CN107942069A - A kind of NGAL latex immunoturbidimetries detection kit and preparation method thereof - Google Patents
A kind of NGAL latex immunoturbidimetries detection kit and preparation method thereof Download PDFInfo
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- CN107942069A CN107942069A CN201711132885.0A CN201711132885A CN107942069A CN 107942069 A CN107942069 A CN 107942069A CN 201711132885 A CN201711132885 A CN 201711132885A CN 107942069 A CN107942069 A CN 107942069A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—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
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
Abstract
The invention discloses a kind of neutrophil gelatinase-associated lipocalin latex immunoturbidimetry detection kit and preparation method thereof, selects sealer of six ammonia of polyethylene glycol as antibody binding latex particle；It is good that neutrophil gelatinase-associated lipocalin detection high specificity, high sensitivity, stabilization of kit are carried out using the present invention, NGAL contents that can be in efficient detection urine, blood plasma, serum, and testing result is related to fluorescence immune chromatography method and enzyme linked immunosorbent assay good.
Description
Technical field
The present invention relates to a kind of NGAL latex immunoturbidimetries detection kit and preparation method thereof, specifically, it is related to
It is a kind of using six ammonia of polyethylene glycol as NGAL latex immunoturbidimetry detection kits of sealer and preparation method thereof.
Background technology
Acute injury of kidney (AKI) is a kind of higher disease of morbidity and mortality, since the disease pathogenesis is unknown,
And lack accurately and reliably early diagnosis marker, therefore effective medicine clinically not yet occur.Traditional injury of kidney
Marker (such as serum creatinine, bladder chalone C, 1 microglobulins of α, β2-microglobulin and RBP ELISA) is mostly feature mark
Will thing, but the structure change of overwhelming majority AKI occurs before functional disorder, and therefore, structural injury of kidney marker is to AKI
Diagnosis it is more sensitive, wherein study most fully, most significant amplitude of variation be exactly neutrophil leucocyte gelatinase related lipid
Transporter (NGAL).
NGAL is a kind of small-sized and more covert polypeptide, can directly be detected from urine.Numerous studies prove, cis-platinum
About 2h can just detect NGAL in urine after induction AKI, and detect substantially changeing for serum creatinine and take 3-4 days.In addition, children's heart
The blood of 2h, urine NGAL concentration are significantly raised after surgery by the patient of generation acute renal failure after dirty operation, and serum creatinine is significantly raised
After 1-3d.Since NGAL concentration had not only been avoided that the trouble brought by extreme oliguresis in measure blood, but also can mitigate because using profit
Urine medicine carries out treating caused interference, and therefore, the early diagnosis that it is measured to AKI is stronger.To sum up, pass through NGAL
The early clinical diagnosis of AKI is carried out, there is important value to improving the therapeutic effect of AKI, reducing the death rate.
Mainly exempt from present for the detection method of NGAL including enzyme-linked immunization (ELISA), traditional immunization turbidimetry, latex
Epidemic disease turbidimetry etc., wherein, latex immunoturbidimetry in the NGAL antigens in sample and kit by will be connected to latex particle
On specific anti-human NGAL antibody occur immune response, formed antigenantibody complex, make latex particle connect networking
Shape, so as to add the turbidity of reaction solution system.Turbidity height is proportionate within the specific limits with NGAL antigen concentrations, leads to
The absorbance of the reaction solution of measure specific wavelength (540nm-700nm) is crossed, can be calculated with reference to calibration curve in sample
The content of NGAL.For enzyme-linked immunization, latex immunoturbidimetry flux is big, takes short, it can be achieved that automating, and
Influence of the human factor to testing result can be reduced；And compared with traditional immunoturbidimetry, it can increase examination using latex particle
The sensitivity and accuracy of agent；Therefore, latex immunoturbidimetry has preferable application prospect.
However, according to healthy population physical examination data, NGAL contents are generally in below 10ng/mL in urine, and according to document
Record, occurs NGAL during acute injury of kidney in urine up to ng/mL thousands of or even up to ten thousand, so needing ensureing that reagent is linear
On the basis of scope, increase the sensitivity of reagent, otherwise need diluted sample just to can guarantee that high concentration NGAL's during clinical examination
Test accuracy, use are very inconvenient.
In addition, BIOPORTO companies of Denmark are the companies for studying NGAL diagnostic reagents earliest in the world, its NGAL ELISA
And the application of latex immunoturbidimetry detection reagent clinically has obtained worldwide accreditation.It is and domestic existing self-produced
The correlation for the detection reagent that NGAL reagents are produced with BIOPORTO companies is relatively low, significantly limit domestic reagent in clinic
On application.
It would therefore be highly desirable to improved on the basis of the preparation of conventional NGAL latex immunoturbidimetry detection kits, with
The sensitivity and accuracy of NGAL latex immunoturbidimetry reagents are improved, improves the stability of reagent.
The content of the invention
Based on the deficiencies of the prior art, the present invention provides a kind of NGAL latex using six ammonia of polyethylene glycol as sealer to exempt from
Epidemic disease turbidimetry detection kit and preparation method thereof, with improve the sensitivity of latex immunoturbidimetry detection reagent, specificity with
And the stability of antibody binding latex particle.
The present invention is adopted the technical scheme that to achieve these goals：
A kind of neutrophil gelatinase-associated lipocalin latex immunoturbidimetry detection kit, including reagent
R1, reagent R2 and calibration object, wherein, the reagent R2 includes antibody, latex particle and R2 buffer solutions, it is characterised in that：It is described
Sealer used in reagent R2 preparation process is six ammonia of polyethylene glycol, and the chemical structural formula of six ammonia of polyethylene glycol is H- (O-
CH2-CH2)n-(NH)5-NH2。
The reagent R1 includes the conventional use of buffer solution of latex immunoturbidimetry detection kit, such as phosphate-buffered
Liquid, HEPES buffer solution, MES buffer solutions, glycine buffer, borate buffer, acetate buffer solution or citrate buffer solution etc.,
Its concentration is 10-250mM.
Reagent R1 may also include conventional electrolyte, surfactant, nonspecific reaction blocking agent, coagulant, anti-corrosion
The components such as agent, biostability.
Further, nonspecific reaction blocking agent can be selected anti-HAMA antibody, anti-RF antibody, sheep blood serum, mouse serum or
Rabbit anteserum etc..
The antibody is specific anti-human NGAL antibody, and monoclonal antibody or polyclonal antibody can be selected.
Preferably, the antibody is mouse anti-human monoclonal's antibody, rabbit anti-human polyclonal antibody or sheep anti-human polyclonal antibody
In any one.
Preferably, the latex particle particle diameter is 100-400nm, surface charge 0.05-0.2mmol/g.
Preferably, the R2 buffer solutions include buffer solution, and phosphate buffer, HEPES bufferings can be selected in the buffer solution
Liquid, MES buffer solutions, glycine buffer, borate buffer, acetate buffer solution or citrate buffer solution etc., its concentration are
10-500mM, pH 5.0-10.0.
It is further preferred that the pH of buffer is 6.0-8.0.
The R2 buffer solutions may also include the reagents such as electrolyte, surfactant, preservative, biostability, above-mentioned thing
Matter is dissolved in buffer solution when in use, for the resuspension and dilution of the latex particle for being coated with antibody.
The calibration object is NGAL recombinant proteins or natural NGAL albumen.
A kind of preparation side of neutrophil gelatinase-associated lipocalin latex immunoturbidimetry detection kit
Method, wherein, the preparation of the reagent R2 includes the following steps：
(1) latex particle and antibody are diluted respectively using coating buffer solution, by the latex particle after dilution with resisting
Body is uniformly mixed, and forms the mixed liquor of latex particle and antibody；
(2) EDC is added into the mixed liquor of latex particle and antibody to be activated, centrifuged after stirring and remove supernatant；
(3) six ammonia of polyethylene glycol is added in the precipitation after being centrifuged to step (2), is centrifuged after being stirred overnight and removes supernatant；
(4) R2 buffer solutions are added in the precipitation after centrifuging to step (3) to be resuspended, ultrasonic disperse is uniform, and in 25-60
DEG C curing 16-72 it is small when, obtain the coated latex particle of stable antibody；
(5) the coated latex particle of antibody that step (4) obtains is diluted to 0.05-0.2%wt, 4- using R2 buffer solutions
10 DEG C of preservations.
Preferably, when the mixing time in the step (2) is 2-3 small；Mixing time in the step (3) is 2-4
Hour.
Preferably, phosphate buffer, HEPES buffer solution, MES buffer solutions, glycine can be selected in the coating buffer solution
Buffer solution, borate buffer, acetate buffer solution or citrate buffer solution etc., its concentration are 10-500mM, pH 5.0-
10.0。
It is further preferred that the coating pH of buffer is 6.0-8.0.
Preferably, the concentration of six ammonia of polyethylene glycol is 0.1-0.5%wt, the use of six ammonia of polyethylene glycol
Volume and latex particle in step (2) are identical with the mixeding liquid volume of antibody.
Preferably, the temperature of whipping process is 25-55 DEG C in the step (1), (2), (3).
It is further preferred that the temperature of whipping process is 25-45 DEG C in the step (1), (2), (3).
Preferably, curing temperature is 30-50 DEG C in the step (4).
Beneficial effect：The invention discloses a kind of neutrophil gelatinase-associated lipocalin latex immunoturbidimetry
Method detection kit and preparation method thereof, selects sealer of six ammonia of polyethylene glycol as antibody binding latex particle；Poly- second two
Six ammonia of alcohol contains polyglycol chain and 6 amino, its amino can be combined with the carboxyl and epoxy group being activated on latex particle, made
Particle surface has more hydrophily, so as to prevent the absorption of biological origin material, reduces nonspecific reaction；Meanwhile poly- second two
Six ammonia of alcohol also helps the D structure of the antibody after being combined with latex particle and/or the stability in its direction, and then improves anti-
The stability of performance after body combination latex particle.
In conclusion application present invention progress neutrophil gelatinase-associated lipocalin detection high specificity,
High sensitivity, stabilization of kit are good, NGAL contents that can be in efficient detection urine, blood plasma, serum, and testing result with it is glimmering
Light immunochromatographic method is related to enzyme linked immunosorbent assay good.
Brief description of the drawings
Fig. 1 show the Sensitivity comparison of the how anti-coating reagent of seven kinds of blocking agents；
Fig. 2 show the Sensitivity comparison of seven kinds of blocking agent monoclonal antibody coating reagents；
Fig. 3 show the serum contributive rate of six kinds of blocking agent reagents；
Fig. 4 show NGAL latex immunoturbidimetries detection kit (J＆W) of the present invention and 1 calibration object curve of comparative example；
Fig. 5 show J＆W and comparative example 1 and fluorescence immunoassay when being detected to 64 groups of NGAL range of normal value urine specimens
Chromatography testing result relevant comparative schemes；
Fig. 6 show J＆W and comparative example 1 and fluorescence immunoassay when being detected to 71 groups of NGAL exceptional value scope urine specimens
Chromatography testing result relevant comparative schemes；
Fig. 7 show BIOPORTO companies ELISA reagent calibration curves；
Fig. 8 show J＆W to scheme with enzyme linked immunosorbent assay testing result relevant comparative；
Fig. 9 show the anti-interference test result of NGAL in J＆W detection serum；
Figure 10 show the anti-interference test result of NGAL in J＆W detection urines；
Figure 11 show J＆W4-8 DEG C of storage stability curve；
Figure 12 show 37 DEG C of storage stability curves of J＆W.
Embodiment
With reference to embodiment, the invention will be further described：
Embodiment 1：Increasing sense using six ammonia of polyethylene glycol as the NGAL latex immunoturbidimetry detection kits of sealer is made
With
1. the preparation of NGAL latex immunoturbidimetries detection kit of the present invention
Reagent R1 includes：25mM HEPES buffer solutions (pH7.5), 150mM sodium chloride, 10mMEDTA-2Na, 1%wt
BSA, 1%wt Macrogol 6000,0.01%wt Tween-20s, 1%wt rabbit anteserums or mice serum, 0.1%wt Sodium azides.
Reagent R2 includes：Rabbit anti-human polyclonal antibody or mouse anti-human monoclonal's antibody (5mg/mL), 10%wt latex particles
(particle diameter 300nm, surface charge 0.15mmol/g), six ammonia of 0.5%wt polyethylene glycol (molecular weight polyethylene glycol for 2000 or
5000), 0.1%wt BSA, 0.1%wt Sodium azide and 50mM HEPES buffer solutions (pH7.5).
Calibration object includes：10mM HEPES buffer solutions (pH7.5), 1%wtBSA, 0.1%wt Tween-20s, 0.1%wt are folded
Nitrogen sodium and NGAL recombinant proteins, wherein NGAL recombinant proteins concentration are respectively 150ng/mL, 600ng/mL, 1500ng/mL,
3000ng/mL, 5000ng/mL.
Wherein, when prepared by how anti-coating reagent R2：
Rabbit anti-human polyclonal antibody is diluted using 50mM HEPES buffer solutions so that the final concentration of 1mg/mL of antibody, volume
For 10mL, stirred 5 minutes under room temperature environment.Use 50mM HEPES buffer solution diluted latex particles so that latex particle is dense eventually
Spend for 2%wt, volume 10mL, stirred 5 minutes under room temperature environment.
Diluted rabbit anti-human polyclonal antibody is mixed with diluted latex particle, when 25 DEG C of stirring in water bath 2 are small.To latex
1%wt EDC 0.6mL are added in the mixed liquor of particle and antibody to be activated, and are centrifuged after 25 DEG C of stirring in water bath 2h and are removed supernatant；To
Add the diluted six ammonia 20mL of 0.5%wt polyethylene glycol of 50mM HEPES buffer solutions in precipitation after centrifugation to be closed, 25 DEG C
Stirring in water bath centrifuges after overnight and removes supernatant；Added in precipitation after centrifugation and be mixed with 0.1%wt BSA and 0.1%wt Sodium azides
50mM HEPES buffer solutions, 1%wt is diluted to by latex reaction solution, and ultrasonic disperse is uniform, and when 37 DEG C of curings 20 are small, is obtained
To the stable coated latex particle of antibody；Delayed using the 50mM HEPES for being mixed with 0.1%wt BSA and 0.1%wt Sodium azides
The coated latex particle of obtained antibody is diluted to 0.1%wt, 4-10 DEG C of preservation by fliud flushing.
When prepared by monoclonal antibody coating reagent R2：
Mouse anti-human monoclonal's antibody 1, mouse anti-human monoclonal's antibody 2 are diluted respectively using 50mM HEPES buffer solutions so that
Each final concentration of 1mg/mL of antibody, volume 10mL, are stirred 5 minutes under room temperature environment.Diluted using 50mM HEPES buffer solutions
Latex particle so that latex particle final concentration of 2%wt, volume 10mL, are stirred 5 minutes under room temperature environment.
Diluted mouse anti-human monoclonal's antibody is mixed with diluted latex particle respectively, when 25 DEG C of stirring in water bath 2 are small.To
1%wt EDC 0.6mL are added in the mixed liquor of latex particle and antibody to be activated, and are centrifuged and are gone after 25 DEG C of stirring in water bath 2h
Clearly；The diluted six ammonia 20mL of 0.5%wt polyethylene glycol of 50mM HEPES buffer solutions is added into the precipitation after centrifugation to be closed,
25 DEG C of stirring in water bath centrifuge after overnight and remove supernatant；Added in precipitation after centrifugation and be mixed with 0.1%wt BSA and 0.1%wt nitrine
The 50mM HEPES buffer solutions of sodium, 1%wt is diluted to by latex reaction solution, and ultrasonic disperse is uniform, and small in 42 DEG C of curings 20
When, obtain the coated latex particle of stable antibody；The coated latex particle of two kinds of antibody is mixed, using being mixed with 0.1%
Two kinds of coated latex particles of antibody are diluted to by the 50mM HEPES buffer solutions of wtBSA and 0.1%wt Sodium azides respectively
0.04%wt, the i.e. coated latex particle total concentration of two kinds of antibody are 0.08%wt, and 4-10 DEG C preserves.
2. sensibilization is tested
Experimental group：With six ammonia of polyethylene glycol (MW5000) (PEG (5000) -6N) for sealer or with polyethylene glycol
(MW2000) six ammonia (PEG (2000) -6N) are sealer；
Control group：Replaced for sealer real with BSA, N101, PEG1000, mPEG-N-5000, mPEG-N-20000 respectively
Six ammonia of polyethylene glycol in group is tested, wherein：N101 main components are 2- methacryloxyethyls phosphocholine and methyl-prop
The copolymer of olefin(e) acid butyl ester polymer (MPC-BMA).
The influence of seven kinds of blocking agent monoclonal antibodies or how anti-coating reagent to NGAL low values (150ng/mL) reactivity is contrasted,
2 μ LNGAL samples and 240 μ L reagents R1 are mixed, and 5 minutes are kept the temperature at 37 DEG C, then add reagent R260 μ L, 37 DEG C of insulations
540nm absorbance As 1 are recorded after 20 seconds, record absorbance A 2, spline calibrations after five minutes.
Experimental result as shown in Figs. 1-2, compared with BSA is closed, in how anti-coating reagent, is tried after PEG (5000) -6N closings
Agent sensitivity higher, monoclonal antibody are coated with reagent, reagent sensitivity higher after PEG (2000) -6N closings, it can be seen that, poly- second two
Six ammonia of alcohol has good increasing sense effect.
Embodiment 2：Serum shadow using six ammonia of polyethylene glycol as the NGAL latex immunoturbidimetry detection kits of sealer
The rate of sound
The preferable six kinds of sealers of contrast reactivity (BSA, N101, PEG (5000) -6N, PEG (2000) -6N,
PEG1000, mPEG-N-5000) influence to reagent measured value accuracy.Specific method is：It is normal to 0.9%NaCl, people's origin
The human neutrophil genatinase associated lipocalin recombinant antigen that concentration is 20000ng/mL is added in serum (EXA-N), people's origin Abnormal Serum (EXA-A) respectively,
Make the final concentration of 200ng/mL of NGAL of each matrix.The sample of these three matrix is carried out using the reagent of above-mentioned blocking agent
Detection, is compared using 0.9%NaCl testing results as control.
Experimental result is as shown in figure 3, compared with traditional sealer BSA, PEG (5000) -6N, PEG1000, mPEG-N-
For the serum contributive rate of 5000 closing monoclonal antibody coating reagents closer to 1.00, measured value is more accurate.Further, with PEG (5000)-
The how anti-coating reagent of 6N, PEG1000, mPEG-N-5000 closing is detected the sample of these three matrix, and PEG (5000)-
For the serum contributive rate of the how anti-coating reagent of 6N closings closer to 1.00, measured value is more accurate.
Embodiment 3：Specificity using six ammonia of polyethylene glycol as the NGAL latex immunoturbidimetry detection kits of sealer
1. the preparation of NGAL latex immunoturbidimetries detection kit (J＆W) of the present invention
Reagent R1 includes：50mM HEPES buffer solutions (pH8.0), 150mM sodium chloride, 10mMEDTA-2Na, 1%wt
BSA, 0.8%wt Macrogol 6000,0.01%wt Tween-20s, 1%wt rabbit anteserums and 0.1%wt Sodium azides.
Reagent R2 includes：Rabbit anti-human polyclonal antibody (5mg/mL), 10%wt latex particles (particle diameter 200nm, surface charge
0.2mmol/g), six ammonia of 0.5%wt polyethylene glycol (molecular weight polyethylene glycol 5000), 0.1%wtBSA, 0.1%wt Sodium azides
With 50mM HEPES buffer solutions (pH8.0).
Calibration object includes：10mM HEPES buffer solutions (pH8.0), 1%wtBSA, 0.1%wt Tween-20s, 0.1%wt are folded
Nitrogen sodium and NGAL recombinant proteins, wherein NGAL recombinant proteins concentration are respectively 150ng/mL, 600ng/mL, 1500ng/mL,
3000ng/mL, 5000ng/mL.
Reagent R2 is prepared and is detected the method for sample as described in Example 1.
2. comparative example is set
Comparative example 1：Other producer's latex immunoturbidimetry (LETIA) reagents
Reagent R1：PH7.3, main component are：Glycine 50mM, NaCl 0.15M, EDTA 10mM, BSA0.1%wt, rabbit
IgG 1%wt；
Reagent R2：PH7.0, main component are：HEPES 20mM, Sodium azide 0.1%wt, small grain size latex 2%wt, big grain
Footpath latex 0.5%wt.
Calibration object：HEPES 50mM, NaCl 0.15M, EDTA 10mM, Sodium azide 0.1%wt, BSA0.5%wt, sucrose
0.5%wt, remaining is deionized water.
Comparative example 2：Commercial optical immunochromatography (FCIA) kit is detected
3. the correlation test with fluorescence immune chromatography method
NGAL latex immunoturbidimetries detection kit (J＆W) of the present invention and 1 calibration object curve of comparative example are as shown in Figure 4.
Urine sample is carried out using NGAL latex immunoturbidimetries detection kit (J＆W) of the present invention and comparative example 1,2 reagents
This detection, NGAL ranges of normal value are below 100ng/mL in people's urine sample of three sets of reagent suggestions.Normal value is detected respectively
Scope sample (≤100ng/mL) and exceptional value sample (>100ng/mL), and by the detection result in comparative example 2 respectively with J＆
W and comparative example 1 are associated.
As seen in figs. 5-6, either range of normal value sample (≤100ng/mL) or exceptional value sample (>100ng/
ML), J＆W kits of the present invention are more preferable with FCIA method measured value correlations, when especially detecting range of normal value sample, PEG
(5000) the latex immunologic function test reagent of -6N closings can effectively avoid false positive reaction.
4. the correlation test with enzyme linked immunosorbent assay
Use NGAL latex immunoturbidimetries detection kit (J＆W) of the present invention and BIOPORTO companies ELISA reagents
(BIOPORTO Diagnostics, NGAL Rapid ELISAKit, KIT037RUO, Lot.NR-1309-02) carries out 20 groups of blood
Clear pattern detection, Fig. 7 show BIOPORTO companies ELISA reagent calibration curves；As shown in figure 8, use NGAL glue of the present invention
Newborn immunoturbidimetry detection kit (J＆W) reachable R related to BIOPORTO companies ELISA reagents2>0.90(R>0.95).By
This is as it can be seen that the latex immunologic function test reagent of PEG (5000) -6N closings is related to enzyme linked immunosorbent assay detection good.
5. anti-interference test
The human neutrophil genatinase associated lipocalin recombinant antigen that concentration is 20000ng/mL is added into normal human serum or normal human urine, it is another to add
The interference factor dilution of each high concentration interference factor or same volume, makes the final concentration of 200ng/ of NGAL in serum or urine
ML, interference factor are as shown in table 1.Contrast with the addition of the measured value of dilution (-) or interference factor (+) sample.
Table 1
Shown compared with (-) such as Fig. 9-10, measured value can be controlled within ± 5%, it is seen then that J＆W antijamming capabilities are excellent
It is different.
6. stability test
The stability of 4-8 DEG C and 37 DEG C preservation is carried out to NGAL latex immunoturbidimetries detection kit (J＆W) of the present invention
Test, measure its signal under NGAL low values (150ng/mL) and high level (600ng/mL) respectively, with 0 day measure signal into
Row contrast.As depicted in figs. 11-12,4-8 DEG C of preservation two-and-a-half years of the present invention have good stability, and 37 DEG C can preserve at least 33 days.
It the above is only the preferred embodiment of the present invention, protection scope of the present invention is not limited to reality shown in this article
Example is applied, all technical solutions belonged under thinking of the present invention belong to protection scope of the present invention.It should be pointed out that led for this technology
For the those of ordinary skill in domain, some modifications and retouching without departing from the principles of the present invention also should be regarded as the present invention's
Protection domain.
Claims (9)
1. a kind of neutrophil gelatinase-associated lipocalin latex immunoturbidimetry detection kit, including reagent
R1, reagent R2 and calibration object, wherein, the reagent R2 includes antibody, latex particle and R2 buffer solutions, it is characterised in that：It is described
Sealer used in reagent R2 is six ammonia of polyethylene glycol, and the chemical structural formula of six ammonia of polyethylene glycol is H- (O-CH2-CH2)n-(NH)5-
NH2。
A kind of 2. neutrophil gelatinase-associated lipocalin latex immunoturbidimetry inspection according to claim 1
Test agent box, it is characterised in that：The antibody is monoclonal antibody or polyclonal antibody.
A kind of 3. neutrophil gelatinase-associated lipocalin latex immunoturbidimetry inspection according to claim 1
Test agent box, it is characterised in that：The latex particle particle diameter is 100-400nm, surface charge 0.05-0.2mmol/g.
A kind of 4. neutrophil gelatinase-associated lipocalin latex immunoturbidimetry inspection according to claim 1
Test agent box, it is characterised in that：The R2 buffer solutions include buffer solution, and the buffer solution includes phosphate buffer, HEPES delays
In fliud flushing, MES buffer solutions, glycine buffer, borate buffer, acetate buffer solution or citrate buffer solution any one or
It is several；The pH of buffer is 5.0-10.0.
A kind of 5. neutrophil gelatinase-associated lipocalin latex immunoturbidimetry inspection according to claim 1
Test agent box, it is characterised in that：The R2 buffer solutions are further included in electrolyte, surfactant, preservative, biostability
Any one or a few, any one or a few use in the electrolyte, surfactant, preservative, biostability
When be mixed in the buffer solution.
6. a kind of preparation method of neutrophil gelatinase-associated lipocalin latex immunoturbidimetry detection kit,
It is characterized in that：The preparation of the reagent R2 includes the following steps：
(1) latex particle and antibody are diluted respectively using coating buffer solution, the latex particle after dilution is mixed with antibody
Close uniform, the mixed liquor of formation latex particle and antibody；
(2) EDC is added into the mixed liquor of latex particle and antibody to be activated, centrifuged after stirring and remove supernatant；
(3) six ammonia of polyethylene glycol is added in the precipitation after being centrifuged to step (2), is centrifuged after being stirred overnight and removes supernatant；
(4) add R2 buffer solutions in the precipitation after centrifuging to step (3) to be resuspended, ultrasonic disperse is uniform, and ripe in 25-60 DEG C
When change 16-72 is small, the coated latex particle of stable antibody is obtained；
(5) the coated latex particle of antibody that step (4) obtains is diluted to 0.05-0.2%wt using R2 buffer solutions, 4-10 DEG C
Preserve.
A kind of 7. neutrophil gelatinase-associated lipocalin latex immunoturbidimetry inspection according to claim 6
The preparation method of test agent box, it is characterised in that：The coating buffer solution includes phosphate buffer, HEPES buffer solution, MES
Any one or a few in buffer solution, glycine buffer, borate buffer, acetate buffer solution or citrate buffer solution；Institute
It is 5.0-10.0 to state coating pH of buffer.
A kind of 8. neutrophil gelatinase-associated lipocalin latex immunoturbidimetry inspection according to claim 6
The preparation method of test agent box, it is characterised in that：The concentration of six ammonia of polyethylene glycol is 0.1-0.5%wt, described poly-
The use volume of six ammonia of ethylene glycol and latex particle in step (2) are identical with the mixeding liquid volume of antibody.
A kind of 9. neutrophil gelatinase-associated lipocalin latex immunoturbidimetry inspection according to claim 6
The preparation method of test agent box, it is characterised in that：The temperature of whipping process is 25-55 DEG C in the step (1), (2), (3).
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Cited By (12)
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| CN109738626A (en) * | 2019-02-19 | 2019-05-10 | 上海复星长征医学科学有限公司 | NGAL latex immunoturbidimetry detection kit and preparation method thereof |
| CN109738626B (en) * | 2019-02-19 | 2021-11-26 | 复星诊断科技(上海)有限公司 | NGAL latex immunoturbidimetry detection kit and preparation method thereof |
| CN110794131A (en) * | 2019-09-12 | 2020-02-14 | 苏州普瑞斯生物科技有限公司 | Interleukin-6 detection kit and preparation method thereof |
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| CN113402606A (en) * | 2020-03-17 | 2021-09-17 | 江苏众红生物工程创药研究院有限公司 | Neutrophil gelatinase-associated lipocalin detection kit and clinical application thereof |
| CN113402606B (en) * | 2020-03-17 | 2023-06-06 | 江苏众红生物工程创药研究院有限公司 | Neutrophil gelatinase-associated lipocalin detection kit and clinical application thereof |
| CN113820493A (en) * | 2020-06-19 | 2021-12-21 | 亳州市新健康科技有限公司 | Sandwich immunoassay kit |
| CN112014572A (en) * | 2020-08-24 | 2020-12-01 | 北京世纪沃德生物科技有限公司 | Preparation method and application of latex particles for detecting KL-6 |
| CN112014576A (en) * | 2020-09-03 | 2020-12-01 | 北京安图生物工程有限公司 | Reagent for detecting human serum amyloid A and preparation method thereof |
| CN113267635A (en) * | 2021-04-29 | 2021-08-17 | 广东优尼德生物科技有限公司 | Adiponectin antibody nano latex particle and kit for detecting adiponectin |
| CN113267635B (en) * | 2021-04-29 | 2022-02-11 | 广东优尼德生物科技有限公司 | Adiponectin antibody nano latex particle and kit for detecting adiponectin |
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