CN107918020B - 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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- 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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- 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 belongs to medicine and technological field of biochemistry, 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: latex particle, biological buffer, sealer, preservative and the water for being coated with neutrophil gelatinase-associated lipocalin antibody.The neutrophil gelatinase-associated lipocalin detection kit is measured using latex-enhanced turbidimetry, relative to traditional NGAL detection kit, have many advantages, such as 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, specifically a kind of measurement neutrophil leucocyte gelatinase correlation rouge
The kit of matter transporter.
Background technique
In recent years, as living standards of the people improve and living habit and eating habit change, the disease incidence of nephrosis by
Year increases.The big multi-pathogenesis complexity of kidney trouble, protracted course, and there is the risk for increasing cardiovascular disease, have become
Major global public health problem.Kidney trouble can be divided into two main syndromes --- acute kidney injury (acute kidney
Injury, AKI) and chronic kidney disease (chronic kidney disease, CKD).AKI refers to burst 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 clinical common one of 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 etc. delivered first on " lancet " magazine
National CKD cross-section survey shows that Chinese CKD illness rate is 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 it finds a kind of reliable
Biomarker, kidney trouble develop early stage carry out efficient diagnosis, it is treated and prognosis have highly important clinic
Meaning.
Research in recent years discovery, a newcomer --- the neutrophil leucocyte gelatinase correlation rouge in lipocalin protein family
Matter transporter (neutrophil gelatinase-associated lipocalin, NGAL), it is close with AKI and CKD
Correlation is the biological indicator predicted a novel markings object of AKI, while being also monitoring CKD progress and kidney function damage.
NGAL was had found that mankind's NGAL gene is monocistron, positioning by Kjeldsen etc. in research human neutrophils' granulocyte in 1993
In on Chromosome 9 long-armed (9q34), overall length 5869bp, 5 ' end nontranscribed domains, the 3 ' ends of 178bp including 1695bp are non-
The original transcriptional domain of transcriptional domain and 3696bp.Similar to other members of lipocalin protein family, NGAL albumen has a guarantor
The tertiary structure kept, the structure make NGAL have the ability to combine some compounds mainly formed by hydrophobic small molecules, even
It can also be in cell surface binding specificity receptor.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, inhibits the processes such as occurrence and development, the infiltration metastasis of Apoptosis and tumour；By with iron content
Siderophore combine, formed NGAL- siderophore-iron complexes, participate in the generation, development and repair process of kidney.Normal
Under physiological status, other than 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,
Especially in the proximal renal tubular epithelial cells of damage.Therefore by the content of detection 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 immune at present
Turbidimetry.Enzyme-linked immunization the degree of automation is not high, and is affected by human factors larger；There is environment dirts for radioimmunology
Dye problem；Though chemoluminescence method sensitivity is high, the measurement range of linearity is smaller and testing cost is higher, and specified chemical is needed to send out
Optical detector；Common turbidimetry haves the shortcomings that sensitivity is inadequate.The above method is difficult to meet clinical labororatory 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 monoclonal antibody that is crosslinked in high molecular emulsion microsphere surface, and when antigen and crosslinking
After thering is the microballoon of antibody to combine, it can flock together rapidly in a short time, change the absorbance of reaction solution.Moreover, anti-
The concentration of the change and tested antigen of answering liquid absorbance has linear relationship in a certain range, 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 sensitivities, the range of linearity
It is wide, pollution-free, the outstanding features such as have a wide range of application, therefore, latex immunoturbidimetry has great researching value.
NGAL concentration is lower than 10ng/ml in normal human urine, and concentration may be up in severe nephrotic's urine
7000ng/ml or more.Such wide 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 measurement human plasma or urine currently on the market,
But these kits can not meet the wide range of linearity and highly sensitive requirement simultaneously, it is therefore desirable to which diluted sample could measure
High concentration NGAL, this brings inconvenience in use.Existing Immunoturbidimetry, such as 104198732 He of patent CN
Kit detection sensitivity disclosed in patent CN 102680698 is all larger than equal to 10ng/ml, and the range of linearity is in 6000ng/
Ml is hereinafter, be difficult to meet clinical detection demand.
Therefore, it is necessary to develop NGAL latex enhancing immune transmission a kind of while that there is the highly sensitive and wide range of linearity
Than turbid detection kit.
Summary 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.
It include reagent R1, reagent R2 and calibration object the present invention relates to a kind of NGAL detection kit, wherein reagent R1 packet
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 partial size
It is 0.5-1.5 μm, preferably 0.8-1.3 μm.
Wherein, the biological buffer is in trishydroxymethylaminomethane, MES, phosphate, MOPSO, DIPSO
It is one or more of；Coagulant is selected from one or more of 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, in every 1 liter of reagent R1 each component dosage are as follows: biological buffer 5-100g, coagulant 2-50g, surface
Activating agent 1-20ml, osmotic pressure regulator 5-20g；The dosage of each component in every 1 liter of reagent R2 are as follows: coating neutrophil leucocyte is bright
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 in every 1 liter of reagent R1 are as follows: trishydroxymethylaminomethane
12.114g, Macrogol 6000 4g, polysorbas20 10ml, sodium chloride 15g；The dosage of each component in every 1 liter of reagent R2 are as follows: packet
By the latex particle 8mg of neutrophil gelatinase-associated lipocalin antibody, trishydroxymethylaminomethane 12.114g,
Bovine serum albumin 1g, sodium azide 1g.
It preferably, also include polyhexamethylene guanide and benzyl alcohol in reagent R2.
In a preferred embodiment, the dosage of polyhexamethylene guanide is 0.5g/l, the use of benzyl alcohol in reagent R2
Amount is 0.2g/l.
The present invention also provides the systems of the latex particle of coating neutrophil gelatinase-associated lipocalin antibody
Preparation Method, the preparation method are chemical crosslink technique:
(1) surface is diluted with carboxy functional group, 0.5-1.5 μm of diameter of ps particle with MES buffer
To final concentration of 0.5-3% (wt), 50-90mM EDAC is added, is uniformly mixed, room temperature concussion reaction 30-80 minutes；
(2) mixed liquor obtained by step (1) is centrifuged, abandons supernatant, twice with MES buffer solution for cleaning, final present latex particulate
It is resuspended in MES buffer, ultrasound is dispersed, and particle dispersion liquid is obtained；
(3) with micro- in MES buffer dilution neutrophil gelatinase-associated lipocalin antibody, with step (2)
Grain dispersion liquid mixing, in room temperature reaction 2-4 hours；
(4) by after the centrifugation of reaction solution 18000rpm obtained by step (3) 30min, particle, room temperature is resuspended with Tris buffer
Capping 1-3 hours；
(5) reaction solution obtained by step (4) is centrifuged, abandons supernatant with Tris buffer solution for cleaning latex 3 times and finally uses R2
Reagent preservation liquid is resuspended latex and precipitates and be diluted to 1%, is acquisition coating neutrophil leucocyte gelatinase phase after ultrasound is fully dispersed
Close the latex particle of lipocalin protein antibody.
Specifically, inventive concept of the invention is: (1) affine with antigen after big partial size latex surface coated antibody
Higher, antigen-antibody reaction rate when greatly improving low concentration NGAL of property, 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 lower, so phenomena such as aggregation crosslinking will not occur,
Turbidity variation is still directly proportional to the concentration of NGAL, to improve the range of linearity of reagent；(3) large-sized particle is being used
And keep the concentration of particle in reagent lower, while having achieved the purpose that the highly sensitive and wide range of linearity；(4) by reagent
R2 adds polyhexamethylene guanide and benzyl alcohol, significantly improves the stability of the reagent under normal temperature conditions.
In conclusion compared with prior art, the present invention having the following advantages that.
(1) kit of the present invention is by using big partial size latex particle, and antigen-antibody is anti-when improving low concentration NGAL
Rate is answered, to reduce the minimum detectability of kit and improve the detection sensitivity of kit.
(2) present invention by optimization buffer system and latex particle concentration, make kit high sensitivity, stability it is good,
High specificity can be used for detecting the content of neutrophil gelatinase-associated lipocalin in human serum or urine, be applicable in
In automatic clinical chemistry analyzer.
(3) present invention is coated with the concentration of the latex particle of NGAL antibody by optimization, so that NGAL is dense in sample to be tested
When spending higher, phenomena such as aggregation is crosslinked will not occur, turbidity variation is still directly proportional to the concentration of NGAL, to improve examination
The range of linearity of agent box detection.
(4) present invention is by adding polyhexamethylene guanide and benzyl alcohol creatively in reagent, so that the kit exists
Stability under normal temperature condition significantly improves, and increases practicability of the kit of the present invention for clinical diagnosis.
Detailed description of the invention
Fig. 1 NGAL kit standard curve of the present invention；
Fig. 2 NGAL kit of the present invention and well-known kit measured value correlation；
Stability under Fig. 3 NGAL kit normal temperature condition of the present invention；
Now in conjunction with drawings and examples, the invention will be further described:
Specific 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 examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Art technology
Personnel should be understood that without departing from the spirit and scope of the invention can details and shape to technical solution of the present invention
Formula is modified or is replaced, but these modifications and replacement are fallen within the 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 preparation 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) surface is diluted to end with carboxy functional group, 0.8 μm of diameter of granules of polystyrene with MES buffer
Concentration is 0.5-3% (wt), and 50-90mM EDAC is added, and is uniformly mixed, room temperature concussion reaction 30-80 minutes；
(2) mixed liquor obtained by step (1) is centrifuged, abandons supernatant, twice with MES buffer solution for cleaning, final present latex particulate
MES buffer is resuspended in final concentration of 2%, ultrasound is dispersed, and particle dispersion liquid is obtained；
(3) NGAL antibody is diluted with MES buffer, is mixed in equal volume with particle dispersion liquid in step (2), it is anti-in room temperature
It answers 2-4 hours；
(4) after step (3) reaction solution 18000rpm being centrifuged 30min, particle, room temperature closing is resuspended with Tris buffer
Reaction 1-3 hours；
(5) centrifugation step (4) reaction solution abandons supernatant, with Tris buffer solution for cleaning latex 3 times, is finally protected with R2 reagent
Liquid storage is resuspended latex and precipitates and be diluted to 1%, and it is stand-by that NGAL antibody latex particle is obtained after ultrasound is fully dispersed.
(6) reagent preparation R2:
Trishydroxymethylaminomethane 100mM
Bovine serum albumin(BSA) 1g/l
NGAL antibody latex particle 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 NGAL sterling is added in above-mentioned buffer by concentration required for reference standard product, is made
The reference standard product of the NGAL reference standard product of 8000ng/ml concentration, remaining concentration can be buffered by 8000ng/ml standard items
Liquid dilutes to obtain.
2 kit of embodiment preparation 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) surface is diluted to end with carboxy functional group, 1.0 μm of diameter of granules of polystyrene with MES buffer
Concentration is 0.5-3% (wt), and 50-90mM EDAC is added, and is uniformly mixed, room temperature concussion reaction 30-80 minutes；
(2) mixed liquor obtained by step (1) is centrifuged, abandons supernatant, twice with MES buffer solution for cleaning, final present latex particulate
MES buffer is resuspended in final concentration of 2%, ultrasound is dispersed, and particle dispersion liquid is obtained；
(3) NGAL antibody is diluted with MES buffer, is mixed in equal volume with particle dispersion liquid in step (2), it is anti-in room temperature
It answers 2-4 hours；
(4) after step (3) reaction solution 18000rpm being centrifuged 30min, particle, room temperature closing is resuspended with Tris buffer
Reaction 1-3 hours；
(5) centrifugation step (4) reaction solution abandons supernatant, with Tris buffer solution for cleaning latex 3 times, is finally protected with R2 reagent
Liquid storage is resuspended latex and precipitates and be diluted to 1%, and it is stand-by that NGAL antibody latex particle is obtained after ultrasound is fully dispersed.
(6) reagent preparation R2:
Trishydroxymethylaminomethane 100mM
Bovine serum albumin(BSA) 1g/l
NGAL antibody latex particle 8mg/l
Sodium azide 1g/l
Polyhexamethylene guanide 0.5g/l
Benzyl alcohol 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 NGAL sterling is added in above-mentioned buffer by concentration required for reference standard product, is made
The reference standard product of the NGAL reference standard product of 8000ng/ml concentration, remaining concentration can be buffered by 8000ng/ml standard items
Liquid dilutes to obtain.
3 kit of embodiment preparation 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) surface is diluted to end with carboxy functional group, 1.0 μm of diameter of granules of polystyrene with MES buffer
Concentration is 0.5-3% (wt), and 50-90mM EDAC is added, and is uniformly mixed, room temperature concussion reaction 30-80 minutes；
(2) mixed liquor obtained by step (1) is centrifuged, abandons supernatant, twice with MES buffer solution for cleaning, final present latex particulate
MES buffer is resuspended in final concentration of 2%, ultrasound is dispersed, and particle dispersion liquid is obtained；
(3) NGAL antibody is diluted with MES buffer, is mixed in equal volume with particle dispersion liquid in step (2), it is anti-in room temperature
It answers 2-4 hours；
(4) after step (3) reaction solution 18000rpm being centrifuged 30min, particle, room temperature closing is resuspended with Tris buffer
Reaction 1-3 hours；
(5) centrifugation step (4) reaction solution abandons supernatant, with Tris buffer solution for cleaning latex 3 times, is finally protected with R2 reagent
Liquid storage is resuspended latex and precipitates and be diluted to 1%, and it is stand-by that NGAL antibody latex particle is obtained after ultrasound is fully dispersed.
(6) reagent preparation R2:
Trishydroxymethylaminomethane 100mM
Bovine serum albumin(BSA) 1g/l
NGAL antibody latex particle 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 NGAL sterling is added in above-mentioned buffer by concentration required for reference standard product, is made
The reference standard product of the NGAL reference standard product of 8000ng/ml concentration, remaining concentration can be buffered by 8000ng/ml standard items
Liquid dilutes to obtain.
4 kit of embodiment preparation 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) surface is diluted to end with carboxy functional group, 1.0 μm of diameter of granules of polystyrene with MES buffer
Concentration is 0.5-3% (wt), and 50-90mM EDAC is added, and is uniformly mixed, room temperature concussion reaction 30-80 minutes；
(2) mixed liquor obtained by step (1) is centrifuged, abandons supernatant, twice with MES buffer solution for cleaning, final present latex particulate
MES buffer is resuspended in final concentration of 2%, ultrasound is dispersed, and particle dispersion liquid is obtained；
(3) NGAL antibody is diluted with MES buffer, is mixed in equal volume with particle dispersion liquid in step (2), it is anti-in room temperature
It answers 2-4 hours；
(4) after step (3) reaction solution 18000rpm being centrifuged 30min, particle, room temperature closing is resuspended with Tris buffer
Reaction 1-3 hours；
(5) centrifugation step (4) reaction solution abandons supernatant, with Tris buffer solution for cleaning latex 3 times, is finally protected with R2 reagent
Liquid storage is resuspended latex and precipitates and be diluted to 1%, and it is stand-by that NGAL antibody latex particle is obtained after ultrasound is fully dispersed.
(6) reagent preparation R2:
Trishydroxymethylaminomethane 100mM
Bovine serum albumin(BSA) 1g/l
NGAL antibody latex particle 8mg/l
Sodium azide 1g/l
Benzyl alcohol 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 NGAL sterling is added in above-mentioned buffer by concentration required for reference standard product, is made
The reference standard product of the NGAL reference standard product of 8000ng/ml concentration, remaining concentration can be buffered by 8000ng/ml standard items
Liquid dilutes to obtain.
Embodiment 5NGAL kit standard curve
Using standard items, select 7 points of calibrations, recombination NGAL content is 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 institute
Show).Each point represents the standard items of a content on curve in Fig. 1, and wherein X-axis indicates that the content of NGAL, y-axis indicate 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.
The measurement of 6 accuracy of embodiment
30 parts of human serums are measured using kit of the invention and the well-known kit of control, 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 present invention is related to contrast agents box as the result is shown
Property is very high.
7 minimum detection limit of embodiment
By taking embodiment 2 as an example, using physiological saline as blank sample, continuous detection 20 times.Calculate the equal of 20 results
Value and standard deviation SD, add the detection of twice of standard deviation method for reporting to limit: (+2SD) with blank mean value.
Test result
It is obtained by test data, the lowest detection of NGAL detection kit 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, is calculated average value (M) and is marked
Quasi- poor (SD) is greater than the sample concentration of blank absorbency+3SD as NGAL detection kit 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 reagent 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 be measured analysis to calibration object after 12 months.NGAL of the invention detects examination
Agent box is with good stability, after 2-8 DEG C is stored 1 year, keeps 95% or more reactivity.
Stability under the conditions of (2) 37 DEG C
NGAL detection kit reagent R1, R2 of embodiment 1,2,3,4 are placed in 37 DEG C of water-baths, respectively before placement,
37 DEG C are measured analysis to calibration object after water-bath 3 days, 7 days and 10 days.NGAL detection kit of the invention has good
Stability at 37 DEG C after water-bath 10 days, keeps 95% or more reactivity.
(3) stability under normal temperature condition
NGAL detection kit reagent R1, R2 of embodiment 1,2,3 and 4 are placed 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 benzyl alcohol into reagent R2, so that the kit is under normal temperature conditions
Stability significantly improves, and increases practicability of the kit of the present invention for clinical diagnosis.
10 interference experiment of embodiment
A certain amount of ascorbic acid, bilirubin, triglycerides and hemoglobin are respectively added in normal human serum, simultaneously
Isometric deionized water is added as noiseless object serum, using the NGAL detection kit of embodiment 1,2,3,4, simultaneously
Measure the concentration of these samples.It is that 5% to be measurement system endure limit to the highest of chaff interferent with annoyance level, it is anti-bad in serum
Hematic acid in 1500mg/l hereinafter, bilirubin in 775mg/l hereinafter, triglycerides in 30g/l hereinafter, hemoglobin is in 5.5g/l
Hereinafter, not influencing measurement result.
The technical means disclosed in the embodiments of the present invention is not limited to the technical means disclosed in the above technical means, and further includes
Technical solution consisting of any combination of the above technical features.The foregoing is a specific embodiment of the present invention, should refer to
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 modifications and embellishments are also considered to be within the scope of the present invention.
Claims (4)
1. a kind of neutrophil gelatinase-associated lipocalin detection kit, it is characterised in that: by independent of each other
Reagent R1 and reagent R2 composition；The dosage of each component in every 1 liter of reagent R1 are as follows: trishydroxymethylaminomethane 12.114g gathers
Ethylene glycol 6000 4g, polysorbas20 10ml, sodium chloride 15g；The dosage of each component in every 1 liter of reagent R2 are as follows: coating is neutral
The latex particle 8mg of granulocyte gelatinase associated lipocalin antibody, trishydroxymethylaminomethane 12.114g, cow's serum
Albumen 1g, sodium azide 1g, polyhexamethylene guanide 0.5g, benzyl alcohol 0.2g；The latex particle is 0.8-1.3 μm of partial size
Polystyrene latex particulate.
2. detection kit described in accordance with the claim 1, which is characterized in that the coating neutrophil leucocyte gelatinase correlation rouge
The latex particle of matter transporter antibody the preparation method comprises the following steps:
(1) surface is diluted to end with carboxy functional group, 0.5-1.5 μm of diameter of ps particle with MES buffer
Concentration is 0.5-3% (wt), and 50-90mM EDAC is added, and is uniformly mixed, room temperature concussion reaction 30-80 minutes；
(2) mixed liquor obtained by step (1) is centrifuged, abandons supernatant, twice with MES buffer solution for cleaning, final present latex particulate is resuspended
In MES buffer, ultrasound is dispersed, and obtains particle dispersion liquid；
(3) with particle point in MES buffer dilution neutrophil gelatinase-associated lipocalin antibody, with step (2)
Dispersion liquid mixing, in room temperature reaction 2-4 hours；
(4) by after the centrifugation of reaction solution 18000rpm obtained by step (3) 30min, particle is resuspended with Tris buffer, room temperature closing is anti-
It answers 1-3 hours；
(5) reaction solution obtained by step (4) is centrifuged, abandons supernatant, with Tris buffer solution for cleaning latex 3 times, finally use R2 reagent
Preservation liquid is resuspended latex and precipitates and be diluted to 1%, is acquisition coating neutrophil leucocyte gelatinase correlation rouge after ultrasound is fully dispersed
The latex particle of matter transporter antibody.
3. according to the detection kit of claims 1 or 2, it is characterised in that: the pH value range of reagent R1 is 6.5-8.5.
4. according to the detection kit of claims 1 or 2, it is characterised in that: its when in use, detection dominant wavelength be 570nm,
The a length of 800nm of complementary wave, measuring method use end-point method: 3 μ l samples are added in 150 μ l reagent R1, are added after 37 DEG C of incubation 5min
50 μ l reagent R2 36s start to read, and read again after 4.5min, then calculate absorbance and calculate difference.
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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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| JP2014518216A (en) * | 2011-06-17 | 2014-07-28 | ハロザイム インコーポレイテッド | Stable formulation of hyaluronan degrading enzyme |
| CN102662064A (en) * | 2012-04-26 | 2012-09-12 | 苏州照康生物技术有限公司 | Immunonephelometry kit for detecting lipid carrier protein related to neutrophils gelatinase and preparation method thereof |
| CN104198732B (en) * | 2014-08-28 | 2015-08-19 | 宁波瑞源生物科技有限公司 | A kind of neutrophil gelatinase-associated lipocalin reagent box for detecting content |
| CN104407154B (en) * | 2014-12-05 | 2016-03-30 | 重庆中元生物技术有限公司 | A kind of high sensitivity latex enhancing immune turbidimetry kit |
| CN107942067B (en) * | 2017-11-15 | 2019-01-11 | 浙江夸克生物科技有限公司 | Cyclic citrullinated peptid assay kit |
| CN107942068B (en) * | 2017-11-15 | 2019-01-15 | 浙江夸克生物科技有限公司 | β2Microglobulin assay kit |
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