CN107918020A - Neutrophil gelatinase-associated lipocalin assay kit - Google Patents
Neutrophil gelatinase-associated lipocalin assay kit Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
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Abstract
The invention belongs to medicine and technological field of biochemistry, is specifically a kind of neutrophil gelatinase-associated lipocalin detection kit.The kit is made of reagent R1 and reagent R2, and reagent R1 includes：Biological buffer, coagulant, surfactant, osmotic pressure regulator and water；Reagent R2 includes：It is coated with latex particle, biological buffer, sealer, preservative and the water of neutrophil gelatinase-associated lipocalin antibody.The neutrophil gelatinase-associated lipocalin detection kit is measured using latex-enhanced turbidimetry, relative to traditional NGAL detection kits, have the advantages that rapid sensitive, accuracy it is good, it is specific it is high, stability is good.
Description
Technical field
The present invention relates to medicine and technological field of biochemistry, is specifically a kind of measure neutrophil leucocyte gelatinase correlation fat
The kit of matter transporter.
Background technology
In recent years, as living standards of the people improve, and habits and customs and eating habit change, the incidence of nephrosis by
Year increases.The big multi-pathogenesis of kidney trouble is complicated, course of disease delay, and with the danger of increase angiocardiopathy, has become
Major global public health problem.Kidney trouble can be divided into two main syndromes --- acute injury of kidney (acute kidney
Injury, AKI) and chronic kidney disease (chronic kidney disease, CKD).AKI refers to happen suddenly and lasting kidney function
The one group of clinical syndrome that can decline, shows as azotemia, Water-Electrolyte and disturbance of acid-base balance and each system disease of whole body
Shape, can be one of clinical common Severe acute disease with oliguresis or anuria.And AKI has significantly with the CKD then occurred
Ground correlation, AKI may cause the generation of CKD.2012, professor Wang Haiyan waited《Lancet》That is delivered on magazine is first
National CKD cross-section surveys show, Chinese CKD illness rates are up to 10.8%, and patient estimated nearly 1.2 hundred million, but CKD fall ill it is past
Toward concealment, most of patient, which experienced, before there are obvious clinical symptoms longer hidden attacks the stage.Therefore find a kind of reliable
Biomarker, kidney trouble develop early stage carry out efficient diagnosis, it is treated and prognosis has highly important clinic
Meaning.
Research in recent years finds, a newcomer --- the neutrophil leucocyte gelatinase correlation fat in lipocalin protein family
Matter transporter (neutrophil gelatinase-associated lipocalin, NGAL), it is close with AKI and CKD
Correlation, is a novel markings thing for predicting AKI, while is also monitoring CKD progress and the biological indicator of kidney function damage.
NGAL is had found that mankind NGAL genes are monocistron, positioning by Kjeldsen when studying human neutrophils' granulocyte in 1993
In on Chromosome 9 long-armed (9q34), total length 5869bp, including 5 ' the end nontranscribed domains of 1695bp, the 3 ' ends of 178bp are non-
The original transcriptional domain of transcriptional domain and 3696bp.Other members are similar to lipocalin protein family, and NGAL albumen has a guarantor
The tertiary structure kept, the structure cause NGAL to have the ability to combine some compounds mainly formed by hydrophobic small molecules, even
Can also be in cell surface binding specificity acceptor.NGAL can transport some lipophilic molecules of inducing inflammatory reaction, as blood is small
Plate activation factor (PAF), leukotriene B4 (LTB4) and lipopolysaccharides (LPS) etc.；MMP-9 activity is adjusted, and may participate in removing scorching
Disease medium, adjusts immune response, suppresses the processes such as occurrence and development, the infiltration metastasis of Apoptosis and tumour；By with iron content
Siderophore combine, form NGAL- siderophores-iron complexes, participate in the generation, development and repair process of kidney.Normal
Under physiological status, in addition to neutrophil leucocyte, NGAL much organizes such as bronchus, stomach, small intestine, pancreas, kidney in the mankind
Epithelial cell in have low expression level.And under pathological state, the epithelial cell of these tissues then has a large amount of NGAL to express,
Particularly in the proximal renal tubular epithelial cells of damage.Therefore by detecting the content of NGAL, it can predict and examine well
Disconnected AKI and CKD.
The detection method of NGAL mainly has enzyme-linked immunization, radioimmunology, chemoluminescence method and latex to be immunized at present
Turbidimetry.Enzyme-linked immunization the degree of automation is not high, and is affected by human factors larger；Radioimmunology is dirty there is environment
Dye problem；Though chemoluminescence method sensitivity is high, the measure range of linearity is smaller and testing cost is higher, it is necessary to which specified chemical is sent out
Optical detector；Common turbidimetry there are sensitivity it is inadequate the shortcomings that.The above method is difficult to meet that clinical labororatory is fast
Speed, the demand of mass detection.
Latex-enhanced turbidimetry is a kind of relatively stable, the accurate homogeneous immunoturbidimetry inspection of body fluid albumen occurred in recent years
Survey method.Latex-enhanced turbidimetry is by the crosslinked monoclonal antibody of high molecular emulsion microsphere surface, and when antigen and crosslinking
After the microballoon for having antibody combines, it can rapidly flock together in a short time, change the absorbance of reaction solution.It is moreover, anti-
The change and the concentration of tested antigen for answering liquid absorbance have linear relationship within the specific limits, can be used to reflect tested antigen
Concentration.Latex immunoturbidimetry is compared with other above-mentioned several methods, and there are easy to operate, high sensitivity, the range of linearity
It is wide, pollution-free, the outstanding feature such as have a wide range of application, therefore, latex immunoturbidimetry has great researching value.
NGAL concentration is less than 10ng/ml in normal human urine, and concentration may be up in severe nephrotic's urine
More than 7000ng/ml.So broad concentration range, proposes detection kit sensitivity and linear measurement range higher
It is required that.Have the kit of NGAL concentration in Latex-enhanced immunoturbidimetric assay measure human plasma or urine currently on the market,
But these kits can not meet the wide range of linearity and highly sensitive requirement at the same time, it is therefore desirable to which diluted sample could measure
High concentration NGAL, this is using above bringing inconvenience.Existing Immunoturbidimetry, such as 104198732 Hes of patent CN
Kit detection sensitivity disclosed in patent CN 102680698 is all higher than being equal to 10ng/ml, and the range of linearity is in 6000ng/
Below ml, is difficult to meet clinical detection demand.
Therefore, it is necessary to develop NGAL latex enhancing immunes transmission a kind of while that there is high sensitivity and the wide range of linearity
Than turbid detection kit.
The content of the invention
For existing neutrophil gelatinase-associated lipocalin existing drawbacks described above during the test, this
Invention provide a kind of rapid sensitive, accuracy it is good, it is specific it is high, stability is good, liquid double reagent is easy to operate is suitable for facing
Bed is full-automatic or the neutrophil gelatinase-associated lipocalin assay kit of semiautomatic biochemistry analysis.
The present invention relates to a kind of NGAL detection kits, include reagent R1, reagent R2 and calibration object, wherein reagent R1 bags
Containing biological buffer, coagulant, surfactant, osmotic pressure regulator and water；Reagent R2 includes coating neutrophil leucocyte gelatin
Latex particle, biological buffer, sealer, preservative and the water of enzyme associated lipocalin antibody；Calibration object includes neutrality
Granulocyte gelatinase associated lipocalin, sodium chloride, bovine serum albumin(BSA), sucrose and Proclin300.
Wherein, in the reagent R2 latex particle of neutrophil gelatinase-associated lipocalin antibody particle diameter
It it is preferably 0.8-1.3 μm for 0.5-1.5 μm.
Wherein, the biological buffer is in trishydroxymethylaminomethane, MES, phosphate, MOPSO, DIPSO
It is one or more of；One or more of the coagulant in polyethylene glycol -200-10000 or Gentran 40 00-12000；Surface
Activating agent is Tween-20；Osmotic pressure regulator is sodium chloride；Sealer is bovine serum albumin；Preservative is sodium azide.
Preferably, the dosage of each component is in every 1 liter of reagent R1：Biological buffer 5-100g, coagulant 2-50g, surface
Activating agent 1-20ml, osmotic pressure regulator 5-20g；The dosage of each component is in every 1 liter of reagent R2：It is bright to be coated with neutrophil leucocyte
The latex particle 5-20mg of glue enzyme associated lipocalin antibody, biological buffer 5-100g, sealer 0.2-10g, anti-corrosion
Agent 0.2-10g.
In a specific embodiment, the dosage of each component is in every 1 liter of reagent R1：Trishydroxymethylaminomethane
12.114g, Macrogol 6000 4g, polysorbas20 10ml, sodium chloride 15g；The dosage of each component is in every 1 liter of reagent R2：Bag
By the latex particle 8mg of neutrophil gelatinase-associated lipocalin antibody, trishydroxymethylaminomethane 12.114g,
Bovine serum albumin 1g, sodium azide 1g.
Preferably, polyhexamethylene guanide and phenmethylol are also included in reagent R2.
In a preferred embodiment, the dosage of polyhexamethylene guanide is 0.5g/l in reagent R2, the use of phenmethylol
Measure as 0.2g/l.
Present invention also offers the system of the latex particle of coating neutrophil gelatinase-associated lipocalin antibody
Preparation Method, the preparation method are chemical crosslink technique：
(1) ps particle of the surface with carboxy functional group, 0.5-1.5 μm of diameter is diluted with MES buffer solutions
To final concentration of 0.5-3% (wt), 50-90mM EDAC are added, are uniformly mixed, room temperature concussion reaction 30-80 minutes；
(2) mixed liquor obtained by step (1) is centrifuged, abandons supernatant, with MES buffer solution for cleaning twice, final present latex particulate
MES buffer solutions are resuspended in, ultrasound is disperseed, and obtains particle dispersion liquid；
(3) with micro- in MES buffer solutions dilution neutrophil gelatinase-associated lipocalin antibody, with step (2)
Grain dispersion liquid mixing, when room temperature reaction 2-4 is small；
(4) by after reaction solution 18000rpm centrifugations 30min obtained by step (3), particulate, room temperature is resuspended with Tris buffer solutions
When capping 1-3 is small；
(5) reaction solution obtained by step (4) is centrifuged, abandons supernatant, with Tris buffer solution for cleaning latex 3 times, finally use R2
Reagent preserves liquid resuspension latex and precipitates and be diluted to 1%, is obtained after ultrasound is fully dispersed and is coated with neutrophil leucocyte gelatinase phase
Close the latex particle of lipocalin protein antibody.
Specifically, inventive concept of the invention is：(1) after big particle diameter latex surface coated antibody, it is affine with antigen
Higher, antigen-antibody reaction speed when drastically increasing low concentration NGAL of property, so as to improve the sensitivity of reagent；(2) when
When NGAL concentration is higher in sample to be tested, because the concentration of latex particle is relatively low, so phenomena such as aggregation crosslinking will not occur, its
Turbidity change is still directly proportional to the concentration of NGAL, so as to improve the range of linearity of reagent；(3) large-sized particle is being used
And make the concentration of particle in reagent relatively low, while achieveed the purpose that high sensitivity and the wide range of linearity；(4) by reagent
R2 adds polyhexamethylene guanide and phenmethylol, surprisingly so that stability of the reagent under normal temperature condition significantly improves.
In conclusion compared with prior art, the present invention have the following advantages that.
(1) kit of the present invention is by using big particle diameter latex particle, and antigen-antibody is anti-when improving low concentration NGAL
Speed is answered, so as to reduce the minimum detectability of kit and improve the detection sensitivity of kit.
(2) present invention is by optimizing the concentration of buffer system and latex particle, make kit high sensitivity, stability it is good,
High specificity, available for the content of neutrophil gelatinase-associated lipocalin in detection human serum or urine, is applicable in
In automatic clinical chemistry analyzer.
(3) concentration that the present invention passes through the latex particle of optimization coating NGAL antibody so that NGAL is dense in sample to be tested
When degree is higher, phenomena such as aggregation is crosslinked will not occur, the change of its turbidity is still directly proportional to the concentration of NGAL, so as to improve examination
The range of linearity of agent box detection.
(4) present invention is by adding polyhexamethylene guanide and phenmethylol creatively in reagent so that the kit exists
Stability under normal temperature condition significantly improves, and adds the practicality that kit of the present invention is used for clinical diagnosis.
Brief description of the drawings
Fig. 1 NGAL kit standards curves of the present invention；
Fig. 2 NGAL kits of the present invention and well-known kit measured value correlation；
Stability under Fig. 3 NGAL kits normal temperature conditions of the present invention；
In conjunction with drawings and examples, the invention will be further described：
Embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But these embodiments are only exemplary, do not form any restrictions to the scope of the present invention.Art technology
Personnel should be understood that without departing from the spirit and scope of the invention can be to the details and shape of technical solution of the present invention
Formula is modified or replaced, but these modifications and replacement are each fallen within protection scope of the present invention.
Test condition and method
Instrument：7080 automatic clinical chemistry analyzer of Hitachi
Parameter：
| Dominant wavelength | 570nm | Sample (S) | 3μL |
| Secondary wavelength | 800nm | Reagent 1 (R1) | 150μL |
| Reaction temperature | 37℃ | Reagent 2 (R2) | 50μL |
| The Direction of Reaction | Rise reaction | Reaction type | End-point method |
Operating procedure：
1 kit of embodiment prepares 1
Reagent R1：pH7.4
Trishydroxymethylaminomethane 100mM
Tween-20 1%
Macrogol 6000 4g/l
Sodium chloride 15g/l
The preparation process of reagent R2 is as follows：
(1) granules of polystyrene of the surface with carboxy functional group, 0.8 μm of diameter is diluted to end with MES buffer solutions
Concentration is 0.5-3% (wt), adds 50-90mM EDAC, is uniformly mixed, room temperature concussion reaction 30-80 minutes；
(2) mixed liquor obtained by step (1) is centrifuged, abandons supernatant, with MES buffer solution for cleaning twice, final present latex particulate
MES buffer solutions are resuspended in final concentration of 2%, ultrasound is disperseed, and obtains particle dispersion liquid；
(3) with MES buffer solutions dilution NGAL antibody, mixed in equal volume with particle dispersion liquid in step (2), it is anti-in room temperature
When answering 2-4 small；
(4) after step (3) reaction solution 18000rpm being centrifuged 30min, particulate, room temperature closing is resuspended with Tris buffer solutions
When reaction 1-3 is small；
(5) centrifugation step (4) reaction solution, abandons supernatant, with Tris buffer solution for cleaning latex 3 times, is finally protected with R2 reagents
Liquid storage is resuspended latex and precipitates and be diluted to 1%, and it is stand-by that NGAL antibody latex particles are obtained after ultrasound is fully dispersed.
(6) reagent preparation R2：
Trishydroxymethylaminomethane 100mM
Bovine serum albumin(BSA) 1g/l
NGAL antibody latex particles 8mg/l
Sodium azide 0.8g/l
Standard items：
Standard items buffer components are as follows：
Sodium chloride 0.15M
Bovine serum albumin(BSA) 0.5%
Sucrose 5%
Proclin300 0.02%
Remaining is deionized water, and concentration adds NGAL sterlings in above-mentioned buffer solution as required for normative reference product, is made
The NGAL normative reference product of 8000ng/ml concentration, the normative reference product of remaining concentration can be buffered by 8000ng/ml standard items
Liquid dilutes to obtain.
2 kit of embodiment prepares 2
Reagent R1：pH7.4
Trishydroxymethylaminomethane 100mM
Tween-20 1%
Macrogol 6000 4g/l
Sodium chloride 15g/l
The preparation process of reagent R2 is as follows：
(1) granules of polystyrene of the surface with carboxy functional group, 1.0 μm of diameter is diluted to end with MES buffer solutions
Concentration is 0.5-3% (wt), adds 50-90mM EDAC, is uniformly mixed, room temperature concussion reaction 30-80 minutes；
(2) mixed liquor obtained by step (1) is centrifuged, abandons supernatant, with MES buffer solution for cleaning twice, final present latex particulate
MES buffer solutions are resuspended in final concentration of 2%, ultrasound is disperseed, and obtains particle dispersion liquid；
(3) with MES buffer solutions dilution NGAL antibody, mixed in equal volume with particle dispersion liquid in step (2), it is anti-in room temperature
When answering 2-4 small；
(4) after step (3) reaction solution 18000rpm being centrifuged 30min, particulate, room temperature closing is resuspended with Tris buffer solutions
When reaction 1-3 is small；
(5) centrifugation step (4) reaction solution, abandons supernatant, with Tris buffer solution for cleaning latex 3 times, is finally protected with R2 reagents
Liquid storage is resuspended latex and precipitates and be diluted to 1%, and it is stand-by that NGAL antibody latex particles are obtained after ultrasound is fully dispersed.
(6) reagent preparation R2：
Trishydroxymethylaminomethane 100mM
Bovine serum albumin(BSA) 1g/l
NGAL antibody latex particles 8mg/l
Sodium azide 1g/l
Polyhexamethylene guanide 0.5g/l
Phenmethylol 0.2g/l
Standard items：
Standard items buffer components are as follows：
Sodium chloride 0.15M
Bovine serum albumin(BSA) 0.5%
Sucrose 5%
Proclin300 0.02%
Remaining is deionized water, and concentration adds NGAL sterlings in above-mentioned buffer solution as required for normative reference product, is made
The NGAL normative reference product of 8000ng/ml concentration, the normative reference product of remaining concentration can be buffered by 8000ng/ml standard items
Liquid dilutes to obtain.
3 kit of embodiment prepares 3
Reagent R1：pH7.4
Trishydroxymethylaminomethane 100mM
Tween-20 1%
Macrogol 6000 4g/l
Sodium chloride 15g/l
The preparation process of reagent R2 is as follows：
(1) granules of polystyrene of the surface with carboxy functional group, 1.0 μm of diameter is diluted to end with MES buffer solutions
Concentration is 0.5-3% (wt), adds 50-90mM EDAC, is uniformly mixed, room temperature concussion reaction 30-80 minutes；
(2) mixed liquor obtained by step (1) is centrifuged, abandons supernatant, with MES buffer solution for cleaning twice, final present latex particulate
MES buffer solutions are resuspended in final concentration of 2%, ultrasound is disperseed, and obtains particle dispersion liquid；
(3) with MES buffer solutions dilution NGAL antibody, mixed in equal volume with particle dispersion liquid in step (2), it is anti-in room temperature
When answering 2-4 small；
(4) after step (3) reaction solution 18000rpm being centrifuged 30min, particulate, room temperature closing is resuspended with Tris buffer solutions
When reaction 1-3 is small；
(5) centrifugation step (4) reaction solution, abandons supernatant, with Tris buffer solution for cleaning latex 3 times, is finally protected with R2 reagents
Liquid storage is resuspended latex and precipitates and be diluted to 1%, and it is stand-by that NGAL antibody latex particles are obtained after ultrasound is fully dispersed.
(6) reagent preparation R2：
Trishydroxymethylaminomethane 100mM
Bovine serum albumin(BSA) 1g/l
NGAL antibody latex particles 8mg/l
Sodium azide 1g/l
Polyhexamethylene guanide 0.5g/l
Standard items：
Standard items buffer components are as follows：
Sodium chloride 0.15M
Bovine serum albumin(BSA) 0.5%
Sucrose 5%
Proclin300 0.02%
Remaining is deionized water, and concentration adds NGAL sterlings in above-mentioned buffer solution as required for normative reference product, is made
The NGAL normative reference product of 8000ng/ml concentration, the normative reference product of remaining concentration can be buffered by 8000ng/ml standard items
Liquid dilutes to obtain.
4 kit of embodiment prepares 4
Reagent R1：pH7.4
Trishydroxymethylaminomethane 100mM
Tween-20 1%
Macrogol 6000 4g/l
Sodium chloride 15g/l
The preparation process of reagent R2 is as follows：
(1) granules of polystyrene of the surface with carboxy functional group, 1.0 μm of diameter is diluted to end with MES buffer solutions
Concentration is 0.5-3% (wt), adds 50-90mM EDAC, is uniformly mixed, room temperature concussion reaction 30-80 minutes；
(2) mixed liquor obtained by step (1) is centrifuged, abandons supernatant, with MES buffer solution for cleaning twice, final present latex particulate
MES buffer solutions are resuspended in final concentration of 2%, ultrasound is disperseed, and obtains particle dispersion liquid；
(3) with MES buffer solutions dilution NGAL antibody, mixed in equal volume with particle dispersion liquid in step (2), it is anti-in room temperature
When answering 2-4 small；
(4) after step (3) reaction solution 18000rpm being centrifuged 30min, particulate, room temperature closing is resuspended with Tris buffer solutions
When reaction 1-3 is small；
(5) centrifugation step (4) reaction solution, abandons supernatant, with Tris buffer solution for cleaning latex 3 times, is finally protected with R2 reagents
Liquid storage is resuspended latex and precipitates and be diluted to 1%, and it is stand-by that NGAL antibody latex particles are obtained after ultrasound is fully dispersed.
(6) reagent preparation R2：
Trishydroxymethylaminomethane 100mM
Bovine serum albumin(BSA) 1g/l
NGAL antibody latex particles 8mg/l
Sodium azide 1g/l
Phenmethylol 0.2g/l
Standard items：
Standard items buffer components are as follows：
Sodium chloride 0.15M
Bovine serum albumin(BSA) 0.5%
Sucrose 5%
Proclin300 0.02%
Remaining is deionized water, and concentration adds NGAL sterlings in above-mentioned buffer solution as required for normative reference product, is made
The NGAL normative reference product of 8000ng/ml concentration, the normative reference product of remaining concentration can be buffered by 8000ng/ml standard items
Liquid dilutes to obtain.
Embodiment 5NGAL kit standard curves
Using standard items, select 7 points of calibrations, restructuring NGAL contents are respectively 0,500,1000,2000,4000,6000,
8000ng/mL, by taking the kit of embodiment 2 as an example, the calibration curve of the NGAL obtained according to said determination step is (such as Fig. 1 institutes
Show).Each point represents the standard items of a content on curve in Fig. 1, and wherein X-axis represents the content of NGAL, and y-axis represents extinction
Degree.As seen from the figure, the range of linearity of kit of the invention detection is 0-8000ng/ml.
The kit of embodiment 1,3,4 has the identical detection range of linearity.
6 accuracy of embodiment measures
30 parts of human serums are measured using the kit and the well-known kit of control of the present invention, phase is carried out to measured value
The analysis of closing property, measurement result are shown in Fig. 2, and X, Y-axis are measured value in figure, and the results show present invention is related to contrast agents box
Property is very high.
7 minimum detection limit of embodiment
It is continuous to detect 20 times using physiological saline as blank sample by taking embodiment 2 as an example.Calculate the equal of 20 results
Value and standard deviation SD, the test limit of twice of standard deviation method for reporting is added with blank average：(+2SD).
Result of the test
Drawn by test data, the lowest detection of NGAL detection kits of the present invention is limited to 0.025ng/ml.Implement
The kit of example 1,3,4 has identical minimum detection limit.
8 sensitivity of embodiment
Blank solution and 4 difference NGAL levels samples are measured, each sample is surveyed 10 times, calculates average value (M) and mark
Accurate poor (SD), is more than the sample concentration of blank absorbency+3SD as NGAL detection kits most using sample absorbance -3SD
Low detection sensitivity.By taking the kit in embodiment 2 as an example, from following table as it can be seen that the sensitivity of detection kit of the present invention is
1ng/ml.The kit of embodiment 1,3,4 has identical sensitivity.
9 stability of embodiment
(1) stability under the conditions of 2-8 DEG C
NGAL detection kit reagents R1, R2 of embodiment 1,2,3,4 are placed in 2-8 DEG C of freezer, stored respectively
Before, 2-8 DEG C of freezer store 3 months, 6 months and calibration object be measured after 12 months analysis.The NGAL detection examinations of the present invention
Agent box is with good stability, after 2-8 DEG C is stored 1 year, keeps more than 95% reactivity.
Stability under the conditions of (2) 37 DEG C
NGAL detection kit reagents R1, R2 of embodiment 1,2,3,4 are placed in 37 DEG C of water-baths, respectively before placement,
37 DEG C of water-baths are measured calibration object analysis after 3 days, 7 days and 10 days.The NGAL detection kits of the present invention have good
Stability, after 37 DEG C of water-baths 10 days, keeps more than 95% reactivity.
(3) stability under normal temperature condition
NGAL detection kit reagents R1, R2 of embodiment 1,2,3 and 4 are positioned under normal temperature condition, stored respectively
Before, storage 2 months, 4 months, 6 months after calibration object is measured, do parallel laboratory test three times, measurement result as shown in figure 3,
From the figure 3, it may be seen that by adding polyhexamethylene guanide and phenmethylol into reagent R2 so that the kit is under normal temperature condition
Stability significantly improves, and adds the practicality that kit of the present invention is used for clinical diagnosis.
10 interference experiment of embodiment
A certain amount of ascorbic acid, bilirubin, triglycerides and hemoglobin are each added in normal human serum, at the same time
Isometric deionized water is added as noiseless thing serum, using the NGAL detection kits of embodiment 1,2,3,4, at the same time
Measure the concentration of these samples.It is that measurement system endures limit to the highest of chaff interferent using annoyance level as 5%, serum moderate resistance is bad
Hematic acid is in below 1500mg/l, and bilirubin is in below 775mg/l, and triglycerides is in below 30g/l, and hemoglobin is in 5.5g/l
Hereinafter, measurement result is not influenced.
Technological means disclosed in the present invention program is not limited only to the technological means disclosed in above-mentioned technological means, further includes
Formed technical solution is combined by above technical characteristic.The above is the embodiment of the present invention, should be referred to
Go out, for those skilled in the art, without departing from the principle of the present invention, if can also make
Dry improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.
Claims (10)
- A kind of 1. neutrophil gelatinase-associated lipocalin detection kit, it is characterised in that：By independent of each other Reagent R1 and reagent R2 compositions；The component of the reagent R1 includes：Biological buffer, coagulant, surfactant, osmotic pressure tune Save agent and water；The component of the reagent R2 includes：It is coated with the latex of neutrophil gelatinase-associated lipocalin antibody Particle, biological buffer, sealer, preservative and water；The latex particle is the polystyrene latex of 0.5-1.5 μm of particle diameter Particle.
- 2. detection kit described in accordance with the claim 1, it is characterised in that：The biological buffer is trihydroxy methyl amino One or more in methane, MES, phosphate, MOPSO, DIPSO；The coagulant for polyethylene glycol -200-10000 or One or more in Gentran 40 00-12000；The surfactant is Tween-20；The osmotic pressure regulator is Sodium chloride；The sealer is bovine serum albumin；The preservative is the one or more in sodium azide or thimerosal.
- 3. detection kit described in accordance with the claim 2, it is characterised in that：The dosage of each component is in every 1 liter of reagent R1：It is raw Thing buffer 5-100g, coagulant 2-50g, surfactant 1-20ml, osmotic pressure regulator 5-20g；It is each in every 1 liter of reagent R2 The dosage of component is：The latex particle 5-20mg of neutrophil gelatinase-associated lipocalin antibody is coated with, biology is slow Electuary 5-100g, sealer 0.2-10g, preservative 0.2-10g.
- 4. detection kit described in accordance with the claim 3, it is characterised in that：The dosage of each component is in every 1 liter of reagent R1：Three Hydroxymethyl aminomethane 12.114g, Macrogol 6000 4g, polysorbas20 10ml, sodium chloride 15g；Each group in every 1 liter of reagent R2 Point dosage be：It is coated with the latex particle 8mg of neutrophil gelatinase-associated lipocalin antibody, trihydroxy methyl amino Methane 12.114g, bovine serum albumin 1g, sodium azide 1g.
- 5. detection kit described in accordance with the claim 1, it is characterised in that：In the reagent R2 also have polyhexamethylene guanide and Phenmethylol.
- 6. according to the detection kit described in claim 5, it is characterised in that：The dosage of polyhexamethylene guanide in the reagent R2 For 0.5g/l, the dosage of phenmethylol is 0.2g/l.
- 7. detection kit described in accordance with the claim 1, it is characterised in that the coating neutrophil leucocyte gelatinase correlation fat The preparation method of the latex particle of matter transporter antibody is：(1) ps particle of the surface with carboxy functional group, 0.5-1.5 μm of diameter is diluted to end with MES buffer solutions Concentration is 0.5-3% (wt), adds 50-90mM EDAC, is uniformly mixed, room temperature concussion reaction 30-80 minutes；(2) mixed liquor obtained by step (1) is centrifuged, abandons supernatant, with MES buffer solution for cleaning twice, final present latex particulate is resuspended In MES buffer solutions, ultrasound is disperseed, and obtains particle dispersion liquid；(3) with particulate point in MES buffer solutions dilution neutrophil gelatinase-associated lipocalin antibody, with step (2) Dispersion liquid mixes, when room temperature reaction 2-4 is small；(4) by after reaction solution 18000rpm centrifugations 30min obtained by step (3), particulate is resuspended with Tris buffer solutions, room temperature closing is anti- When answering 1-3 small；(5) reaction solution obtained by step (4) is centrifuged, abandons supernatant, with Tris buffer solution for cleaning latex 3 times, finally with R2 reagents Preserve liquid resuspension latex to precipitate and be diluted to 1%, obtained after ultrasound is fully dispersed and be coated with neutrophil leucocyte gelatinase correlation fat The latex particle of matter transporter antibody.
- 8. according to the detection kit of claim 1-6 any one, it is characterised in that：The pH value range of reagent R1 is 6.5- 8.5。
- 9. according to the detection kit of claim 1-6 any one, it is characterised in that：Its when in use, detection dominant wavelength be 570nm, a length of 800nm of complementary wave, assay method use end-point method：150 μ l reagents R1 add 3 μ l samples, and 5min is incubated in 37 DEG C After add 50 μ l reagent R2 36s and start reading, reading again after 4.5min, then calculates absorbance and calculates difference.
- 10. the detection kit according to claim 1-6 any one is measuring neutrophil leucocyte gelatin in human serum or urine Application in the content of enzyme associated lipocalin.
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| CN201711131536.7A CN107918020B (en) | 2017-11-15 | 2017-11-15 | Neutrophil gelatinase-associated lipocalin assay kit |
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| CN201711131536.7A CN107918020B (en) | 2017-11-15 | 2017-11-15 | Neutrophil gelatinase-associated lipocalin assay kit |
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| CN110133276A (en) * | 2019-04-01 | 2019-08-16 | 芜湖森爱驰生物科技有限公司 | A kind of neutrophil gelatinase-associated lipocalin detection kit |
| CN114859048A (en) * | 2022-05-27 | 2022-08-05 | 美康生物科技股份有限公司 | Chitinase 3-like protein 1 kit |
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|---|---|
| CN107918020B (en) | 2019-01-01 |
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