CN102628868B - Latex enhanced immunoturbidimetry kit for detection of asymmetric dimethylarginine content - Google Patents
Latex enhanced immunoturbidimetry kit for detection of asymmetric dimethylarginine content Download PDFInfo
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Abstract
The invention relates to a latex enhanced immunoturbidimetry kit for the detection of asymmetric dimethylarginine content. Specifically, the invention relates to a latex enhanced immunoturbidimetry kit for the detection of asymmetric dimethylarginine (ADMA) content in human blood samples. The kit comprises a mouse anti-human monoclonal antibody solution, an ADMA-human Serum albumin complex crosslinked latex particle expansion and an ADMA calibrator. According to the method, by the utilization of competitive binding of free ADMA in blood and ADMA crosslinked on the surface of latex particles to ADMA monoclonal antibody, turbidity formed by the reaction between the ADMA-crosslinked latex particles and ADMA monoclonal antibody is reduced. Therefore, the content of ADMA is detected through the reduction degree of turbidity. The kit provided by the invention can be applied in a biochemical analyzer commonly-used in clinic, is convenient and rapid to operate and has strong specificity. Both sensitivity and detection range of the kit can satisfy clinic application needs.
Description
Technical field
The invention provides a kind of kit that utilizes immunoturbidimetry method to measure Ng,Ng-Dimethylarginine content in human serum, plasma sample, be specifically related to utilize Ng,Ng-Dimethylarginine in human serum, plasma sample and crosslinkedly have the latex suspension competition of Ng,Ng-Dimethylarginine-human serum albumins complex in conjunction with mouse-anti Ng,Ng-Dimethylarginine monoclonal antibody, by detecting the decline of immune turbidity, reach higher sensitivity and wider sensing range, belong to medical immunology in-vitro diagnosis field.
Background technology
Ng,Ng-Dimethylarginine (asymmetric diethylarginine, ADMA) is a kind of natural amino acid, is extensively present in tissue and cell.It can suppress the biological effect synthetic and that produce therefrom of nitric oxide synthetase (nitric oxide synthase, NOS), stops the synthetic of endothelial cell nitric oxide (NO), causes function of vascular endothelium to reduce.The order of severity of the vascular lesionses such as the variation of plasma ADM A concentration and function of vascular endothelium infringement, atherosclerotic, cerebral infarction, hypertension, high lipoprotein abnormalities, diabetes, renal failure is closely related, is one of emphasis problem of current clinical research.Can say, the concentration of plasma ADM A is the risk factor of endothelial dysfunction and angiocardiopathy.
ADMA is generated by arginine methyltransferase in vivo, arginine methyltransferase has two types: I type methylate histone and nuclear RNA-in conjunction with albumen, generate NG-monomethyl-L-arginine (NG-monomethyl-L-arginine, L-NMMA) and ADMA; The II type myelin major protein that only methylates produces L-NMMA and SDMA (symmetricdimethylarginine, SDMA).L-NMMA and ADMA all can reduce the activity of NOS, but the former plasma concentration is approximately 1/20 of ADMA, and therefore, great majority research is all for the detection of ADMA.The concentration of SDMA in blood plasma is similar to ADMA, but NOS is not had to effect.
The method of measuring ADMA in blood plasma comprises high performance liquid chromatography, enzyme linked immunological and immunofluorescence etc.But the mensuration of ADMA is more loaded down with trivial details in biological sample, because exist arginine and SDMA and ADMA to show cross reactivity in detection.ADMA and arginic different two methyl that are only on a guanidine radicals nitrogen.In addition, in normal human serum, arginic concentration is approximately 100 times of ADMA concentration.SDMA is than arginine closer to ADMA, and difference is only the position of a methyl.In normal human serum, the concentration of SDMA is suitable with the concentration of ADMA, is approximately 1uM.
Moreover,, all there is the feature that sample preparation is loaded down with trivial details, hand-manipulated, time-consuming is long in the method for above-mentioned detection ADMA.Therefore, invent ADMA in a kind of specific detection human plasma, can be applied to kit on self-reacting device becomes very and needs simultaneously.
Summary of the invention
Therefore, technical purpose of the present invention is to provide a kind of Ng,Ng-Dimethylarginine detection kit that can be applied on full-automatic biochemical analyzer, can detect fast and accurately the Ng,Ng-Dimethylarginine content in human plasma sample.
Therefore, a first aspect of the present invention relates to a kind of latex enhancing immune turbidimetry kit of measuring Ng,Ng-Dimethylarginine content, it is characterized in that, comprising:
1) Ng,Ng-Dimethylarginine R1 reagent;
2) Ng,Ng-Dimethylarginine R2 reagent;
3) Ng,Ng-Dimethylarginine calibration object;
Described Ng,Ng-Dimethylarginine R1 reagent comprises Ng,Ng-Dimethylarginine monoclonal antibody specific, damping fluid, stabilizing agent, set accelerator and antiseptic;
Described Ng,Ng-Dimethylarginine R2 reagent comprises latex particle, damping fluid, stabilizing agent and the antiseptic that Ng,Ng-Dimethylarginine-inert protein complex is coated;
Described Ng,Ng-Dimethylarginine calibration object is comprised of Ng,Ng-Dimethylarginine, damping fluid, stabilizing agent and antiseptic.
Preferably, described Ng,Ng-Dimethylarginine-inert protein complex is will after Ng,Ng-Dimethylarginine and inert protein coupling, to obtain by coupling agent.Preferably, described coupling agent is selected from a kind of in carbodiimides (EDAC), glutaraldehyde, two isocyanic acid compounds and dihalide dinitro benzene.
Preferably, described inert protein is selected from a kind of in human serum albumins, bovine serum albumin(BSA), ox transferrins, hemocyanin.
Preferably, described latex particle is a kind of nucleocapsid structure, and newborn core is poly styrene polymer, and newborn shell is comprised of styrene, n-butyl acrylate and methacrylic acid copolymer.
Preferably, described latex particle according to its surface with chemical group difference can be selected from a kind of that carboxyl, amino, hydroxyl, hydrazide group, chloromethyl modify.
Preferably, described latex particle diameter range is 50-250nm, and working concentration is selected from 0.05-0.5%.
Preferably, described Ng,Ng-Dimethylarginine-inert protein complex can be combined in latex particle surface by chemical bonded refractory by the chemical group carrying with latex particle itself, conventional chemical group comprises carboxyl, amino, hydroxyl, hydrazide group, chloromethyl etc., the present invention is by the Chemical Crosslinking Methods optimized by the compound specific carboxyl that is combined in latex particle surface of Ng,Ng-Dimethylarginine-inert protein, and the Chemical Crosslinking Methods of described optimization may be summarized to be " the two pH of a step " method.
Preferably, described damping fluid is selected from 3-[N, N-bis-(hydroxyethyl) amino] one or more in-2-hydroxy-propanesulfonic acid-sodium hydrate buffer solution, 4-(2-hydroxyethyl)-1-piperazine propane sulfonic acid-sodium hydrate buffer solution, trishydroxymethylaminomethane-HCl damping fluid, phosphate buffer, glycine buffer and barbitol buffer solution, working concentration is selected from 10-200mM.
Preferably, described stabilizing agent be selected from 0.1-5% concentration range bovine serum albumin(BSA), the sodium chloride of 0.5-1% concentration range, the Tween-20 of the EDTA of 2-50mM, 0.1-1% concentration range, the glycerine of 1-10% concentration range one or more.
Preferably, described antiseptic is selected from one or more in Sodium azide, thimerosal, phenol and ethyl mercury sodium thiosulfate, and working concentration is selected from 0.02%-0.1%.
Preferably, described set accelerator is selected from a kind of in PEG-4000, PEG-6000, PEG-8000, sodium dextran sulfate, and working concentration scope is selected from 1-6%.
Most preferably, the latex enhancing immune turbidimetry kit of described mensuration Ng,Ng-Dimethylarginine content is comprised of following component:
R1:0.2mg/mL ADMA monoclonal antibody, 0.9% sodium chloride, 5%PEG-6000,0.2%BSA, 20mM EDTA, 0.1% sodium azide, 50mM Tris-HCl pH of buffer 7.2;
R2: polystyrene latex particle 0.25%, 50mM glycine buffer pH8.0,0.9% sodium chloride, 0.8%BSA, 20mM EDTA, 0.1% polysorbas20,0.1% sodium azide that Ng,Ng-Dimethylarginine-human serum albumins complex is coated, wherein said polystyrene latex particle surface is 150nm with carboxyl, diameter;
Ng,Ng-Dimethylarginine calibration object: concentration is respectively 0,0.5,2,5,10, the Ng,Ng-Dimethylarginine of 20umol/L, 0.9% sodium chloride, 1%BSA, 50mM EDTA, 0.1% sodium azide, 50mM Tris-HCl pH of buffer 7.2.
In other words, the present invention is based on a specificity for the monoclonal antibody of ADMA and the crosslinked latex particle suspending liquid that has ADMA-human serum albumins, in specific reaction system, there is antigen-antibody reaction and form certain turbidity, can be detected based on spectrophotometric Biochemical Analyzer.And if in human plasma, have the ADMA of free state to exist, can turbidity be declined with the ADMA competition that is fixed on latex particle surface in conjunction with monoclonal antibody, by the reduction degree of system turbidity after assaying reaction, ADMA is carried out to quantitative test.
The present invention has solved specificity and the low problem of automaticity that ADMA detects simultaneously, can wide popularization and application.
Particularly, to achieve these goals, the technical scheme that the present invention takes is: by use, be coupled at the coated latex particle of ADMA in inert protein and be suspended in and in specific damping fluid, make R2 reagent.As coated latex particle diameter range, be wherein 100-200nm, concentration is 1-5mg/mL.Because ADMA molecular weight is very little, be difficult to be combined with latex particle.Therefore in the present invention, be used as coated ADMA and first by chemical bond, be coupled on inertia high molecular weight protein, the inert protein that molecular weight is larger can be better combined with latex particle, and it is 0.1-1mg/mL that coupling has the inert protein concentration of ADMA.Coupling has the inert protein of ADMA further by the Chemical Crosslinking Methods of optimizing, to be coated on latex particle surface, Chemical Crosslinking Methods used is " the two pH of a step " method, be that first latex particle is activated agent activation under low pH condition, mix with the ADMA-inert protein complex in high pH solution again, mixed pH value of solution is neutral, this method can so that coupling have the inert protein of ADMA efficient, special with latex particle surface conjunction.The buffer suspension liquid being used as is the glycocoll-NaOH damping fluid that contains stabilizing agent and antiseptic, and concentration is 20-100mM, and pH scope is 7-9.The stabilizing agent being used as is that bovine serum albumin(BSA) 0.1-1%, sodium chloride concentration are that 0.7-0.9%, ethylenediamine tetraacetic acid concentration are that 5-50mM, polysorbas20 concentration are 0.1-1%.Antiseptic used is Sodium azide 0.05-0.1%.
By adding specificity to make R1 reagent for the monoclonal antibody of ADMA, in specific damping fluid, to add inorganic salts, set accelerator, protein and antiseptic in specific damping fluid.Wherein selected monoclonal antibody be specificity for the antibody of ADMA, can prepare by art-recognized hybridoma method, also can buy commercial product, such as Abcam, Mlilipore etc.First selected ADMA monoclonal antibody will detect by indirect ELISA method, confirms can not produce cross reaction with SDMA, L-NMMA and arginine.Wherein require the surge capability of damping fluid for regulating pH scope between 7-9, can select various damping fluids, preferably HEPES, glycocoll, Tris etc., compare with other damping fluids, has that surge capability is strong, solubleness is high, good biological fitness and a reactionlessness.Wherein Tris-HCl can the long period control constant pH scope, therefore more preferably Tirs-HCl, and its concentration range is 10-100mM, pH scope is 7-8.Set accelerator is selected PEG-6000, can accelerate immune response speed, shortens detection time, and its final concentration is 2-6%.Protein is selected bovine serum albumin(BSA), and final concentration is 0.1-1%.Sodium chloride concentration is 0.7-0.9%, and Sodium azide concentration is 0.05-0.1%.
By add ADMA concentration in specific damping fluid, be respectively 0,0.5,2,5,10,20umol/L makes multiple spot calibration object, wherein require the surge capability of damping fluid for regulating pH scope between 7-9, can select various damping fluids, preferred HEPES, glycocoll, Tris etc., compare with other damping fluids, have that surge capability is strong, solubleness is high, good biological fitness and a reactionlessness.Wherein Tris-HCl can the long period control constant pH scope, therefore more preferably Tirs-HCl, and its concentration range is 10-200mM, pH scope is 7-8.Wherein contain specific protein stabiliser if bovine serum albumin(BSA) concentration is 0.1-1%, contain specific antiseptic if Sodium azide concentration is 0.05-0.1%, contain specific antioxidant as ethylenediamine tetraacetic acid concentration be 5-50mM.
The invention provides for measuring at human plasma, Ng,Ng-Dimethylarginine immunoturbidimetry assay method in blood serum sample, it is characterized in that, by containing the human plasma of Ng,Ng-Dimethylarginine, serum sample, compete binding specificity for the monoclonal antibody of ADMA with the Ng,Ng-Dimethylarginine that latex particle is coated, cause turbidity to decline, the degree and the human plasma that decline, Ng,Ng-Dimethylarginine content in serum is directly proportional, adopt automatic clinical chemistry analyzer device can calculate human plasma, the content of Ng,Ng-Dimethylarginine in serum sample.
The present invention compared with prior art, has following features:
1) can be applied on automated chemical analyser, detection speed is fast;
2) detection specificity is strong, does not produce cross reaction with L-NMMA, SDMA and arginine, has very high correlativity with HPLC method;
3) detection sensitivity and sensing range reach other just at the kit peer-level of clinical practice, can meet clinical practice needs.
Accompanying drawing explanation
Fig. 1 is the linear graph of the prepared kit of the present invention.
Fig. 2 is that the prepared kit of the present invention and HPLC method kit detect the correlativity that clinical sample is compared.
Embodiment
To further illustrate the present invention by following non-limiting example below, as well known to those skilled in the art, without departing from the spirit of the invention, can make many modifications to the present invention, such modification also falls into scope of the present invention.
Following experimental technique if no special instructions, is conventional method, and the experiment material of using if no special instructions, all can easily be obtained from commercial company.
Embodiment
Embodiment 1: the human serum albumins of preparation coupling Ng,Ng-Dimethylarginine
After 10mg human serum albumins is diluted with 1mL 0.1M phosphate buffer (pH7.8), add Ng,Ng-Dimethylarginine, add again 50mg EDAC, 4 ℃ of reactions 24 hours, use again 0.1M phosphate buffer (pH7.8) to dialyse 24 hours under 4 ℃ of conditions, be made into the human serum albumins (Ng,Ng-Dimethylarginine-human serum albumins complex) of coupling ADMA.
Embodiment 2: prepare Ng,Ng-Dimethylarginine R2 reagent
Surface, with polystyrene latex solution (concentration 10%) (purchased from the Merck) 0.5mL of carboxyl, diameter 150nm, added the 0.05M MES damping fluid (pH6.0) of 4.5mL, then adds 5mg EDAC, 37 ℃ of reactions 1 hour.Again 0.5mg coupling is had to the human serum albumins of ADMA with after 5mL 0.05M borate buffer solution (pH9.2) dilution, join immediately in above-mentioned latex solution, 37 ℃ of reactions 6 hours.Finally add glycine buffer (pH8.5) the stirring 1h of 1mL 0.1M to carry out cessation reaction, the centrifugal supernatant that removes, with the glycine buffer (pH8.0) of 20mL 50mM, wash 3 times, in the glycine buffer of 50mM, contain 0.9% sodium chloride, 20mM EDTA, 0.8%BSA, 0.1% polysorbas20,0.1% sodium azide, last with the glycine buffer of same 20mL, be dispersed into milky latex suspension again, for R2 reagent, in final R2 reagent, the coated polystyrene latex granule density of Ng,Ng-Dimethylarginine-human serum albumins complex is 0.25%.
Embodiment 3: prepare Ng,Ng-Dimethylarginine R1 reagent
In the Tris-HCl of 50mM pH7.2 damping fluid, add the ADMA monoclonal antibody (purchased from Abcam) of 0.2mg/mL, add sodium chloride 0.9%, PEG-60005%, BSA 0.2%, EDTA20mM, sodium azide 0.1% simultaneously, stir as R1 reagent.
Embodiment 4: prepare Ng,Ng-Dimethylarginine calibration object
In the Tris-HCl of 50mM pH7.2 damping fluid, add that concentration is respectively 0,0.5,2,5,10, the Ng,Ng-Dimethylarginine of 20umol/L, add in addition sodium chloride 0.9%, BSA1%, EDTA 50mM, sodium azide 0.1%, stir as Ng,Ng-Dimethylarginine multiple spot calibration object.
Embodiment 5: the Performance Detection of Ng,Ng-Dimethylarginine agent box
1. the usage of detection kit:
Hitachi's 7180 Biochemical Analyzers of take are example: measure wavelength 570nm, first add R1 reagent 150uL, 37 ℃ of reactions added ADMA calibration object 10uL after 30 seconds, react again and after 60 seconds, add R2 reagent 50uL, assaying reaction the 10th second, absorbance (the A1 of 70 seconds, A2), calculate absorbance difference Δ A=A2-A1, every pipe replication 2 times, the absorbance difference Δ A that each calibration QC is recorded for 2 times is ordinate, corresponding concentration is horizontal ordinate, make " concentration-absorbance difference " calibration curve, get sample to be tested, be measured in the same method the absorbance difference of sample, substitution calibration curve, can calculate the content of ADMA in sample to be tested.
2. repeatability detects:
With normal saline dilution human serum sample, compound concentration is respectively 0.1,0.5,1, the sample of 2umol/L, the kit forming with above-mentioned Ng,Ng-Dimethylarginine R1, Ng,Ng-Dimethylarginine R2 and Ng,Ng-Dimethylarginine calibration object, compares Repeatability with the ELISA reagent h box of A company.The reagent fundamental analysis principle of A company is; first use acylting agent by the ADMA acyl group in sample; then add ELISA Plate; with the ADMA competitive binding ADMA polyclonal antibody being combined in solid phase; adopt standard items and the enzymic-labelled antibody competition combination of varying level; then absorbance, production standard curve are read in colour developing.After the same step operation of sample and standard items, on typical curve, obtain corresponding A/D MA concentration.Kit of the present invention is measured 10 times according to method described in 1, calculates the coefficient of variation of different ADMA content pattern detection, with A company commercial ELISA kit comparative result as shown in following table 1 and 2:
The detection repeatability of table 1 kit of the present invention:
| 0.1uM | 0.5uM | 1uM | 2uM | |
| 1 | 0.14 | 0.52 | 1.01 | 2.12 |
| 2 | 0.13 | 0.47 | 1.02 | 2.18 |
| 3 | 0.18 | 0.48 | 1.11 | 2.07 |
| 4 | 0.14 | 0.49 | 1.07 | 2.09 |
| 5 | 0.15 | 0.45 | 1.08 | 2.08 |
| 6 | 0.12 | 0.46 | 1.04 | 2.1 |
| 7 | 0.17 | 0.49 | 0.98 | 2.12 |
| 8 | 0.19 | 0.55 | 1.01 | 2.04 |
| 9 | 0.12 | 0.58 | 1.02 | 2.08 |
| 10 | 0.17 | 0.47 | 1.07 | 2.09 |
| Mean value | 0.151 | 0.496 | 1.041 | 2.097 |
| SD | 0.025144 | 0.0416867 | 0.0401248 | 0.0374314 |
| CV | 16.7％ | 8.4％ | 3.9％ | 1.8％ |
The detection repeatability of the ELISA kit of table 2, A company:
| 0.1uM | 0.5uM | 1uM | 2uM | |
| 1 | 0.08 | 0.50 | 1.08 | 2.09 |
| 2 | 0.07 | 0.54 | 1.05 | 2.18 |
| 3 | 0.19 | 0.45 | 1.11 | 2.07 |
| 4 | 0.14 | 0.44 | 1.07 | 2.11 |
| 5 | 0.11 | 0.51 | 0.97 | 2.08 |
| 6 | 0.12 | 0.43 | 1.04 | 2.02 |
| 7 | 0.17 | 0.48 | 0.98 | 2.12 |
| 8 | 0.14 | 0.52 | 1.04 | 2.02 |
| 9 | 0.11 | 0.55 | 1.07 | 2.08 |
| 10 | 0.15 | 0.42 | 1.03 | 2.09 |
| Mean value | 0.128 | 0.484 | 1.044 | 2.086 |
| SD | 0.0376534 | 0.0469515 | 0.0432563 | 0.0467143 |
| CV | 29.42％ | 9.70％ | 4.14％ | 2.24％ |
From upper table 1 and 2, can find out, the repeatability of kit of the present invention is better than the ELISA kit of A company, is more conducive to the application of clinical diagnosis.
3. linearity test:
Screening Ng,Ng-Dimethylarginine concentration is about the sample of 20umol/L, of physiological saline, make the dilution of 10 different proportions, according to the method described in 1, detect each concentration, every concentration replication 3 times, mean value and the theoretical concentration of measuring concentration are carried out to recovery analysis, result error is all less than 10%, proves that linearity can reach 20umol/L, the results are shown in Table 3 and Fig. 1.
The linearity of table 3 kit of the present invention
| Dilution ratio | Theoretical concentration [um/L] | Measure concentration [um/L] | The recovery [%] |
| 0/10 | 0.0 | 0.0 | |
| 1/10 | 2.00 | 2.07 | 101.0％ |
| 2/10 | 4.00 | 4.12 | 104.0％ |
| 3/10 | 6.00 | 6.04 | 103.3％ |
| 4/10 | 8.00 | 8.08 | 101.3％ |
| 5/10 | 10.00 | 10.13 | 104.2％ |
| 6/10 | 12.00 | 12.07 | 101.7％ |
| 7/10 | 14.00 | 14.11 | 100.6％ |
| 8/10 | 16.00 | 16.08 | 99.8％ |
| 9/10 | 18.00 | 18.13 | 100.2％ |
| 10/10 | 20.00 | 20.02 | 104.0％ |
4. kit of the present invention and HPLC detect the correlativity comparison of clinical sample
With kit of the present invention and HPLC, 34 routine patient's sample are detected simultaneously, testing result is shown in Fig. 2, the sample ADMA result that the HPLC method of take detects is horizontal ordinate, the result that the kit of the present invention of take detects is done regretional analysis as ordinate, dependent equation is Y=0.9965X+0.018, and correlation coefficient r=0.999 shows through statistical procedures result, it is good that the inventive method detects clinical sample measured value correlativity with HPLC method, shows that the detection specificity of kit of the present invention is strong.
Claims (12)
1. a latex enhancing immune turbidimetry kit of measuring Ng,Ng-Dimethylarginine content, is characterized in that, comprising:
1) Ng,Ng-Dimethylarginine R1 reagent;
2) Ng,Ng-Dimethylarginine R2 reagent;
3) Ng,Ng-Dimethylarginine calibration object;
Described Ng,Ng-Dimethylarginine R1 reagent comprises: Ng,Ng-Dimethylarginine monoclonal antibody specific, 10-200mM damping fluid, stabilizing agent, 1-6% set accelerator and 0.02%-0.1% antiseptic;
Described Ng,Ng-Dimethylarginine R2 reagent comprises: latex particle, 10-200mM damping fluid, stabilizing agent and 0.02%-0.1% antiseptic that 0.05-0.5% Ng,Ng-Dimethylarginine-inert protein complex is coated;
Described Ng,Ng-Dimethylarginine calibration object is comprised of Ng,Ng-Dimethylarginine, 10-200mM damping fluid, stabilizing agent and 0.02%-0.1% antiseptic;
Wherein, described stabilizing agent is selected from the bovine serum albumin(BSA) of 0.1-5% concentration range, one or more in the polysorbas20 of the sodium chloride of 0.5-1% concentration range, 2-50mM EDTA, 0.1-1% concentration range and the glycerine of 1-10% concentration range;
The chemical group that described Ng,Ng-Dimethylarginine-inert protein complex carries by latex particle itself is combined in latex particle surface via chemical bonded refractory, described chemical group is selected from carboxyl, amino, hydroxyl, hydrazide group, chloromethyl, and described latex particle diameter range is 50-250nm;
By Chemical Crosslinking Methods, Ng,Ng-Dimethylarginine-inert protein complex is combined on latex particle surface, described Chemical Crosslinking Methods comprises step:
First latex particle is activated agent and activates under low pH condition, then mixes with the Ng,Ng-Dimethylarginine-inert protein complex in high pH solution, makes Ng,Ng-Dimethylarginine-inert protein complex and latex particle surface conjunction.
2. the latex enhancing immune turbidimetry kit of mensuration Ng,Ng-Dimethylarginine content according to claim 1, it is characterized in that, described Ng,Ng-Dimethylarginine-inert protein complex is will after Ng,Ng-Dimethylarginine and inert protein coupling, to obtain by coupling agent.
3. the latex enhancing immune turbidimetry kit of mensuration Ng,Ng-Dimethylarginine content according to claim 1, it is characterized in that, described inert protein is selected from a kind of in human serum albumins, bovine serum albumin(BSA), ox transferrins and hemocyanin.
4. the latex enhancing immune turbidimetry kit of mensuration Ng,Ng-Dimethylarginine content according to claim 1, it is characterized in that, described latex particle is a kind of nucleocapsid structure, breast core is poly styrene polymer, and newborn shell is comprised of styrene, n-butyl acrylate and methacrylic acid copolymer.
5. the latex enhancing immune turbidimetry kit of mensuration Ng,Ng-Dimethylarginine content according to claim 1, it is characterized in that described low pH is 6.0, and described high pH is 9.2.
6. the latex enhancing immune turbidimetry kit of mensuration Ng,Ng-Dimethylarginine content according to claim 1, is characterized in that, the chemical group that described latex particle itself carries is carboxyl.
7. the latex enhancing immune turbidimetry kit of mensuration Ng,Ng-Dimethylarginine content according to claim 1, it is characterized in that, described damping fluid is selected from 3-[N, N-bis-(hydroxyethyl) amino] a kind of in-2-hydroxy-propanesulfonic acid-sodium hydrate buffer solution, 4-(2-hydroxyethyl)-1-piperazine propane sulfonic acid-sodium hydrate buffer solution, trishydroxymethylaminomethane-HCl damping fluid, phosphate buffer, glycine buffer and barbitol buffer solution.
8. the latex enhancing immune turbidimetry kit of mensuration Ng,Ng-Dimethylarginine content according to claim 1, is characterized in that, described antiseptic is selected from one or more in Sodium azide, thimerosal, phenol and ethyl mercury sodium thiosulfate.
9. the latex enhancing immune turbidimetry kit of mensuration Ng,Ng-Dimethylarginine content according to claim 1, is characterized in that, described set accelerator is selected from a kind of in PEG-4000, PEG-6000, PEG-8000 and sodium dextran sulfate.
10. the latex enhancing immune turbidimetry kit of mensuration Ng,Ng-Dimethylarginine content according to claim 1, is characterized in that it is comprised of following component:
The Tris-HCl damping fluid that R1:0.2mg/mL ADMA monoclonal antibody, 0.9% sodium chloride, 5%PEG-6000,0.2%BSA, 20mM EDTA, 0.1% sodium azide, 50mM pH value are 7.2;
R2: polystyrene latex particle 0.25%, the glycine buffer pH8.0 of 50mM, 0.9% sodium chloride, 0.8%BSA, 20mM EDTA, 0.1% polysorbas20,0.1% sodium azide that Ng,Ng-Dimethylarginine-human serum albumins complex is coated, wherein said polystyrene latex particle surface is 150nm with carboxyl, diameter;
Ng,Ng-Dimethylarginine calibration object: the Tris-HCl damping fluid that concentration is respectively 0,0.5,2,5,10, the Ng,Ng-Dimethylarginine of 20umol/L, 0.9% sodium chloride, 1%BSA, 50mM EDTA, 0.1% sodium azide, 50mM pH value are 7.2.
The latex enhancing immune turbidimetry kit of 11. mensuration Ng,Ng-Dimethylarginine content according to claim 1, wherein
By Chemical Crosslinking Methods, Ng,Ng-Dimethylarginine-inert protein complex is combined on latex particle surface, described Chemical Crosslinking Methods comprises step:
A) the polystyrene latex solution of 0.5mL concentration 10% is added in the MES damping fluid that the 0.05MpH value of 4.5mL is 6.0, then add 5mg EDAC, 37 ℃ of reactions 1 hour;
B) 0.5mg coupling is had after the borate buffer solution dilution that the human serum albumins of Ng,Ng-Dimethylarginine is 9.2 by 5mL0.05M pH value, join immediately in step latex solution a), 37 ℃ of reactions 6 hours, Ng,Ng-Dimethylarginine-inert protein complex is combined on latex particle surface;
Wherein said polystyrene latex surface is 150nm with carboxyl, diameter.
The latex enhancing immune turbidimetry kit of 12. mensuration Ng,Ng-Dimethylarginine content according to claim 11, wherein said Chemical Crosslinking Methods is at step b) also comprise afterwards step:
C) add the glycine buffer that 1mL0.1M pH value is 8.5 to stir 1h cessation reaction, the centrifugal supernatant that removes, the glycine buffer that is 8.0 by 20mL50mM pH value washing 3 times, obtains the coated latex particle of Ng,Ng-Dimethylarginine-inert protein complex.
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| PCT/CN2012/086510 WO2013097607A1 (en) | 2011-12-30 | 2012-12-13 | Latex enhanced immunoturbidimetry kit for detecting asymmetric dimethylarginine content |
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