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CN105753969A - Asymmetric dimethylarginine immunogen, antibody, detecting reagent and preparation method - Google Patents

Asymmetric dimethylarginine immunogen, antibody, detecting reagent and preparation method Download PDF

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CN105753969A
CN105753969A CN201610209278.9A CN201610209278A CN105753969A CN 105753969 A CN105753969 A CN 105753969A CN 201610209278 A CN201610209278 A CN 201610209278A CN 105753969 A CN105753969 A CN 105753969A
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dimethyl
arginine
antibody
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immunogen
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付卫
虞留明
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SUZHOU EVERMED CO Ltd
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Abstract

The invention discloses asymmetric dimethylarginine immunogen, an antibody, a detecting reagent and a preparation method. The asymmetric dimethylarginine immunogen is high in immunogenicity, can be induced to obtain an anti-asymmetric dimethylarginine specific antibody with high valence, and does not have any cross reaction with common 62 medicines. The asymmetric dimethylarginine immunogen detecting reagent prepared from the antibody can be used for precisely and quickly determining the content of asymmetric dimethylarginine in biological samples such as blood serum, blood plasma and urea. Compared with a current detecting reagent on the market, the detecting reagent has the advantages of being simple and convenient to operate, high in sensitivity, strong in specificity and accurate in result, further can effectively reduce the detecting cost of asymmetric dimethylarginine, and is beneficial for clinical large-scale popularization and use.

Description

Dimethyl-L-arginine immunogen, antibody and detectable and preparation method
Technical field
The invention belongs to biological technical field, relate to Dimethyl-L-arginine immunogen, anti-Dimethyl-L-arginine specific antibody and Dimethyl-L-arginine detectable and preparation method thereof.
Background technology
Dimethyl-L-arginine (Asymmetricdimethylarginine, ADMA), its structural formula is such as shown in formula II:
Formula II
Dimethyl-L-arginine is a kind of endogenous molecule that can detect in human serum, blood plasma and urine, it is a kind of methylated arginine, methylated under protein-arginine methyltransferase catalysis by the arginine residues forming intracellular protein, generate then through hydrolysis.Having been demonstrated that, the internal ADMA of existence regulates the endogenetic mechanisms that nitric oxide (NO) generates, i.e. the activity of ADMA energy competitive inhibition nitric oxide synthetase (NOS), thus reducing the synthesis of nitric oxide in vascular endothelial cells.ADMA is widely present in tissue and cell, and in normal human, every day produces about 300 μm of ol(and is about 60mg), bulk concentration is significantly larger than other can produce the inhibiting endogenous arginine of NOS equally, therefore is considered as most important no inhibitor.After ADMA enters blood circulation from cell, sub-fraction is directly decomposed through cracking processes is discharged with urine by kidney, but major part is by being distributed widely in DDAH (DDAH) catalytic degradation of kidney, liver and cardiovascular system.A large amount of clinical researches both at home and abroad prove: ADMA content extremely can cause vascular endothelial dysfunction and cause the generation of multiple cardiovascular disease, such as atherosclerosis, coronary heart disease, hyperlipemia, cerebral infarction, hypertension, heart failure and apoplexy etc..Therefore, ADMA has been acknowledged as a kind of new cardiovascular disease important symbol thing.There is research to point out at present: ADMA concentration raises not only relevant with cardiovascular function exception, but also develop closely related with the generation of the disease such as chronic renal failure, diabetes, preeclampsia, hyperhomocysteinemiainjury, liver failure, erection disturbance.
Given this, set up the ADMA detection method being applicable to clinic to be significant, but ADMA concentration level is relatively low in blood, and there is arginine and SDMA (SDMA) to the ADMA cross reaction detected interference, therefore ADMA detection difficulty is bigger.At present, the method measuring ADMA specifically includes that high performance liquid chromatography, tablets by HPLC-MS and euzymelinked immunosorbent assay (ELISA) etc..The common feature of high performance liquid chromatography and HPLC-MS method is that accuracy is good, specificity is high, shortcoming is complicated operation, sample treatment is loaded down with trivial details, test length consuming time, need special expensive instrument, cannot be carried out high throughput analysis, it is difficult to be applied to clinic.Euzymelinked immunosorbent assay (ELISA) is slightly convenient relative to chromatography, and its shortcoming is in that to need manual operations, length consuming time, is affected by human factors big, it is difficult to realize automatization, be currently mainly applied to experimentation.In addition, document there was reported for the fluorescence polarization immunoassay of ADMA mensuration, radioimmunoassay, latex enhancing immune turbidimetry, cloned enzyme donor immunoassay, the equiprobable detection method of enzyme process, but in the market but without the matured product of larger scale clinical application.Therefore, research and development one can measure ADMA content in human sample, sensitivity and specificity easily and fast, exactly and reach clinical requirement, can be applied to the method for full automatic biochemical apparatus simultaneously and test kit has become the urgent needs in Clinical Laboratory field.
Summary of the invention
The defect that the present invention exists to overcome prior art, adopting unique Dimethyl-L-arginine to prepare the strong Dimethyl-L-arginine immunogen of immunogenicity and antibody thereof, the Dimethyl-L-arginine homogeneous enzyme immunoassay detectable prepared with this antibody can be implemented on automatic clinical chemistry analyzer Dimethyl-L-arginine high flux, rapid detection.This detectable has the advantages such as easy and simple to handle, highly sensitive, high specificity, result are accurate, moreover it is possible to effectively reduces Dimethyl-L-arginine testing cost, is conducive to clinical expansion to use.
It is an object of the present invention to provide the Dimethyl-L-arginine immunogen that a kind of immunogenicity is strong.
A kind of immunogenic preparation method of Dimethyl-L-arginine of offer is provided.
Another purpose of the present invention is in that to provide the anti-Dimethyl-L-arginine specific antibody of the high specificity using Dimethyl-L-arginine immunogen of the present invention to prepare.
It is yet a further object of the present invention to provide a kind of Dimethyl-L-arginine detectable and preparation method thereof.
The Dimethyl-L-arginine immunogen of the present invention, immunogenicity is high, it is possible to induction obtains the anti-Dimethyl-L-arginine specific antibody of high-titer.This antibody specificity is high, strong with the adhesion of Dimethyl-L-arginine.The Dimethyl-L-arginine detectable prepared by this antibody, it is possible to determine the Dimethyl-L-arginine content in sample quickly and accurately.The present invention is achieved by the following technical solutions:
A kind of Dimethyl-L-arginine immunogen, its structural formula is such as shown in formula I:
Formula I
Carrier for having immunogenic protein or polypeptide, the one in serum albumin, hemocyanin or Elityran.It is more preferably serum albumin, more preferably bovine serum albumin.
Described Dimethyl-L-arginine immunogen is formed by connecting by Dimethyl-L-arginine and above-mentioned carrier, and the chemical constitution of Dimethyl-L-arginine is such as shown in formula II:
Formula II
The immunogenic preparation method of this Dimethyl-L-arginine is as follows:
(1) carrier protein 100～300mg is dissolved in the phosphate buffer of 25～75ml0.2M, pH8.5；
(2) following chemicals is joined stirring and dissolving in small beaker: 100～300mg Dimethyl-L-arginine, 1.75～5.25ml dimethylformamide, 1.75～5.25ml ethanol, 3.5～10.5ml10mM, the kaliumphosphate buffer of pH5.0,100～300mg1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 25～75mgN-hydroxy thiosuccinimide, react these chemicals at room temperature stirring and dissolving to 30～60min；
(3) solution dissolved is dropped in carrier protein solution, and stir at 2～8 DEG C overnight, obtain antigen；Synthetic antigen is purified through dialysis, obtains Dimethyl-L-arginine immunogen.
A kind of anti-Dimethyl-L-arginine specific antibody, it is by the complete antibody molecule produced after above-mentioned Dimethyl-L-arginine immunogen immune laboratory animal, or is antibody fragment or the antibody derivatives of reservation and Dimethyl-L-arginine specific binding capacity.
Described complete antibody molecule, antibody fragment or antibody derivatives, for the polyclonal antibody adopting single Dimethyl-L-arginine immunogen that animal booster immunization is obtained, or the monoclonal antibody for obtaining through somatic hybridization after immunity.Described laboratory animal is the one of rabbit, goat, mice, sheep, Cavia porcellus or horse, it is preferred to rabbit.
Described anti-Dimethyl-L-arginine specific antibody is adopted conventional method inoculation experiments animal by above-mentioned prepared Dimethyl-L-arginine immunogen, takes antiserum after booster immunization.
The preparation method of anti-Dimethyl-L-arginine specific antibody, specifically comprises the following steps that
(1) with PBS, the BSA-Dimethyl-L-arginine immunogen of above-mentioned synthesis is diluted to 0.1～3.0mg/ml, obtains antigenic solution, then mix with equivalent Freund's complete adjuvant with 0.5～5.0ml antigenic solution, laboratory animal is injected；
After (2) 2～3 weeks, then with equivalent incomplete Freund's adjuvant, above-mentioned laboratory animal is injected once with antigenic solution identical for 0.5～5.0ml, inject once every surrounding afterwards, amount to injection 3～6 times；
(3) above-mentioned laboratory animal is taken blood, separate purification and obtain the anti-Dimethyl-L-arginine specific antibody that titer is 1:30000～1:50000.
The present invention provides a kind of Dimethyl-L-arginine detectable, containing above-mentioned anti-Dimethyl-L-arginine specific antibody and indicator, described indicator one in enzyme reagent, radiosiotope reagent, fluorometric reagent or luminescence reagent；Described enzyme reagent is made up of the substrate of Dimethyl-L-arginine enzyme mark conjugate and enzyme, and enzyme mark conjugate is glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark conjugate, and the substrate of enzyme is G-6-P.
The preparation method of Dimethyl-L-arginine detectable, it is characterised in that comprise the steps of
(1) reagent A: 2.018～8.072g, the nicotinamide adenine dinucleotide of 5.625～22.50mM oxidation state and 0.856～3.422g, 5.625～22.50mM G-6-P Tris buffer solution of 0.5～2L55mM, pH=8.0 are made homogeneous zymolyte；Being added in above-mentioned homogeneous zymolyte by described anti-Dimethyl-L-arginine specific antibody, the volume ratio of anti-Dimethyl-L-arginine specific antibody and homogeneous zymolyte is 1:100～1:10000；
(2) volume ratio of reagent B: be added in the Tris buffer of 120mM, pH=8.2 by Dimethyl-L-arginine enzyme mark conjugate, Dimethyl-L-arginine enzyme mark conjugate and Tris buffer is 1:100～1:10000.
The volume ratio of described anti-Dimethyl-L-arginine specific antibody and homogeneous zymolyte is preferably 1:800；
The volume ratio of described Dimethyl-L-arginine enzyme mark conjugate and Tris buffer is preferably 1:2000.
The preparation method of described Dimethyl-L-arginine enzyme mark conjugate comprises the steps of
(1) preparation of glucose-6-phosphate dehydrogenase (G6PD) solution: weighing the glucose-6-phosphate dehydrogenase (G6PD) that 7.5～22.5mg specification is 100KU, room-temperature dissolution contains 72.6mg0.05MTris, 8mg3.3mMMgCl in 6～18mL2With in the solution of 100mgNaCl, pH=9.0；Add the nicotinamide adenine dinucleotide of 112.5～337.5mg reduction-state, 67.5～202.5mg G-6-P and 0.375～1.125mL carbitol in the solution；It is added dropwise over 1～3mL dimethyl sulfoxide again；
(2) activation of Dimethyl-L-arginine: weigh 5～15mg Dimethyl-L-arginine under anhydrous conditions, is dissolved in 300～900 μ L dimethylformamides；Above-mentioned solution temperature is made to drop to-2 ~-8 DEG C；Add 1.5～4.5 μ L tri-n-butylamines；Add 0.75～2.25 μ L isobutyl chlorocarbonate；-2 ~-8 DEG C are stirred 30～60 minutes；
(3) connection of glucose-6-phosphate dehydrogenase (G6PD) and Dimethyl-L-arginine: the Dimethyl-L-arginine dropwise that step (2) activates is joined in the glucose-6-phosphate dehydrogenase (G6PD) solution that step (1) is dissolved；2-8 DEG C of stirring is overnight；
(4) purified product: by G-25 gel chromatography column purification connect product, it is thus achieved that end product be glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, at 2-8 DEG C store.
Dimethyl-L-arginine homogeneous enzyme immunoassay detectable is before the use, in order to avoid the substrate of the enzyme mark conjugate in indicator and enzyme reacts, the substrate of enzyme mark conjugate and enzyme is unmixed and separated, so the substrate of enzyme and above-mentioned anti-Dimethyl-L-arginine specific antibody being mixed.
The Dimethyl-L-arginine immunogens of the present invention is strong, immunogenicity is high, and anti-Dimethyl-L-arginine specific antibody high specificity, the titer prepared are high, and with 62 kinds of common medicines without any cross reaction；Homogeneous enzyme immunoassay detectable containing above-mentioned anti-Dimethyl-L-arginine specific antibody can easily and fast, the Dimethyl-L-arginine content that accurately determines in the biological samples such as serum, and multiple sample can be measured on automatic clinical chemistry analyzer simultaneously, realize the rapid mensuration of high flux of Dimethyl-L-arginine, accuracy is high, high specificity, degree of accuracy is all enhanced before comparing with detection efficiency, it is simultaneously achieved the full-automation of detection process, less demanding to testing staff, it is easy to accomplish with promote the use of.
Accompanying drawing explanation
The ELISA that Fig. 1 is Dimethyl-L-arginine detects response curve；
Fig. 2 is the homogeneous enzyme immunoassay response curve of Dimethyl-L-arginine；
Fig. 3 is Dimethyl-L-arginine homogeneous enzyme immunoassay correlation analysis figure.
Detailed description of the invention
The immunogenic synthesis of embodiment one Dimethyl-L-arginine
Dimethyl-L-arginine immunogen is by bovine serum albumin (BovineSerumAlbumin, BSA) and the Dimethyl-L-arginine shown in formula IIGroup is formed by connecting, and specifically comprises the following steps that
1. bovine serum albumin 200mg is dissolved in the phosphate buffer of 50ml0.2M, pH8.5；
2. following chemicals is joined stirring and dissolving in small beaker: 200mg Dimethyl-L-arginine, 3.5ml dimethylformamide, 3.5ml ethanol, 7.0ml10mM, the kaliumphosphate buffer of pH5.0,200mg1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 50mgN-hydroxy thiosuccinimide, react these chemicals at room temperature stirring and dissolving to 30min；
3. the solution dissolved is dropped in BSA solution, and stir at 2～8 DEG C overnight, obtain antigen；Synthetic antigen is purified through dialysis, obtains Dimethyl-L-arginine immunogen.
Embodiment two: the preparation of anti-Dimethyl-L-arginine specific antibody
Dimethyl-L-arginine immunogen embodiment one prepared adopts conventional method inoculation experiments animal rabbit, takes antiserum, specifically comprise the following steps that after booster immunization
1. with PBS, the Dimethyl-L-arginine immunogen of above-mentioned synthesis is diluted to 1.0mg/ml, obtains antigenic solution, then mix with equivalent Freund's complete adjuvant with 1.0ml antigenic solution, experimental animal rabbit is injected.
After 2.2～3 weeks, then with equivalent incomplete Freund's adjuvant, above-mentioned experimental animal rabbit is injected once with antigenic solution identical for 1.0ml, inject once every surrounding afterwards, amount to injection 4 times.
3. the experimental animal rabbit of pair step 2 takes blood, separates purification and obtains the anti-Dimethyl-L-arginine specific antibody that titer is 1:30000～1:50000.
Embodiment three: the ELISA inspection of Dimethyl-L-arginine
1. the foundation of the ELISA examination criteria curve of Dimethyl-L-arginine
(1) preparation of standard substance
Dimethyl-L-arginine powder (being purchased from Sigma company) is dissolved in methanol solution, prepares into the storage liquid of 1000 μm of ol/L.It is the standard solution of 4.00 μm of ol/L, 2.00 μm of ol/L, 1.00 μm of ol/L, 0.5 μm of ol/L, 0.25 μm of ol/L and 0.00 μm of ol/L with ELISA buffer by storing that liquid dilutes successively.Wherein, ELISA buffer contains 50.0mMTris, the BSA of 145mMNaCl and 0.25%.
(2) the ELISA method of inspection utilizing Dimethyl-L-arginine prepares standard curve
Anti-Dimethyl-L-arginine specific antibody prepared in embodiment two is diluted to the final concentration solution of 1:6000 with PBS, and 100 μ L/ holes are coated on 96 hole elisa plates, place 12-24h for 4 DEG C；After the above-mentioned 96 hole elisa plates being coated with anti-Dimethyl-L-arginine antibody being washed 3 times with PBS, add 200 μ L/ holes 0.5% BSA solution, close for 4 DEG C and place 8-16h.Then wash 3 times with PBS, add the standard substance in 20 μ L/ holes.Add the HRP-Dimethyl-L-arginine conjugate of 100 μ L/ hole working concentrations；After incubated at room temperature 30min, PBS washes plate 5 times；Then every hole adds 100 μ LTMB substrates, incubated at room 30min.Every hole adds 100 μ L stop buffers (2M sulphuric acid) again.Measure the light absorption value of 450nm.The light absorption value calibration of the 450nm corresponding to each standard substance, makes standard curve, and result is as shown in Figure 2.
2. the detection of Dimethyl-L-arginine content in testing sample
(1) testing sample is made
Preparation method: Dimethyl-L-arginine powder (being purchased from Sigma company) is dissolved in methanol solution and makes the storage liquid of 1000 μm of ol/L, and this storage liquid is diluted in blank serum, to final concentration respectively 0.00,0.10,0.80,2.50 μm of ol/L, form the serum sample of concentration blank, basic, normal, high.This blank serum is the Healthy Human Serum without Dimethyl-L-arginine.
(2) method of testing
Utilize the ELISA method of inspection of above-mentioned Dimethyl-L-arginine, the serum sample of above-mentioned blank, basic, normal, high concentration is replaced standard substance, test the serum sample light absorption value at 450nm of above-mentioned blank, basic, normal, high concentration.
(3) test result
The standard curve of the ELISA inspection of comparison Dimethyl-L-arginine shown in Fig. 1, calculate Dimethyl-L-arginine content in each sample, and each sample is carried out 3 multiple holes mensuration, calculating the response rate according to the actual content of Dimethyl-L-arginine in above-mentioned sample, result is as shown in table 1.
The ELISA of table 1 Dimethyl-L-arginine detects recovery experiment
Blood serum sample Blank Low In High
Sample concentration (μm ol/L) 0.00 0.10 0.80 2.50 5 -->
Test 1 0.01 0.09 0.83 2.62
Test 2 0.02 0.13 0.77 2.49
Test 3 0.02 0.11 0.85 2.53
Meansigma methods (μm ol/L) 0.01 0.11 0.82 2.55
The response rate (%) - 110.0 102.5 102.0
From result in table 1: adopt the Dimethyl-L-arginine response rate that the ELISA detectable of Dimethyl-L-arginine of the present invention measures in variable concentrations sample all higher, equal > 90%, illustrate that anti-Dimethyl-L-arginine specific antibody of the present invention may be used for the detection of Dimethyl-L-arginine in sample, and result precision is high.
Embodiment four: the preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing
1. the preparation of glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) solution:
(1) accurately weighing the G6PDH that 15mg specification is 100KU, room-temperature dissolution contains 72.6mg(0.05M in 12mL) Tris, 8mgMgCl2(3.3mM) with in the solution of 100mgNaCl, these pH value of solution=9.0, this step carries out in beaker C.
(2) in above-mentioned beaker C, add the nicotinamide adenine dinucleotide (NADH) of 225mg reduction-state, 135mg G-6-P (G-6-P) and 0.75mL carbitol (Carbitol).
(3) in above-mentioned beaker C, 2mL dimethyl sulfoxide (dimethysulfoxide, DMSO) it is added dropwise over again.
2. the activation of Dimethyl-L-arginine:
(1) weigh the above-mentioned Dimethyl-L-arginine of 10mg under anhydrous conditions, be dissolved in 600 μ LDMF.
(2) above-mentioned solution temperature is made to drop to-2 ~-8 DEG C.
(3) 3 μ L tri-n-butylamine (tributylamine) are added.
(4) 1.5 μ L isobutyl chlorocarbonate (isobutylchloroformate) are added.
(5)-2 ~-8 DEG C are stirred 30 minutes.
The connection of 3.G6PDH and Dimethyl-L-arginine:
(1) the Dimethyl-L-arginine dropwise of above-mentioned activation is joined in the G6PDH solution of above-mentioned dissolving.
(2) 2-8 DEG C of stirring is overnight.
4. purified product:
By the solution in G-25 gel chromatography column purification step 3, it is thus achieved that end product be glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, at 2-8 DEG C store.
Embodiment five: the preparation of Dimethyl-L-arginine homogeneous enzyme immunoassay detectable
1. the preparation of reagent A: by 4.036g(11.25mM) nicotinamide adenine dinucleotide (NAD) of oxidation state, 1.711g(11.25mM) G-6-P (G-6-P) is placed in beaker D, makes homogeneous zymolyte with the Tris buffer solution of 1L55mM, pH=8.0；Being added in above-mentioned homogeneous zymolyte by the anti-Dimethyl-L-arginine specific antibody of above-mentioned preparation, the volume ratio of antibody and homogeneous zymolyte is 1:800.
2. the volume ratio of the preparation of reagent B: glucose-6-phosphate dehydrogenase (G6PD) embodiment four prepared-hapten conjugation thing is added in the Tris buffer of 120mM, pH=8.2, above-mentioned conjugate and Tris buffer is 1:2000.
Embodiment six: the inspection of Dimethyl-L-arginine homogeneous enzyme immunoassay and result
1. obtain standard curve:
(1) auspicious BS-480 automatic clinical chemistry analyzer response parameter (see table 2) advanced in years is set.
(2) operating procedure is: first reagent adding A, adds standard substance, is eventually adding reagent B.After adding reagent B, measure the OD of different time points340Light absorption value, calculates reaction rate during various criterion product concentration, needs constantly to adjust the volume ratio of reagent A and reagent B in actual mechanical process, adjusts light-metering point simultaneously, finally draws comparatively ideal reaction normal curve chart, as shown in Figure 3.
Table 2 steps auspicious BS-480 automatic clinical chemistry analyzer response parameter
2. pattern detection: the standard curve obtained by the homogeneous enzyme immunoassay detectable of the present invention, the basic, normal, high concentration Quality Control sample of replication 10 times, above-mentioned Quality Control sample is: be dissolved in human serum by Dimethyl-L-arginine standard substance, to concentration respectively 0.50,5.00,20.00 μm of ol/L.Detection data and data analysis are in Table 3.
Table 3 sample determination and precision and the response rate are assessed
Blood serum sample Low In High
Sample concentration (μm ol/L) 0.50 5.00 20.00
1 0.56 5.43 21.78
2 0.45 5.39 19.69
3 0.57 4.77 18.72
4 0.54 4.96 20.56
5 0.49 4.91 20.31
6 0.43 5.35 19.77
7 0.55 5.08 19.96
8 0.57 5.76 20.88
9 0.50 5.54 19.98
10 0.52 4.68 21.09
Meansigma methods (μm ol/L) 0.52 5.19 20.27
Standard deviation (SD) 0.05 0.36 0.86
Precision (CV%) 9.62% 6.94% 4.24% 7 -->
Response rate % 104.00 103.80 101.35
Testing result: the accuracy that the homogeneous enzyme immunoassay detectable of the present invention measures is high, and the response rate reaches 95%-105%, precision is high, and CV is below 10%.
Embodiment seven: interfering effects of drug is tested
Choose 62 kinds of Common drugs and carry out Interference Detection, adjust concentration to 1.00 μm of ol/L, adopt the homogeneous enzyme immunoassay method of embodiment six to be measured:
1. reagent A haptoreaction interference medicament to be measured and embodiment five prepared, adds reagent B；
2. detect the OD of above-mentioned mixed solution340Light absorption value, obtains the concentration of respective substance according to the standard curve of embodiment six.
62 kinds of common medicine names and measurement result are referring specifically to table 4.
Table 4 common interference drug monitoring result
ID# Compound title It is equivalent to Dimethyl-L-arginine Concentration (μm ol/L) ID# Compound title It is equivalent to Dimethyl-L-arginine Concentration (μm ol/L)
1 Aspirin 0.0 32 Phenylpropanolamine 0.0
2 β-phenyl-ethylamine 0.0 33 Procainamide 0.0
3 Amphetamines 0.0 34 Procaine 0.0
4 Ampicillin 0.0 35 Quinidine 0.0
5 Bent 0.0 36 Zomepirac 0.0
6 Chlorpromazine 0.0 37 Phyenlephrinium 0.0
7 Clorazepate 0.0 38 Cinnamyl Ai Kening 0.0
8 Xylene oxygen heptan Acid 0.0 39 Ecgonine 0.0
9 Fenoprofen 0.0 40 The West, ground 0.0
10 Methamphetamine 0.0 41 Cotinine 0.0
11 Gentisic acid 0.0 42 Atenolol 0.0
12 Gemfibrozil 0.0 43 Propranolol 0.0
13 Hydrocodone 0.0 44 Glutethimide 0.0
14 Ibuprofen 0.0 45 Bute 0.0
15 Imipramine 0.0 46 Lysergic acid diethyl Amide 0.0
16 Diaminourea hexichol Sulfone 0.0 47 Cannabinol 0.0
17 Naproxen 0.0 48 Loperamide 0.0
18 Hydrochlorothiazide 0.0 49 Isoxsuprine 0.0
19 Pethidine 0.0 50 Phenylalanine 0.0
20 Allylnoroxymorphone Ketone 0.0 51 Fluoxetine Hydrochloride 0.0
21 Ephedrine 0.0 52 Salbutamol 0.0
22 Nicotiamide 0.0 53 Penicillin 0.0
23 Ranitidine 0.0 54 Methyl diethanolamine 0.0
24 Amobarbital 0.0 55 Dimethylene dioxygen Amfetamine 0.0 8 -->
25 Methylene dioxy benzene Propylamine 0.0 56 The many hilas of succinic acid Quick 0.0
26 Tetrahydrocannabinol 0.0 57 Nalbuphine 0.0
27 Nystatin 0.0 58 Normorphine 0.0
28 Acetylmorphine 0.0 59 Oxycodone 0.0
29 Benzfetamine 0.0 60 KET 0.0
30 Promethazine 0.0 61 Diphenhydramine 0.0
31 Aspartame 0.0 62 Duromine 0.0
Measurement result shows: the concentration that above-mentioned 62 kinds of Common drugs are equivalent to Dimethyl-L-arginine is respectively less than 0.01 μm of ol/L.As can be seen here, the antibody of the present invention is the specific antibody of anti-Dimethyl-L-arginine, with other medicines no cross reaction.
Embodiment eight: correlation analysis
The homogeneous enzyme immunoassay reagent that 100 example clinical samples use high performance liquid chromatography and the present invention respectively carries out correlation analysis, and the data of mensuration are referring to table 5.
Table 5 clinical sample measured value
Catalogue number(Cat.No.) Homogeneous enzyme immunoassay method measured value (μm ol/L) High effective liquid chromatography for measuring value (μm ol/L)
1 3.37 3.43
2 1.41 1.42
3 0.65 0.67
4 2.24 2.31
5 0.99 1.03
6 1.23 1.20
7 0.37 0.36
8 1.52 1.56
9 6.98 7.11
10 3.09 3.03
11 1.00 0.94
12 1.14 1.17
13 0.75 0.76
14 5.43 5.52
15 1.33 1.35
16 2.15 2.21
17 4.66 4.54
18 3.02 3.15
19 0.93 0.95
20 0.67 0.70
21 1.35 1.32
22 0.52 0.53
23 0.80 0.77 9 -->
24 4.89 4.98
25 3.41 3.43
26 5.52 4.69
27 7.25 7.39
28 2.40 2.20
29 5.66 5.95
30 5.64 5.49
31 1.33 1.36
32 2.69 2.72
33 4.58 4.47
34 2.50 2.58
35 1.54 1.56
36 0.77 0.80
37 0.91 0.93
38 3.30 3.33
39 6.95 6.86
40 4.17 4.24
41 2.38 2.45
42 5.25 5.51
43 0.46 0.43
44 3.92 3.96
45 5.35 5.49
46 0.80 0.79
47 2.55 2.58
48 0.79 0.85
49 1.74 1.80
50 0.67 0.66
51 1.21 1.21
52 4.04 4.16
53 2.53 2.62
54 1.39 1.40
55 6.55 6.79
56 3.38 3.50
57 6.72 6.85
58 2.35 2.43
59 0.83 0.86
60 0.91 0.92
61 3.24 3.42
62 2.60 2.63 10 -->
63 4.52 4.57
64 3.80 3.83
65 2.81 2.86
66 0.66 0.67
67 1.94 2.00
68 7.20 7.49
69 0.63 0.62
70 2.96 3.04
71 3.65 3.51
72 5.32 5.50
73 0.89 0.89
74 1.14 1.14
75 2.72 2.79
76 4.66 4.83
77 5.90 6.26
78 6.00 5.72
79 1.91 1.89
80 0.50 0.51
81 0.64 0.67
82 0.98 1.02
83 5.83 5.61
84 1.45 1.41
85 5.00 5.24
86 3.15 3.30
87 2.67 2.72
88 3.95 3.90
89 6.40 6.87
90 4.12 4.49
91 2.91 2.80
92 0.39 0.40
93 1.55 1.51
94 5.07 5.27
95 2.34 2.43
96 4.78 5.10
97 0.82 0.85
98 6.92 6.46
99 2.44 2.35
100 6.81 6.52
Above-mentioned data are mapped, and referring to Fig. 3, the linear equation obtained is: y=1.0073x+0.0102, coefficient R2=0.9941, it was shown that the accuracy that the detectable of the present invention measures Dimethyl-L-arginine clinical samples is high.
It should be noted that; the foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every equivalent structure utilizing description of the present invention and accompanying drawing content to do or equivalence flow process conversion; or directly or indirectly it is used in other correlative technology fields, all in like manner include in the scope of patent protection of the present invention.

Claims (8)

1. a Dimethyl-L-arginine immunogen, its structural formula is such as shown in formula I:
Formula I
Carrier for having immunogenic protein or polypeptide, the one in serum albumin, hemocyanin or Elityran.
2. the immunogenic preparation method of Dimethyl-L-arginine as claimed in claim 1, it is characterised in that comprise the steps of
(1) carrier protein 100～300g is dissolved in the phosphate buffer of 25～75ml0.2M, pH8.5；
(2) following chemicals is joined stirring and dissolving in small beaker: 100～300mg Dimethyl-L-arginine, 1.75～5.25ml dimethylformamide, 1.75～5.25ml ethanol, 3.5～10.5ml10mM, the kaliumphosphate buffer of pH5.0,100～300mg1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 25～75mgN-hydroxy thiosuccinimide, react above-mentioned chemicals at room temperature stirring and dissolving to 30～60min；
(3) solution dissolved is dropped in carrier protein solution, and stir at 2～8 DEG C overnight, obtain antigen；Synthetic antigen is purified through dialysis, obtains Dimethyl-L-arginine immunogen.
3. an anti-Dimethyl-L-arginine specific antibody, the complete antibody molecule produced after Dimethyl-L-arginine immunogen immune laboratory animal described in claim 1, or be antibody fragment or the antibody derivatives of reservation and Dimethyl-L-arginine specific binding capacity.
4. the anti-Dimethyl-L-arginine specific antibody of one according to claim 3, it is characterized in that described complete antibody molecule, antibody fragment or antibody derivatives, for the polyclonal antibody adopting single Dimethyl-L-arginine immunogen that animal booster immunization is obtained, or the monoclonal antibody for obtaining through somatic hybridization after immunity.
5. the preparation method of the anti-Dimethyl-L-arginine specific antibody as according to any one of claim 3-4, it is characterised in that comprise the steps of
(1) with PBS, Dimethyl-L-arginine immunogen is diluted to 0.1～3.0mg/ml, obtains antigenic solution, then mix with equivalent Freund's complete adjuvant with 0.5～5.0ml antigenic solution, laboratory animal is injected；
After (2) 2～3 weeks, then with equivalent incomplete Freund's adjuvant, above-mentioned laboratory animal is injected once with antigenic solution identical for 0.5～5.0ml, inject once every surrounding afterwards, amount to injection 3～6 times；
(3) laboratory animal of step (2) is taken blood, separate purification and obtain the anti-Dimethyl-L-arginine specific antibody that titer is 1:30000～1:50000.
6. a Dimethyl-L-arginine detectable, containing the anti-Dimethyl-L-arginine specific antibody described in claim 3 or 4 and indicator, described indicator one in enzyme reagent, radiosiotope reagent, fluorometric reagent or luminescence reagent；Described enzyme reagent is made up of the substrate of Dimethyl-L-arginine enzyme mark conjugate and enzyme, and enzyme mark conjugate is glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark conjugate, and the substrate of enzyme is G-6-P.
7. the preparation method of a Dimethyl-L-arginine detectable as claimed in claim 6, it is characterised in that comprise the steps of
(1) reagent A: 2.018～8.072g, the nicotinamide adenine dinucleotide of 5.625～22.50mM oxidation state and 0.856～3.422g, 5.625～22.50mM G-6-P Tris buffer solution of 0.5～2L55mM, pH=8.0 are made homogeneous zymolyte；Being added in above-mentioned homogeneous zymolyte by the anti-Dimethyl-L-arginine specific antibody described in claim 3 or 4, the volume ratio of anti-Dimethyl-L-arginine specific antibody and homogeneous zymolyte is 1:100～1:10000；
(2) volume ratio of reagent B: be added in the Tris buffer of 120mM, pH=8.2 by Dimethyl-L-arginine enzyme mark conjugate, Dimethyl-L-arginine enzyme mark conjugate and Tris buffer is 1:100～1:10000.
8. the preparation method of Dimethyl-L-arginine detectable according to claim 7, it is characterised in that the preparation method of described Dimethyl-L-arginine enzyme mark conjugate comprises the steps of
(1) preparation of glucose-6-phosphate dehydrogenase (G6PD) solution: weighing the glucose-6-phosphate dehydrogenase (G6PD) that 7.5～22.5mg specification is 100KU, room-temperature dissolution contains 72.6mg0.05MTris, 8mg3.3mMMgCl in 6～18mL2With in the solution of 100mgNaCl, pH=9.0；Add the nicotinamide adenine dinucleotide of 112.5～337.5mg reduction-state, 67.5～202.5mg G-6-P and 0.375～1.125mL carbitol in the solution；It is added dropwise over 1～3mL dimethyl sulfoxide again；
(2) activation of Dimethyl-L-arginine: weigh 5～15mg Dimethyl-L-arginine under anhydrous conditions, is dissolved in 300～900 μ L dimethylformamides；Above-mentioned solution temperature is made to drop to-2 ~-8 DEG C；Add 1.5～4.5 μ L tri-n-butylamines；Add 0.75～2.25 μ L isobutyl chlorocarbonate；-2 ~-8 DEG C are stirred 30～60 minutes；
(3) connection of glucose-6-phosphate dehydrogenase (G6PD) and Dimethyl-L-arginine: the Dimethyl-L-arginine dropwise that step (2) activates is joined in the glucose-6-phosphate dehydrogenase (G6PD) solution that step (1) is dissolved；2-8 DEG C of stirring is overnight；
(4) purified product: by G-25 gel chromatography column purification connect product, it is thus achieved that end product be glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, at 2-8 DEG C store.
CN201610209278.9A 2016-04-06 2016-04-06 Asymmetric dimethylarginine immunogen, antibody, detecting reagent and preparation method Pending CN105753969A (en)

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