkaline phosphatase
Epidioxy ethane substrate, it is necessary to be kept in dark place under the conditions of 2~8 DEG C, the used time slowly mixes again.
6. cleaning fluid
Trishydroxymethylaminomethane 1211.4mg, NaCl 9g, polysorbas20 1g, water 900mL are added into container successively, is mixed
It is even to be dissolved to each component, pH value of solution is adjusted to 8.0 ± 0.5 with sodium hydroxide solution, is added water and is settled to 1000mL, mixes 30 points
Clock, 0.22 μm of filtering obtain cleaning fluid.
Embodiment 7
The present embodiment provides a kind of detection kit, including the first liquid storage component, the second liquid storage component, the 3rd liquid storage group
Part, the 4th liquid storage component, the 5th liquid storage component, the 6th liquid storage component, the 7th liquid storage component.Wherein, the first liquid storage component is used for
L-FABP calibration objects are stored, the second liquid storage component is used to store enzyme conjugates working solution, and the 3rd liquid storage component is used to store magnetic bead
Working solution, the 4th liquid storage component are used to store cleaning fluid, and the 5th liquid storage component is used to store substrate solution, and the 6th liquid storage component is used
In storage pre-treatment reagent, the 7th liquid storage component is used to store L-FABP quality-control products.
As one embodiment of the present of invention, magnetic bead working solution includes the carboxyl magnetic bead for being marked with L-FABP antibody；Magnetic bead
Particle diameter be 3 μm, the concentration for being marked with the carboxyl magnetic bead of L-FABP antibody is 1mg/ml；Enzyme conjugates working solution includes alkaline phosphorus
The anti-L-FABP antibody of sour enzyme mark, the concentration of the anti-L-FABP antibody of alkali phosphatase enzyme mark is 1 μ g/ml；L-FABP is calibrated
Product and L-FABP quality-control products are the Tris solution containing L-FABP antigens；Substrate solution is enzyme-catalyzed chemical luminescence substrate solution；Before
Reagent treatment is to include the buffer solution of NaCl, sucrose, glycerine, polysorbas20 and preservative.
The present embodiment provides a kind of liver fatty acid binding protein detection kit component collocation method, specific as follows：
1. calibration object and quality-control product configuration
L-FABP antigens are determined into concentration by tracing to the source, L-FABP calibration objects and L- are prepared with calibration object diluted
FABP quality-control products.Such as the concentration of calibration object is respectively：5,10,20,40,80,160ng/ml, quality-control product concentration is 100ng/ml
The specific composition of calibration object dilution is：50mM Tris, 250mmol/L NaCl, 1% horse serum, 1% excipient
With 1% preservative, pH 8.0；Excipient is preferably mannitol；Preservative is preferably Proclin-300.
2. pre-treatment reagent
Pre-treatment reagent is buffer solution, contains 100mM Tris；150mM NaCl；5% sucrose；5% glycerine；0.1%BSA
With 0.15% preservative；Preservative is preferably Proclin-300.
3. enzyme conjugates working solution
Enzyme conjugates concentrate in embodiment 1 or embodiment 2 is diluted with enzyme combination diluent, is configured to suitable dense
The kit of degree enzyme conjugates working solution, enzyme conjugates working solution concentration are 1.0 μ g/ml.Enzyme combination diluent specifically into
It is divided into：30mM MOPS, 250mmol/L NaCl, 1%BSA, 5% sucrose, 5% glycerine, 0.1% polysorbas20,0.15mmol/L
ZnCl2、0.15mmol/L MgCl2With 0.15% preservative, pH pH6.5；Preservative is preferably Proclin-300.
4. magnetic bead working solution (M)
Magnetic bead in embodiment 3 or embodiment 4 is preserved into liquid magnetic bead diluted, is configured to the reagent of suitable concentration
Box magnetic bead working solution, magnetic bead working solution concentration are 1mg/ml.The specific composition of magnetic bead dilution is：30mM HEPES,
250mmol/LNaCl, 1%BSA, 1% sucrose, 1% gelatin, 0.1% polysorbas20,1%PVP360 and 3% preservative, pH are
8.0；Preservative is preferably Proclin-300.
5. substrate solution
Accurately weigh 3- (2- spirals adamantane) -4- methoxyl groups -4- (3- phosphorus oxygens acyl)-phenyl -1,2- dioxanes two
Sodium salt (AMPPD) 0.5g, trishydroxymethylaminomethane 18g, NaCl 100g, hexadecyltrimethylammonium chloride 0.02g, water are fixed
It is 9.4 ± 0.05 to hold to 1000mL, adjustment chemical luminous substrate pH value of solution；This chemical luminous substrate is for alkaline phosphatase
Epidioxy ethane substrate, it is necessary to be kept in dark place under the conditions of 2~8 DEG C, the used time slowly mixes again.
6. cleaning fluid
Trishydroxymethylaminomethane 1211.4mg, NaCl 9g, polysorbas20 1g, water 900mL are added into container successively, is mixed
It is even to be dissolved to each component, pH value of solution is adjusted to 8.0 ± 0.5 with sodium hydroxide solution, is added water and is settled to 1000mL, mixes 30 points
Clock, 0.22 μm of filtering obtain cleaning fluid.
Embodiment 8
The present embodiment provides to be contained using liver fatty acid binding protein in embodiment 5-7 any kits detection urine
The step of amount：
1. respectively plus 25 μ l L-FABP calibration objects, L-FABP quality-control products and urine specimen are into corresponding test tube；
2. the mixing of 25 μ l pre-treatment reagents is added in each test tube；
Mixed after 3. 50 μ l enzyme conjugates working solutions and 50 μ l magnetic bead working solutions are added in each test tube；42 DEG C of reactions
5min, obtain the first reaction solution；
4. above-mentioned first reaction solution is carried out into Magneto separate, magnetic bead is collected；300 μ l cleaning fluids cleaning magnetic is added in each test tube
Pearl, repeat 3-5 times, remove cleaning fluid；
5. adding 100 μ l substrate solutions in each test tube, luminous value is detected after mixing.
Embodiment 9
The present embodiment provides to be contained using liver fatty acid binding protein in embodiment 5-7 any kits detection urine
The step of amount：
1. respectively plus 25 μ l L-FABP calibration objects, L-FABP quality-control products and urine specimen are into corresponding test tube；
2. the mixing of 25 μ l pre-treatment reagents is added in each test tube；
Mixed after 3. 50 μ l enzyme conjugates working solutions and 50 μ l magnetic bead working solutions are added in each test tube；37 DEG C of reactions
10min, obtain the first reaction solution；
4. above-mentioned first reaction solution is carried out into Magneto separate, magnetic bead is collected；300 μ l cleaning fluids cleaning magnetic is added in each test tube
Pearl, repeat 3-5 times, remove cleaning fluid；
5. adding 100 μ l substrate solutions in each test tube, luminous value is detected after mixing.
The linear verification of experimental example 1
Kit Plays product or quality-control product are taken, determine 3 times, averages, and is as a result returned with expected concentration at every
Straight line, regression coefficient r ＞ 0.99 are calculated, illustrate the dilution good linearity of kit provided by the invention.
The precision of experimental example 2 is verified
The urine high level quality-control product with traceability, each portion of low value quality-control product are taken, 10 inspections are carried out to every part of quality-control product
Survey, totally 10 testing results will calculate average value, standard value.According to coefficient of variation CV=(standard deviation/average value) ×
100%, it is calculated：CV1(80ng/mL)=4%, CV2(20ng/mL)=2%.It follows that kit provided by the invention
With higher precision.
The sensitivity of experimental example 3 checking,
Take the quality-control product with traceability to be diluted to detection range lower limit (0.5ng/ml) to be nearby measured, replication
3 times, average value is calculated, is compareed afterwards with quality-control product target value.As a result show, kit detected value of the invention connects with target value
Closely, illustrate that kit provided by the invention has higher sensitivity.
The detection range of experimental example 4 is verified
The standard items with traceability are taken, luminescence phenomenon has been detected whether respectively after diluting different multiples, has as a result shown,
In the range of concentration is 0.5-500ng/ml, there is luminescence phenomenon, the kit detection range for showing the present invention is 0.5-
500ng/ml。
Obviously, above-described embodiment is only intended to clearly illustrate example, and is not the restriction to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of change or
Change.There is no necessity and possibility to exhaust all the enbodiments.And the obvious change thus extended out or
Among changing still in the protection domain of the invention.
Claims (11)
- A kind of 1. liver fatty acid binding protein detection kit, it is characterised in that includingFirst liquid storage component, for storing L-FABP calibration objects；Second liquid storage component, for storing enzyme conjugates working solution；3rd liquid storage component, for storing magnetic bead working solution；4th liquid storage component, for storing cleaning fluid；5th liquid storage component, for storing substrate solution；6th liquid storage component, for storing pre-treatment reagent；7th liquid storage component, for storing L-FABP quality-control products.
- 2. liver fatty acid binding protein detection kit according to claim 1, it is characterised in that the magnetic bead work Make the carboxyl magnetic bead that liquid includes being marked with L-FABP antibody；The particle diameter of the magnetic bead is 1-5 μm.
- 3. liver fatty acid binding protein detection kit according to claim 2, it is characterised in that it is described mark have The concentration of the carboxyl magnetic bead of L-FABP antibody is 0.1~1mg/ml.
- 4. liver fatty acid binding protein detection kit according to claim 1, it is characterised in that the enzyme combines Thing working solution includes the anti-L-FABP antibody of alkali phosphatase enzyme mark.
- 5. liver fatty acid binding protein detection kit according to claim 4, it is characterised in that the alkaline phosphorus The concentration of the anti-L-FABP antibody of sour enzyme mark is 0.5~2 μ g/ml.
- 6. liver fatty acid binding protein detection kit according to claim 1, it is characterised in that the L-FABP Calibration object and the L-FABP quality-control products are the Tris buffer solutions containing L-FABP antigens.
- 7. liver fatty acid binding protein detection kit according to claim 1, it is characterised in that the substrate is molten Liquid is enzyme-catalyzed chemical luminescence substrate solution.
- 8. liver fatty acid binding protein detection kit according to claim 1, it is characterised in that the pre-treatment Reagent is the buffer solution for including NaCl, sucrose, glycerine, polysorbas20 and preservative.
- A kind of 9. method for detecting liver fatty acid binding protein, it is characterised in that usage right is required described in any one of 1-8 Detection kit detection, comprise the following steps：(1) sample to be tested, L-FABP calibration objects and L-FABP quality-control products are added separately in pre-treatment reagent, added after mixing Enter magnetic bead working solution, enzyme conjugates working solution, 37 DEG C of -42 DEG C of reaction 5min-10min, obtain the first reaction solution after mixing；(2) first reaction solution is subjected to Magneto separate, collects magnetic bead；The magnetic bead is cleaned by cleaning fluid；(3) substrate solution is added in the magnetic bead after cleaning, obtains mixed liquor；Detect the luminous intensity of the mixed liquor.
- 10. according to the method for claim 9, it is characterised in that the sample to be tested, the L-FABP calibration objects or described The ratio of L-FABP quality-control products and the pre-treatment reagent is 1:1.
- 11. according to the method for claim 9, it is characterised in that the magnetic bead working solution, the enzyme conjugates working solution with The ratio of the substrate solution is 1:1:2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711147897.0A CN107656072A (en) | 2017-11-17 | 2017-11-17 | Liver fatty acid binding protein detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711147897.0A CN107656072A (en) | 2017-11-17 | 2017-11-17 | Liver fatty acid binding protein detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107656072A true CN107656072A (en) | 2018-02-02 |
Family
ID=61121703
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711147897.0A Pending CN107656072A (en) | 2017-11-17 | 2017-11-17 | Liver fatty acid binding protein detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107656072A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN111289758A (en) * | 2020-03-10 | 2020-06-16 | 苏州翊讯生物科技有限公司 | Kit for H-FABP quantitative detection and H-FABP quantitative detection method |
| CN112771377A (en) * | 2018-09-28 | 2021-05-07 | Cmic控股有限公司 | Method for examining liver disease, examination kit therefor, and diagnostic agent therefor |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849469A (en) * | 2015-04-16 | 2015-08-19 | 广州市达瑞生物技术股份有限公司 | Kit for detecting NGAL content and preparation method thereof |
| CN105628914A (en) * | 2016-02-04 | 2016-06-01 | 广州科方生物技术有限公司 | Diluent enabling stability for acridinium ester antigen-antibody conjugate and preparation method of diluent |
| CN105823893A (en) * | 2016-05-17 | 2016-08-03 | 北京豪迈生物工程有限公司 | IV type collagen test kit and test method thereof |
| CN105973878A (en) * | 2016-05-17 | 2016-09-28 | 北京豪迈生物工程有限公司 | Laminin (LN) testing kit and testing method thereof |
| CN106053826A (en) * | 2016-05-12 | 2016-10-26 | 威海威高生物科技有限公司 | III type procollagen N-terminal peptide quantitative measurement kit and preparation method thereof |
| CN106443018A (en) * | 2016-08-31 | 2017-02-22 | 上海科华生物工程股份有限公司 | Myoglobin monoclonal abzyme marking compound and preparation method thereof and detection test kit |
| CN106442482A (en) * | 2016-11-22 | 2017-02-22 | 无锡艾科瑞思产品设计与研究有限公司 | Detection kit for melamine in food |
| CN106501504A (en) * | 2016-09-26 | 2017-03-15 | 王键 | Laminin，LN test kit and preparation method thereof |
| CN106568976A (en) * | 2016-11-04 | 2017-04-19 | 江苏福隆生物技术有限公司 | Neuron-specific enolase stabilizer and preparation method thereof |
| CN106771133A (en) * | 2016-11-30 | 2017-05-31 | 三诺生物传感股份有限公司 | A kind of composition and its application as enzyme mark compound preservation liquid |
| CN106932583A (en) * | 2015-12-29 | 2017-07-07 | 北京大成生物工程有限公司 | Human epidermal growth factor acceptor Her-2/neu immue quantitative detection reagent boxes and preparation method and application |
-
2017
- 2017-11-17 CN CN201711147897.0A patent/CN107656072A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849469A (en) * | 2015-04-16 | 2015-08-19 | 广州市达瑞生物技术股份有限公司 | Kit for detecting NGAL content and preparation method thereof |
| CN106932583A (en) * | 2015-12-29 | 2017-07-07 | 北京大成生物工程有限公司 | Human epidermal growth factor acceptor Her-2/neu immue quantitative detection reagent boxes and preparation method and application |
| CN105628914A (en) * | 2016-02-04 | 2016-06-01 | 广州科方生物技术有限公司 | Diluent enabling stability for acridinium ester antigen-antibody conjugate and preparation method of diluent |
| CN106053826A (en) * | 2016-05-12 | 2016-10-26 | 威海威高生物科技有限公司 | III type procollagen N-terminal peptide quantitative measurement kit and preparation method thereof |
| CN105823893A (en) * | 2016-05-17 | 2016-08-03 | 北京豪迈生物工程有限公司 | IV type collagen test kit and test method thereof |
| CN105973878A (en) * | 2016-05-17 | 2016-09-28 | 北京豪迈生物工程有限公司 | Laminin (LN) testing kit and testing method thereof |
| CN106443018A (en) * | 2016-08-31 | 2017-02-22 | 上海科华生物工程股份有限公司 | Myoglobin monoclonal abzyme marking compound and preparation method thereof and detection test kit |
| CN106501504A (en) * | 2016-09-26 | 2017-03-15 | 王键 | Laminin，LN test kit and preparation method thereof |
| CN106568976A (en) * | 2016-11-04 | 2017-04-19 | 江苏福隆生物技术有限公司 | Neuron-specific enolase stabilizer and preparation method thereof |
| CN106442482A (en) * | 2016-11-22 | 2017-02-22 | 无锡艾科瑞思产品设计与研究有限公司 | Detection kit for melamine in food |
| CN106771133A (en) * | 2016-11-30 | 2017-05-31 | 三诺生物传感股份有限公司 | A kind of composition and its application as enzyme mark compound preservation liquid |
Non-Patent Citations (1)
| Title |
|---|
| 《中国教育装备采购网》: "猪肝脏型脂肪酸结合蛋白（L-FABP）酶联免疫分析（ELISA）试剂盒说明书", 《中国教育装备采购网》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN112771377A (en) * | 2018-09-28 | 2021-05-07 | Cmic控股有限公司 | Method for examining liver disease, examination kit therefor, and diagnostic agent therefor |
| CN111289758A (en) * | 2020-03-10 | 2020-06-16 | 苏州翊讯生物科技有限公司 | Kit for H-FABP quantitative detection and H-FABP quantitative detection method |
| CN111289758B (en) * | 2020-03-10 | 2024-02-09 | 苏州翊讯生物科技有限公司 | Kit for quantitative detection of H-FABP and method for quantitative detection of H-FABP |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102944679A (en) | Kit for performing retinol binding protein detection by using latex turbidimetry | |
| CN107656071A (en) | A kind of NT ProBNP detection kits and its application method | |
| CN102680698B (en) | Neutrophil gelatinase-associated lipocalin (NGAL) measures kit (latex enhancing immune turbidimetry) | |
| CN104849469A (en) | Kit for detecting NGAL content and preparation method thereof | |
| JP4576499B2 (en) | Protein measurement method | |
| CN207248894U (en) | Bladder chalone C time resolution detection card and kit | |
| CN107918022A (en) | A kind of cTnI detection kits and its application method | |
| CN107656072A (en) | Liver fatty acid binding protein detection kit | |
| CN104215769A (en) | Latex enhanced immunoturbidimetry NGAL detection kit | |
| CN107942051A (en) | A kind of D dimer detection kits and its application method | |
| CN107907691A (en) | A kind of myoglobin assay kit and its application method | |
| CN107942047A (en) | Interleukin-18 detection kit | |
| CN107957495A (en) | A kind of CK-MB detection kits and its application method | |
| CN107831163A (en) | A kind of chemiluminescence detection kit of thyroglobulin and preparation method thereof | |
| CN104535770A (en) | Myoglobin determination kit of compound antibody | |
| CN106442967A (en) | Method for detecting affinity of monoclonal antibodies of transmembrane proteins | |
| CN105486875A (en) | Retinol conjugated protein detection kit | |
| CN107831314A (en) | A kind of N middle-ends BGP chemiluminescence detection kit and preparation method thereof | |
| TW201135228A (en) | Analyte measurement apparatus and method | |
| US20090111091A1 (en) | Specimen pretreatment liquid, kit for measuring virus, and method for detecting virus | |
| CN107942072A (en) | 1 detection kit of the injury of kidney factor | |
| CN107918021A (en) | Neutrophil gelatinase-associated lipocalin detection kit | |
| CN108445215A (en) | A kind of kit and preparation method quantitatively detecting myeloperoxidase | |
| CN105181969B (en) | A kind of neutrophil gelatinase-associated lipocalin reagent box for detecting content | |
| CN202631543U (en) | D-dimer fluorescent immune quantitative measurement test strip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180202 |
|
| RJ01 | Rejection of invention patent application after publication |