A kind of ADMA immunization measures reagent and detection methodTechnical field
The present invention relates to little molecule and protein orientation be connected, little molecular chaperones and detection method, belong to medical immunologyIn-vitro diagnosis field.It is based on homogeneous immunology enzymatic assays.Particularly relate to the preparation of a kind of detection method and test kit, specialIt not a kind of ADMA detectable and preparation thereof and detection method.
Background technology
ADMA (NG, NG,dimethyl-L-arginine, asymmetricDimethylarginine, ADMA) also it is Dimethyl-L-arginine.Within 1970, Kakimoto divides first from mankind's urineSeparate out ADMA.ADMA by the protein containing arginine residues at protein arginine transmethylaseMethylate, after methylated Proteolytic enzyme under the effect of (protein arginine methyltransferases, PRMT)ADMA can be discharged.ADMA can make nitric oxide (NO) generate minimizing, in vivo by competitive inhibition nitricoxide synthase (NOS)Play the effect of endogenous NO synthetic inhibitor, cause vascular endothelial function to get muddled, and then cause atherosclerosis etc.The cardiovascular and cerebrovascular disease of series develops.ADMA has significant correlation with cerebral infarction and risk factor thereof, and it is to ischemicThe incidence rate of cerebrovascular and mortality rate have powerful predictive ability.There is clinical table in cardiovascular disease in plasma ADMA levelsJust had begun to before now raise.Therefore, ADMA is perspective as one, and independent cardiovascular diseases's diagnosis index has great meaningJustice.The rising of ADMA level simultaneously not only serves as the tendency of cardiovascular and cerebrovascular disease, moreover it is possible to as heart failure, hypertension, coronary heart disease,Diabetes, renal failure liver failure, a significant risk factor of the diseases such as erection disturbance.Therefore, ADMA is new as oneEndothelial dysfunction and the cardiovascular disease mark of type are significant in terms of clinical diagnosis.
At present, the method for quantitative determination ADMA, there are high performance liquid chromatography (HPLC), Liquid Chromatography/Mass Spectrometry (HPLC-MS), enzymeConnection immunoabsorption (ELISA), chemiluminescence immunoassay (CLIA).All there is operation complexity, the detection time is long, automatizationDegree is low, and repeatability is poor, is unfavorable for automation equipment and standardization.
Also have been reported that, ADMA enzyme process detect, required reaction all 3 steps or more than, the kind of required enzyme also more than 3 kinds,And the key enzyme having such as Citrullinase not disclosed document report, available sources is failed to understand, is difficult to obtain.Add uncertainProperty and unstability, it is not easy to realize automatization and the standardization of instrument.
The preparation difficulty of anti-ADMA antibody is higher, there is the shortcoming that specificity is bad.With NG,N'G-Dimethyl-L-arginine, methylThere is bigger cross reaction, poor specificity, the reagent poor specificity of preparation in arginine and L-arginine etc..Document is had to reportThe hapten preparation method of similar this little molecule of ADMA determines specificity and the affinity of the antibody of preparation.Little molecule is with carryingThe connection site of body protein determines the specificity preparing antibody, it is ensured that the specific epitope of little molecule is not anti-halfFormer coupling process is destroyed.Carrier protein ensure that the abundant exposure of little molecule with hapten suitable linking arm length,If long, can be substantially reduced the immunogenicity of little molecule, the shortest, carrier protein can affect the structure of little molecule.The present invention adoptsUse specific linking arm, by the orthoselection of connection site, carboxyl site or amino sites, fully expose exempting from of ADMAEpidemic disease site, thus obtain the antibody of high-affinity and high specific.The present invention also use specific connection carrier rabbit albumin,Human albumin and Mus albumin, and do not use general connection carrier such as bovine serum albumin, hemocyanin and thyroid ball eggWhite etc., the ADMA being primarily due to couple with bovine serum albumin and Elityran etc. stimulates the antiserum produced also to containFor the antibody of bovine serum albumin and Elityran etc., false positive results may be caused；And the molecular weight of hemocyaninRelatively big, the hapten that coupling obtains easily affects the epitope even shielding little molecule, and the affinity of antibody causing preparation is low.And the rabbit using albumin rabbit serum to do vector immunity is less likely to produce the antibody of the albumen for rabbit self, use MusMouse-anti people's ADMA monoclonal antibody specific that serum albumin vector selection obtains, affinity and specificity are better than other and carryBody.
Lack highly sensitive, the ADMA detectable of high specificity in the market, the suitableeestClose the reagent of Automated inspection.
The present invention uses enzyme coupling ADMA complex, resists with ADMA specificityBody immunological response, after forming antigen antibody complex, the activity of enzyme-ADMA complex is just suppressed.ADMA in sampleWith enzyme-ADMA complex competitive binding anti-ADMA specific antibody.ADMA in sample is the most, in conjunction with antibody the most.The antibody that enzyme-ADMA complex combines is the fewest, by measuring the change of enzymatic activity in reaction system, can obtain in sampleThe content of ADMA.Speed is fast, simple to operate, highly sensitive in detection, high specificity and can be real on automatic clinical chemistry analyzerNow the advantage to the rapid detection of high flux of small-molecule substance, has higher using value.
Summary of the invention
This detection method, based on immunological response, uses the enzymatic determination method of homogeneous immunity, by little molecule ADMA and enzymeThe ADMA to be measured in complex, with sample and anti-ADMA specific antibody competition binding is formed by protein interconnection technique, logicalCross the height of enzymatic activity to realize the detection of ADMA concentration in sample.The method can provide one the most safely but also can be quick, highEffect, sensitive, homogeneous enzyme immunoassay detectable accurately detecting ADMA content in sample to be tested and preparation method thereof.This reagent canIt is applied to widely used various types of automatic biochemistry analyzers in clinic, thus reaches the mensuration requirement of extensive sample,Improve practicality.
The invention provides the test kit of detection ADMA, comprise reagent R1, reagent R2, buffer, stabilizer, ADMA referenceThe liquid reagent box of standard substance or comprise reagent R1, the powdered reagent box of reagent R2, ADMA reference standard product.
Further, a preferred technical scheme is: a kind of ADMA homogeneous immunological detection reagent, it is characterised in that bagInclude: anti-ADMA specific antibody, the detectable preparation being used for detecting anti-ADMA specific antibody-ADMA complex and detection sideMethod;Above-mentioned ADMA homogeneous immunological detection reagent box is based on the principle that the biological sample containing ADMA is multiple with ADMA-G6PDH enzymeCompound competition binding anti-ADMA specific antibody；ADMA-G6PDH multienzyme complex and anti-ADMA specific antibody form immunity combinationAfter thing, the activity of its catalytic substrate G-6-P reduces or loses, and the amount of product NADH also reduces；Measure sampleThe concentration amount of middle ADMA is the highest, and the anti-ADMA specific antibody that competition is arrived is the most, and anti-ADMA specific antibody is to ADMA-G6PDHThe activity suppression of multienzyme complex is the lowest, then the NADH amount reacting generation is the most；Amount according to measuring NADH calculates, thus obtains sampleADMA content in product.
According to this goal of the invention, a further preferred embodiment: wherein prepare ADMA immunogen for preparingAnti-ADMA specific antibody.The carboxylic group preferably utilizing ADMA cross-links with carrier protein, and preferred linking group amino is ownAcid；Carrier protein preferred rabbit albumin and Mouse albumin；The preferred new zealand white rabbit of immune animal and Balb/c mice；ChemistryThe preferred carbodlimide method of coupling mode；Anti-ADMA specific antibody prepared by above-mentioned method for optimizing is high to ADMA affinity, specialProperty is good, the equal no cross reaction of same NG,N'G-Dimethyl-L-arginine, monomethyl arginine, L-arginine.
A preferred embodiment according to this goal of the invention, wherein prepares ADMA-multienzyme complex for measuring ADMAMeasure test kit.Preferably enzyme is glucose-6-phosphate dehydrogenase (G6PD) (EC 1.1.1.49)；Preferably use ADMA amino groupCross-linking with glucose-6-phosphate dehydrogenase (G6PD), linking group is aminocaproic acid；ADMA and the crosslinking of glucose-6-phosphate dehydrogenase (G6PD)The preferred glutaraldehyde method of method；ADMA is the preferred G-6-P of protective agent alive with the enzyme of glucose-6-phosphate dehydrogenase (G6PD) crosslinkingNADH；Above-mentioned protective agent G-6-P preferred concentration 10-100mM, NADH preferred concentration is 10-100mM；Use rationallyEnzymatic protective reagent, preferentially occupy the enzyme active center of G6PDH, it is to avoid in ADMA-G6PDH coupling process activity center destruction andThe enzyme caused is lived and is lost.
A preferred embodiment according to this goal of the invention, during wherein ADMA measures test kit, R1 reagent buffer is excellentSelect glycine buffer；The preferred 4.0-8.0 of PH；The preferred 10-100mM of G-6-P concentration；The preferred 10-of NaCl concentration200mM；The preferred 10-100mM of NAD+ concentration；0.001%-0.01% sodium azide；Tween 20 concentration is preferably 0.01%-0.1%；BSA is preferably 0.1%-0.5%；
Anti-ADMA antibody is preferably 0.01-2%.
A preferred embodiment according to this goal of the invention, during wherein ADMA measures test kit, R2 reagent buffer is excellentSelect TRIS buffer；The preferred 7.0-9.0 of PH.
A preferred embodiment according to this goal of the invention, wherein the stabilizer of R2 is that 0.2%-0.5% Sanguis Bovis seu Bubali is pureAlbumen, 5%-20% glycerol, 1%-5% mannitol, 0.001%-0.01% sodium azide, 0.1-2mM EDTA, 0.1%-1% PEG20000.
A preferred embodiment according to this goal of the invention, wherein said ADMA standard substance are pure for the addition of ADMAProduct human serum or the liquid of other similar serum substrate.Preferably, select with the addition of the liquid of the human serum of ADMA sterling.
A preferred embodiment according to this goal of the invention, utilizes ADMA immunologic function test reagentDetection method, it is characterised in that comprise the following steps:
1) this contacts with anti-ADMA specific antibody will to hold test sample；
2) according to the combination situation of ADMA in sample to be tested Yu anti-ADMA specific antibody；
Measure NADH preferably by automatic analyzer and produce speed, it is judged that the content of ADMA in sample;
Described sample to be tested is serum, blood plasma, saliva or urine.
The present invention and prior art compare, and its technological progress is significant.The present invention connects skill by protein orientationThe ADMA immunogens that art is obtained is strong, immunogenicity is high.Preferred by carrier protein, uses rabbit albumin orientation evenThe connection little molecule of ADMA, it is thus achieved that anti-ADMA polyclonal antibody.Use the Mus albumin orientation little molecule of coupling ADMA, it is thus achieved that anti-ADMA is mono-Clonal antibody.Prepared anti-ADMA specific antibody high specificity, affinity are high.The present invention by by carrier protein withThe directional selectivity of ADMA carboxyl and amino sites connects, middle addition linking arm, substantially increases the spy of ADMA antibodyOpposite sex identification ability, the NG,N'G-Dimethyl-L-arginine similar with structure, monomethyl arginine, arginine no cross reaction.AndThe preparation of the haptenic synthesis of ADMA and ADMA multienzyme complex uses connection site different on ADMA, the homogeneous enzyme of ADMA of preparationExempt to measure reagent and there is the highest specificity.Homogeneous enzyme immunoassay detectable containing above-mentioned anti-ADMA specific antibody can be squareJust determining the ADMA content in sample, quickly and accurately, this reagent is liquid double reagent, can be used for detecting in serum or blood plasmaThe concentration of ADMA, it is adaptable to clinical automatic clinical chemistry analyzer.
Accompanying drawing explanation
Fig. 1: ADMA immunogen structure chart；
The synthesis path of Fig. 2: ADMA derivant 1；
The synthesis path of Fig. 3: ADMA derivant 2；
Fig. 4: ADMA homogeneous enzyme immunoassay reaction normal curve chart；
Fig. 5: ADMA homogeneous enzyme immunoassay correlation analysis figure.
Detailed description of the invention
The synthesis of embodiment one: ADMA derivant 1 and structural confirmation thereof
In following example, the biological chemical structure of the ADMA derivant 1 of use is as shown in the compound 3 in Fig. 2, synthetic routeSuch as Fig. 2.
Concrete synthesis step:
1) weigh 900mg compound 1 (ADMA) to be dissolved in 90ml pyridine, add 360 mg EDC (1-ethyl-3-immediately[3-dimethylaminopropyl] carbodiimide hydrochloride) and 540mg NHS(N-Hydroxysuccinimide), this solution is stirred at room temperature 30 hours, obtains the synthetic solvent of compound 2；
2) in above-mentioned solution, add the beta-mercaptoethanol of 126ul, 30min, the EDC of inactivation excess, desalination to pH are stirred at room temperatureIn the PBS of 7.2；
3) aminocaproic acid taking 15.00g is dissolved in the PBS solution of 10mL pH 7.2, takes the above-mentioned synthetic solvent of 50mL and is slowly dropped toIn the solution of aminocaproic acid, it is stirred at room temperature, after being added dropwise to complete, continues room temperature reaction 2h.Acetic acid is used again after synthetic solvent dilute with waterEthyl ester (EtOAc) extracts, and uses water and salt to rinse organic layer, uses Na2SO4It is dried, makes solvent evaporate by decompression method,Finally obtain compound as white solid 3.
Above-mentioned white solid purified product is carried out Structural Identification
Utilize Chromatography/Mass Spectrometry technology (LCMS) to be analyzed the derivant obtained identifying, use the series connection four of Agilent companyLevel bar mass spectrograph LC/MSD1200 series, ion source uses cation or negative ionization mode.Chromatographic column specification is: SymmetryC18 (70 × 4.6mm, 5 μm), column temperature is 30 DEG C, and flow velocity is 0.6 mL/min, and detection wavelength is 224nm, and flowing is 10-70% mutuallyAcetonitrile.LCMS result shows: purity > 95%；Retention time 3.012min.The above results, it may be determined that this compound as white solidFor above-mentioned ADMA derivant 1.
The synthesis of embodiment two: ADMA derivant 2 and structural confirmation thereof
The biological chemical structure of the ADMA derivant 2 used in following example is as shown in the compound 8 in Fig. 3, and it synthesizes roadLine is as such as Fig. 3.
Concrete synthetic route is as follows:
1) ADMA weighing 100mg is dissolved in concentrated sulphuric acid, adds 1200mg methanol, is heated to reflux 2h.Synthetic solvent dilute with waterAfter, make solvent evaporate by decompression method, finally obtain compound as white solid 4；
2) 500mg compound 5(aminocaproic acid is weighed) it is dissolved in 50ml pyridine, add 400mg EDC (1-ethyl-immediately3-3-dimethylaminopropyl] carbodiimide hydrochloride) and 600mg NHS(N-Hydroxysuccinimide), this solution is stirred at room temperature 30 hours, obtains the synthetic solvent of compound 6；
3) adding the beta-mercaptoethanol of 90ul in above-mentioned solution, 30min is stirred at room temperature, the EDC of inactivation excess, desalination is extremelyIn the PBS of pH7.2；
4) compound 4 of 50mg is dissolved in the PBS solution of 5mL pH7.2, is slowly added dropwise 50mL above-claimed cpd 6 wherein and synthesizes moltenLiquid, is stirred at room temperature, and continues room temperature reaction 2h after being added dropwise to complete.Extract by ethyl acetate (EtOAc) again after synthetic solvent dilute with waterTake, use water and salt to rinse organic layer, use Na2SO4It is dried, makes solvent evaporate by decompression method, finally obtain white solidBody compound 7；
5), during 50mg compound 7 is dissolved in 5mL DMF, adds 5ml methanol, the NaOH of 1g, reacting by heating 2 hours, synthesizedSolution；
6) will extract by ethyl acetate (EtOAc) again after above-mentioned synthetic solvent dilute with water, use water and salt to rinse organic layer,Use Na2SO4It is dried, makes solvent evaporate by decompression method, finally obtain compound as white solid 8.
Above-mentioned white solid purified product is carried out Structural Identification
Utilize Chromatography/Mass Spectrometry technology (LCMS) to be analyzed the derivant obtained identifying, use the series connection four of Agilent companyLevel bar mass spectrograph LC/MSD1200 series, ion source uses cation or negative ionization mode.Chromatographic column specification is: SymmetryC18 (70 × 4.6mm, 5 μm), column temperature is 25 DEG C, and flow velocity is 0.6 mL/min, and detection wavelength is 224nm, and flowing is 10-70% mutuallyAcetonitrile.LCMS result shows: purity > 95%；Retention time 2.336min.The above results, it may be determined that this compound as white solid8 are ADMA derivant 2.
Embodiment three: ADMA immunogen synthesizes
ADMA immunogen is formed by connecting by the linking group of rabbit albumin or Mouse albumin with the ADMA derivant shown in Fig. 1,In the present embodiment, as a example by the linking arm ADMA derivant as aminocaproic acid, this immunogenic synthetic method is described in detail.
Rabbit albumin-ADMA carboxy derivatives A1 immunogen synthesizes:
Specifically comprise the following steps that
1) being dissolved in by 40mg rabbit albumin in the MES buffer of 4mL 0.1M pH 5.0, above-mentioned solution is placed in beaker A；
2) 30mg ADMA derivant 1 is dissolved in the MES buffer of 6mL 0.1M pH 5.0, joins in beaker A；
3) add in 50mg EDC to beaker A, obtain mixed solution, reaction 2 hour is stirred at room temperature；By the mixing after above-mentioned stirringSolution, through neutral PBS dialysis purification, obtains rabbit albumin-ADMA immunogen, is stored in-70 DEG C.
Mus albumin-ADMA carboxy derivatives A1 immunogen synthesizes:
Specifically comprise the following steps that
1) being dissolved in by 40mg Mus albumin in the MES buffer of 4mL 0.1M pH5.0, above-mentioned solution is placed in beaker A；
2) 30mg ADMA derivant 1 is dissolved in the MES buffer of 6mL 0.1M pH5.0, joins in beaker A；
3) add in 50mg EDC to beaker A, obtain mixed solution, reaction 2 hour is stirred at room temperature；By the mixing after above-mentioned stirringSolution, through neutral PBS dialysis purification, obtains Mus albumin-ADMA immunogen, is stored in-70 DEG C.
It is similar to, when ADMA derivant is glycine, lysine, aminocaproic acid, and linking group-(CH2)n-O-CH2-During COO-, ADMA immunogen as shown in Equation 1 can be prepared by same method.Certainly, carrier is still for having immunogenicityProtein.
The synthesis that present invention discloses only the ADMA derivant that linking group is aminocaproic acid is implementedExample has also carried out relevant subsequent experiment, and linking group acts primarily as the interconnection function of small molecule derivative and carrier, but different longDegree linking arm is different to ADMA structure degree of exposure；ADMA derivative molecular structure that immunogenicity is strong and weak with synthesized and selectedKind of carrier is relevant, and different bearer types is the most different to the structure influence of little molecule, additionally uses different linking armsThe antigen prepared, the specific antibody of corresponding preparation is respectively provided with different performances.
The preparation of embodiment four: ADMA-glucose-6-phosphate dehydrogenase (G6PD) complex
In present example, preparation ADMA-G6PDH multienzyme complex is by ADMA derivant 2(embodiment one) with G6PDH enzyme couplingObtain, be different from the embodiment of the present invention three synthesis ADMA derivant 1 used in ADMA immunogen.Lot of documents and test numberAccording to showing, when preparing little molecule polyclonal antibody, the company that a part of antibody is identification carrier albumen and little molecule can be producedMeeting place, therefore this partial antibody with the former combination of small molecule immune, but but can not identify the little molecule measured in sample,Therefore can largely effect on the performance of small molecule immune detectable, and the site utilizing little molecule different carries out immunogen and enzyme is evenThe preparation of connection thing then can avoid this problem to occur.
Glutaraldehyde method prepares ADMA-glucose-6-phosphate dehydrogenase (G6PD) complex
1) weighing the G6PDH that 20mg specification is 300KU, room-temperature dissolution contains 0.1M MES, 3mM MgCl in 5ml2、100mgIn the solution of NADH, 150mg G-6-P (G-6-P) and 0.9% NaCl, these pH value of solution=8.0；
2) weigh the ADMA derivant 2 of 30mg, be dissolved in the dimethyl sulfoxide of 1mL；
3) ADMA derivant 2 solution is mixed with G6PDH solution；
4) the glutaraldehyde 60ul of 1% it is slowly added dropwise；
5) add the PBS of 10mL, and continue stirring reaction 2h；
6) purified product.
By dialysis purification connect product, it is thus achieved that end product be glucose-6-phosphate dehydrogenase (G6PD)-ADMA enzyme be combinedThing, stores at 2-8 DEG C.
The preparation of embodiment five: ADMA immunologic function test reagent
ADMA homogeneous enzyme immunoassay detectable, including: above-mentioned anti-ADMA specific antibody, is used for detecting anti-ADMA specificity and resistsThe enzyme reagent of body-ADMA complex, including: ADMA-G6PDH multienzyme complex and the substrate of enzyme.
ADMA homogeneous enzyme immunoassay detectable before the use, in order to avoid the multienzyme complex in enzyme reagent and the substrate of enzymeReacting, the substrate of multienzyme complex and enzyme is separated, does not mixes, so by special with above-mentioned anti-ADMA for the substrate of enzymeProperty antibody mixes.It is to say, ADMA homogeneous enzyme immunoassay detectable includes two kinds of reagent being provided separately, the most such asUnder:
The preparation of reagent R1: 20 mM NAD, 30 mM G-6-P G6P, the glycine buffer of 100 mM pH=6.0Homogeneous zymolyte is made in dissolving；Add the anti-ADMA monoclonal antibody specific of 0.1%-1% in above-mentioned homogeneous zymolyte, at thisIn embodiment, concrete ratio is 0.5%；Add 0.2%BSA, 0.01% Twen 20,0.01% sodium azide；
The preparation of reagent R2: add ADMA-G6PDH multienzyme complex prepared by 0.01%-1% in the Tris buffer of 0.1M, pH=9,Concrete ratio is 0.05% in the present embodiment；Add 0.2%BSA, 2% glycerol, 1mM EDTA, 0.1%PEG20000, sodium azide0.01%。
The using method of above-mentioned ADMA immunologic function test reagent, comprises the following steps:
1) this contacts with anti-ADMA specific antibody will to hold test sample;
2) according to the combination situation of ADMA in sample to be tested Yu anti-ADMA specific antibody, ADMA in enzyme reagent judgment sample is utilizedContent;
Concrete, during detection, sample to be tested being added in reagent R1, the ADMA in sample to be tested is special with the anti-ADMA in reagent AHeterogenetic antibody occurs specific binding, generates anti-ADMA specific antibody-ADMA complex;Add reagent R2, now reagentAnti-ADMA specific antibody occurs specific binding ADMA-G6PDH multienzyme complex in R2 with reagent R1, not special with anti-ADMAThe ADMA-G6PDH multienzyme complex that heterogenetic antibody combines mixes with substrate, contacts, and enzymatic reaction occurs, and constitutes the anti-ADMA of detection specialThe enzyme reagent of heterogenetic antibody-ADMA complex, enzyme reagent is according to the knot of the above-mentioned anti-ADMA specific antibody of ADMA in sample to be testedConjunction situation judges the content of ADMA in sample to be tested.
Due to ADMA-G6PDH multienzyme complex and the ADMA competitive binding anti-ADMA specific antibody in sample to be tested, instituteWith, in sample to be tested, the amount of ADMA is the most, and the amount of ADMA-G6PDH multienzyme complex free in homogeneous enzymatic solution is the most, and enzymatic is anti-Should be the fastest, cause OD340Rise.Above-mentioned sample to be tested is physiology sample, such as serum, blood plasma, urine, saliva etc..As onePreferably scheme, above-mentioned sample to be tested is serum or blood plasma.
Embodiment six: ADMA homogeneous enzyme immunoassay is checked
1, obtain standard curve: Hitachi 7060 automatic clinical chemistry analyzer response parameter (being shown in Table 1) is set.Operating process is: firstReagent adding R1, adds standard substance, hatches 5min for 37 degree, is eventually adding reagent R2.After adding reagent R2, measure different time pointsOD340 light absorption value, calculate reaction rate during various criterion product concentration, obtain comparatively ideal response curve as shown in Fig. 4.
Table 1 Hitachi 7060 automatic clinical chemistry analyzer response parameter
| Project name | ADMA |
| Reagent R1 | 1600ul |
| Reagent R2 | 40ul |
| Sample size | 15ul |
| Analysis method | Two point rate assay |
| Dominant wavelength | 340nm |
| Commplementary wave length | 405nm |
| Response time | 15min |
| Incubation time | 5min |
| The Direction of Reaction | Upwards |
| Calibrating method | Spline |
| Calibration object concentration | 0、0.63、1.25、2.50、5.00、10.00 µmol/L |
2, the mensuration of sample, precision and response rate assessment
The standard curve obtained by the homogeneous enzyme immunoassay detectable of the present invention, replication basic, normal, high concentration Quality Control sample10 times, in this example, Quality Control sample is that ADMA standard substance are dissolved in human serum, concentration to respectively 0.63,2.50,10.00Mol/L, result is as shown in table 2.
The homogeneous enzyme of table 2 ADMA is excused from an examination the analytical performance evaluation of agent
Testing result shows: the homogeneous enzyme of ADMA prepared by present invention agent precision of being excused from an examination is high, and CV < 6%, accuracy is high, and the response rate existsBetween 95%-105%.
Embodiment seven: chaff interference is tested
Endogenic L-arginine (L-arg), monomethyl arginine (MMA), NG,N'G-Dimethyl-L-arginine (SDMA), cholic acid (CA)It is several important chaff interference affecting ADMA Accurate Determining with chenodeoxycholic acid (CD-CA).With the homogeneous enzyme of the ADMA in the present inventionBe excused from an examination agent measure above-mentioned three kinds of chaff interferences, the results are shown in Table 3
Table 3 chaff interference is tested
| Compound name | Test concentrations | Cross reacting rate |
| ADMA | 10 µmol/L | 100% |
| L-arginine | 100 µmol/L | <0.01% |
| Monomethyl arginine | 100 µmol/L | <0.01% |
| NG,N'G-Dimethyl-L-arginine | 100 µmol/L | <0.09% |
| Cholic acid | 100 µmol/L | <0.05% |
| Chenodeoxycholic acid | 100 µmol/L | <0.08% |
Testing result illustrates that the cross reacting rate of above-mentioned chaff interference is respectively less than 0.1%, shows that the homogeneous enzyme of ADMA prepared by the present invention is exempted fromReagent can the content of Accurate Determining ADMA.
Embodiment eight: correlation analysis
Homogeneous for the ADMA of present invention Enzyme immunoassay reagent is compared with ADMA method in Human Serum by High Performance Liquid Chromatography, knotFruit is shown in Table 4.
The homogeneous Enzyme immunoassay of table 4 ADMA is analyzed with high-performance liquid chromatogram determination results relevance
Correlation curve is shown in Fig. 5, it is seen that the two matching is good.
It should be noted that the foregoing is only embodiments of the invention, not thereby limit the scope of the claims of the present invention,Every equivalent structure utilizing description of the invention and accompanying drawing content to be done or equivalence flow process conversion, or be directly or indirectly used inOther correlative technology fields, are the most in like manner included in the scope of patent protection of the present invention.