CN102621323A - Method for detecting 6-methylmercaptopurine - Google Patents
Method for detecting 6-methylmercaptopurine Download PDFInfo
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Abstract
The invention discloses a homogeneous phase enzyme immuno-detection method for 6-methylmercaptopurine (6-MMP), a method for detecting an enzyme linked immuno-adsorbent and a detection reagent used by the two methods. According to the two detection methods, whether the 6-MMP exists in a sample to be detected can be determined by using the detection reagent developed from an antibody for resisting 6-MMP specificity, and the content of the 6-MMP can be quantitatively determined. Compared with the conventional genotyping and high performance liquid chromatography (HPLC) method, the immuno-detection method has the advantages of simplicity and convenience and quickness in operation, accurate detection result, low cost and the like, and has a very good prospect for large-scale clinical popularization and application in the future, particularly in middle-small hospitals lack of expensive instruments.
Description
Technical field
The invention belongs to biological technical field, relate to 6-(first sulfydryl) purine detection method.
Background technology
(6-Methylmercaptopurine, 6-MMP), its structural formula is suc as formula shown in (I) for 6-(first sulfydryl) purine.
6-MMP is 6-(sulfydryl) purine (6-Mercaptopurine, metabolin 6-MP).6-MP is widely used in the treatment of multiple important diseases clinically, comprising: acute leukemia, organ transplant and some autoimmune diseases etc., if but this medicine improper use can produce serious even can life-threatening hematotoxicity, Mardini
[1]Through measuring the dosage that the concentration of its metabolin 6-MMP in patient blood decides 6-MP, document result shows when using 6-MP in suggestion, and when the 6-MMP concentration in the blood＜0.6 μ g/mL, dosage does not reach relevant curative effect; And when 6-MMP concentration＞5.0 μ g/mL, then can cause toxicity to liver.Therefore, generally need the concentration of metabolin 6-MMP be controlled within 0.6 μ g/mL～5.0 μ g/mL.
The method that tradition check 6-MMP uses is HPLC and LC/MS-MS.These method complicated operations, expense is high, utilizes the immunity inspection of anti-6-MMP specific antibody development then to remedy these shortcomings.Classic method need be carried out complicated pre-treatment to sample, and the time is long, and expense is high; Detection method of the present invention can confirm whether there is 6-MMP in the sample to be tested, can carry out quantitative measurement to the content of 6-MMP equally.Immunity inspection with anti-6-MMP specific antibody development can be through the reasonable standard medication of the direct direct clinical of assaying reaction product 6-MMP.Compare with the HPLC method with Genotyping; Immunologic detection method provided by the invention has advantages such as easy and simple to handle, quick, that testing result is accurate, expense is low; For carrying out clinical large scale application in the future, the middle and small hospital that particularly lacks expensive instrument has good prospect.
Summary of the invention
The present invention is exactly in order to overcome the complicated defective of detection 6-MMP method that prior art exists, to adopt the immunity inspection reagent of 6-MMP specific antibody development to remedy.The invention provides the method for using immunity inspection reagent of the present invention to test.
One of the object of the invention is to provide the homogeneous enzyme immunoassay detection method of a kind of 6-MMP.
Another object of the present invention is to provide the enzyme-linked immunosorbent of a kind of 6-MMP to detect (Enzyme linked Immunosorbent Assay, ELISA) method.
Provided by the invention two kinds the 6-MMP detection method is easy to operate, the result is accurate, highly sensitive, overcome the complicated defective of detection 6-MMP method that prior art exists.The present invention realizes through following technical scheme:
The homogeneous enzyme immunoassay method of inspection of a kind of 6-MMP, the homogeneous enzyme immunoassay testing reagent of use 6-MMP, this detectable is made up of anti-6-MMP specific antibody and 6-MMP enzyme mark conjugate, comprises following operation steps:
(1) adopts automatic clinical chemistry analyzer, sample to be tested and 6-MMP testing reagent are put into sample storehouse and reagent storehouse respectively;
(2) carry out the setting of test item parameter according to table 1;
Table 1 6-MMP homogeneous enzyme immunoassay inspection parameter is provided with table
(3) light absorption value of mensuration OD340nm;
(4) the production standard curve comes the 6-MMP concentration in the quantitative sample.
More than operation (2) step can also be carried out through following preferred version:
6-MMP homogeneous enzyme immunoassay inspection parameter is provided with table
Said anti-6-MMP specific antibody is produced after by 6-MMP immunogen immune animal and is obtained.
Described 6-MMP immunogene, its structural formula is suc as formula shown in (I):
In the formula, R is a linking group, and carrier has immunogenicity.
R can be-(CH
2)
n-COO-,-O-(CH
2)
n-COO-,-S-(CH
2)
n-COO-or-NH-(CH
2)
n-COO-etc., n are the integers between 1 to 20.Preferred R is-(CH
2)
n-COO-, the n value is 1 to 10.More preferably R is-(CH
2)
5-COO-.
Above-mentioned carrier is often selected protein for use for having immunogenic material.Be preferably haemocyanin, hemocyanin and thyroglobulin.Bovine serum albumin more preferably.
When R is-(CH
2)
5During-COO-, immunogenic route of synthesis of this 6-MMP and method are following:
(1) the 6-MMP derivant is synthetic:
With the 6-MMP of 10-100ml organic solvent A dissolving 1.0-10.0g and the carbonate of 1.0-5.0g, obtain solution 1; The 6-bromocaproic acid second fat of 1.0-5.0g is joined in the organic solvent A of 5-50ml, obtain solution 2, solution 1 and 2 is merged carry out stirring reaction.Reacted solution is neutralized with inorganic acid solution, and purifying, drying after the organic solvent B extract and separate obtain white powder 6-MMP derivant.
(2) preparation of carrier solution: will have the 0.2M that immunogenic protein 100-300mg is dissolved in 10-100ml, in pH 8.5 phosphate buffers.
(3) activation of 6-MMP derivant and immunogenic synthetic: the 6-MMP derivant that dissolves 50-500mg with the organic solvent A of 1.0-5.0ml; Through 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (1-Ethyl-3-(3-dimethylaminopropyl) Carbodiimide, method EDAC)
[2]Carry out activation and carry out cross-linking reaction, behind the dialysis purifying, obtain having immunogenic 6-MMP immunogene with carrier solution.
Organic solvent A described in the said method be dimethyl sulfoxide (DMSO) (Dimethyl Sulfoxide, DMSO), dimethyl formamide (N, N-Dimethylformamide, DMF), methyl alcohol or ethanol, preferred DMF.Described organic solvent B is: ethyl acetate, ether or chloroform, ethyl acetate; Described carbonate is Na
2CO
3Or K
2CO
3, preferred Na
2CO
3Described inorganic acid solution is hydrochloric acid solution or sulfuric acid solution, the preferred salt acid solution.
When R is-O-(CH
2)
n-COO-,-S-(CH
2)
n-COO-or-NH-(CH
2)
nDuring-COO-etc., immunogenic route of synthesis of 6-MMP and R are-(CH
2)
nBasic identical during-COO-.
The preparation method of anti-6-MMP specific antibody:
(1) with phosphate buffer synthetic 6-MMP immunogene is diluted to 0.5-5.0mg/ml;
(2) through conventional Freund method animal is injected acquisition antibody, the animal specific antisera is extracted in the injection back, obtains effective antibody.
In the said method, preferably the 6-MMP immunogene is diluted to 1.0-2.0mg/mL with phosphate buffer.
" antibody " of indication not only refers to complete antibody molecule among the present invention, also comprises the antibody fragment or the derivant that keep the complete antibody specific binding capacity.Antibody of the present invention can be for polyclonal antibody can be a monoclonal antibody also, is preferably polyclonal antibody.
Antibody of the present invention can obtain through prior art for preparing.The typical method that obtains polyclonal antibody is to use single immunogene, after adding or not adding adjuvant, carries out immunity at one or more position of animal, and host animal comprises: rabbit, goat, mouse, sheep, cavy or horse.Preferably, above-mentioned host animal is a rabbit.Animal is regularly taken a blood sample and obtains an amount of specific corrosioning anteserum, and antiserum can purifying.Monoclonal antibody can be made through somatocyte hybriding technology.
The preparation method of the 6-MMP enzyme mark conjugate in the homogeneous enzyme immunoassay method of inspection of 6-MMP of the present invention in the used 6-MMP testing reagent is following:
(3) enzyme solutions preparation: take by weighing enzyme; Be selected from beta galactosidase (β-galactosidase) or glucose-6-phosphate dehydrogenase (G6PD) (and Glucose-6-phosphate Dehydrogenase, G6PDH), preferred G6PDH; Be dissolved at ambient temperature in the phosphate buffer, final concentration is 2-6mg/mL;
(4) activation of 6-MMP derivant and conjugate is synthetic: with organic solvent dissolution 6-MMP derivant mentioned above, making its final concentration is 10-50mg/mL, through the tri-n-butylamine method
[3](Wong, Jameson.Chemisty of protein and nucleic acid cross-linking and conjugation.2
NdEdition, the method for putting down in writing among the 368-369) carries out activation, and carry out cross-linking reaction, obtain 6-MMP enzyme mark conjugate after purified and the dialysis with enzyme solutions.
Organic solvent described in the above-mentioned preparation method is selected from DMF, DMSO, methyl alcohol or ethanol.Preferably, organic solvent is DMF.
The preparation method of the 6-MMP testing reagent described in the homogeneous enzyme immunoassay method of inspection of 6-MMP of the present invention is following:
(1) preparation of R1 reagent: above-mentioned anti-6-MMP specific antibody is diluted in the homogeneous phase R1 damping fluid; Homogeneous phase R1 damping fluid contains 50mM Tris; 0.25%BSA; 50mM G-6-P and 50mM nicotine adenine-dinucleotide, the volume ratio of said 6-MMP specific antibody and R1 damping fluid is 1: 1000-1: 10000, the volume ratio of preferred antibody and R1 damping fluid is 1: 1000-1: 3000;
(2) preparation of R2 reagent: enzyme-6MMP conjugate is diluted to R2 damping fluid (100mM Tris; 0.25%BSA); The volume ratio of said enzyme-6MMP conjugate and R2 damping fluid is 1: 1000-1: 10000, and the volume ratio of preferred enzyme-6MMP conjugate and R2 damping fluid is 1: 1000-1: 3000.
The homogeneous enzyme immunoassay method of inspection of 6-MMP of the present invention through automatic clinical chemistry analyzer, adds sample during detection, adds R1 reagent again, adds R2 reagent at last, measures the OD340 light absorption value of different time points, calculates the reaction rate of variable concentrations standard items.Need constantly the ratio of adjustment R1 reagent and R2 reagent in the actual mechanical process, obtain comparatively ideal reaction normal curve.Through the reaction rate value of sample, and obtain the concentration value of sample according to the relational expression of reaction rate in the typical curve and standard items concentration.
This detection method is a kind of competitive reaction; Need not separate through solid phase with free 6-MMP with the 6-MMP of antibodies in the reaction system, its ultimate principle is: free 6-MMP is at war with to the binding site of specific antibody with the 6-MMP derivant that is coupled on the G6PDH in the liquid sample.The 6-MMP enzyme conjugates of emulative replacement of the 6-MMP in the liquid sample and antibodies, and its binding site from antibody is discharged, thus make enzyme recover active.Therefore, the content of 6-MMP is many more in the liquid sample, and free 6-MMP derivant-G6PDH enzyme conjugates is just many more, thereby can obtain stronger signal.Therefore can come the 6-MMP concentration in the quantitative sample through the production standard curve.
The present invention also provides the ELISA detection method of a kind of 6-MMP, uses 6-MMP ELISA testing reagent, this detectable by: anti-6-MMP specific antibody, 6-MMP enzyme mark conjugate and substrate are formed, and comprise following operation steps:
(1) will resist the 6-MMP antibody dilution to become 1 with PBS: 1000-1: 20000 final concentration solution, 100 μ L/ holes are coated on the 96 hole elisa plates, and 4 ℃ are spent the night;
(2) with after the PBS washing 3 times, add 0.5% the BSA solution in 200 μ L/ holes, 4 ℃ of sealings are spent the night, PBS washing 3 times;
(3) standard items in adding 20 μ L/ holes;
(4) the 6-MMP enzyme that adds 100 μ L/ hole working concentrations is marked conjugate;
(5) hatch 30min under the room temperature, PBS washes plate 5 times;
(6) every hole adds 100 μ L substrates, preferred tmb substrate, incubated at room 30min.
(7) every hole adds 100 μ L stop buffers (2M sulfuric acid).
(8) light absorption value of mensuration 450nm.
Preferably will resist the 6-MMP antibody dilution to become 1 in the above operation steps (1): 2000-1 with PBS: 10000 final concentration solution, 100 μ L/ holes are coated on the 96 hole elisa plates, and 4 ℃ are spent the night;
6-MMP ELISA testing reagent of the present invention, contain:
(1) anti-6-MMP specific antibody
(2) enzyme mark conjugate and substrate: the enzyme of enzyme mark conjugate can be a horseradish peroxidase (HRP), (Alkaline phosphatase AP) etc., is preferably HRP to alkaline phosphatase; Substrate is the substrate of corresponding enzyme; Be preferably 3,3 ', 5; 5 '-tetramethyl benzidine (TMB), the wherein preparation method of enzyme mark conjugate:
A) take by weighing enzyme and be dissolved at ambient temperature in the phosphate buffer, final concentration is 2-6mg/ml;
B) activation of 6-MMP derivant and conjugate is synthetic: with organic solvent dissolution 6-MMP derivant, final concentration is 10-50mg/ml, carries out activation through the method for EDAC, and carries out cross-linking reaction with enzyme solutions, behind the dialysis purifying, obtains 6-MMP enzyme mark conjugate.
Organic solvent described in the above-mentioned preparation method is selected from DMF, DMSO, methyl alcohol or ethanol.Preferably, organic solvent is DMF.
This check is a kind of solid phase competitive ELISA reaction of routine.The content of 6-MMP is many more in the liquid sample, and just few more with the 6-MMP derivant-enzyme conjugates of antibodies on the solid support, it is shallow more to develop the color.Therefore can come the 6-MMP concentration in the quantitative sample through the production standard curve.
The ultimate principle of this ELISA method of inspection is: the 6-MMP that dissociates in the liquid sample and the 6-MMP derivant of HRP mark are at war with to the binding site that is coated on the specific antibody on the solid support; Remove unconjugated molecule through washing, the substrate that adds HRP again carries out chromogenic reaction.The content of 6-MMP is many more in the liquid sample, and just few more with the 6-MMP derivant-HRP enzyme conjugates of antibodies on the solid support, it is shallow more to develop the color.Therefore can come the 6-MMP concentration in the quantitative sample through the production standard curve.
Description of drawings
Fig. 1 is a 6-MMP homogeneous enzyme immunoassay detection reaction curve
Fig. 2 is the ELISA detection reaction curve of 6-MMP
Embodiment
The preparation of embodiment 1 anti-6-MMP specific antibody
1.6-MMP derivant is synthetic, its chemical constitution is suc as formula shown in (II).
The route of synthesis and the method for this 6-MMP derivant are following:
(1) with 30ml DMF dissolving 5.0g 6-MMP and 4.45g K
2CO
3, add the DMF solution that 10ml contains 4.06g 6-bromocaproic acid second fat, stirring reaction 30min under 40 ℃ of conditions.
(2) will react back solution subsequently with the neutralization of 1N HCl solution, and use ethyl acetate extraction.Organic phase is through drying, filtration, vacuum drying and purification on normal-phase silica gel purifying.With 25ml dissolve with methanol 1.5g purified product, add the WS that contains 600mg LiOH.H2O subsequently, stirring at room reaction 4 hours.Product is dilute with water, ether washing after concentrating, and aqueous phase solution is with 1N HCl solution adjustment pH to 5.0.Use dichloromethane extraction at last, organic phase is through Na
2SO
4Dry, obtain white solid 6-MMP derivant after filtering and concentrating.LCMS result shows: purity is 99.7%; Molecular weight is 281; Molion is 282 (M+1).
2.6-MMP it is immunogenic synthetic
The 6-MMP immunogene is by haemocyanin, and hemocyanin or thyroglobulin and 6-MMP pass through-(CH
2)
5-COOH group is formed by connecting, with bovine serum albumin (Bovine Serum Albumin is that the concrete synthetic method of example is following BSA):
(1) 200mg BSA is dissolved in 50ml 0.2M, in the phosphate buffer of pH 8.5;
(2) following chemicals is joined stirring and dissolving in the small beaker: 200mg synthetic 6-MMP derivant, 3.5ml DMF, 3.5ml ethanol, 7.0ml 10mM; The kaliumphosphate buffer of pH 5.0,200mg EDAC, 50mg N-hydroxy-succinamide (N-hydroxysuccinimide; Sulfo-NHS); With its stirring and dissolving, at room temperature react 30min;
(3) will dissolve good drips of solution and add in the BSA solution, and, obtain antigen 2～8 ℃ of following stirred overnight; Synthetic good antigen through the dialysis purifying, is obtained the 6-MMP immunogene.
3. the preparation of anti-6-MMP specific antibody
(1) with PBS synthetic 6-MMP immunogene is diluted to 1.5mg/ml, mixes with Freund's complete adjuvant with antigenic solution then, rabbit is injected;
After (2) 2～3 weeks, mix the back with incomplete Freund once with the identical antigenic solution of 1.0ml again to rabbit injection, every afterwards at a distance from around once, totally twice, extract the antiserum of rabbit, obtain effective antibody.
The homogeneous enzyme immunoassay testing reagent preparation of embodiment 2 6-MMP
(1) preparation of R1 reagent: in the R1 damping fluid, homogeneous phase R1 damping fluid contains 50mM Tris with the antibody dilution for preparing, 0.25%BSA, 50Mm G-6-P and 50mM NAD.The volume ratio of antibody and R1 damping fluid is 1: 1000.
(2) preparation of R2 reagent
1) preparation of G6PDH-6MMP
A) take by weighing 15mg G6PDH, be dissolved in 12ml, in the 0.05M Tris damping fluid, add the DMF mixing of 100mg NADH, 0.5ml carbitol and 1ml successively;
B) the 6-MMP derivant with 10mg is dissolved among 420 μ l dimethyl sulfoxide (DMSO)s and the 180 μ l DMF, adds 6 μ l tri-n-butylamines (tributylamine) and the 3 different esters of μ l chloro-carbonic acid (isobutylchloroformate), stirring reaction 30min under 2～8 ℃ of conditions;
C) stirred overnight under 2～8 ℃ of conditions subsequently, and the G6PDH-6MMP that obtains carried out purifying.
2) G6PDH-6MMP for preparing is diluted in the R2 damping fluid.The R2 damping fluid is 100mM Tris, 0.25%BSA.The volume ratio of antibody and R2 damping fluid is 1: 1000.
The homogeneous enzyme immunoassay method of inspection and the calibration result of embodiment 3 6-MMP
Table 1 Hitachi 7180 analyser 6-MMP homogeneous enzyme immunoassay inspection parameter tables
Through automatic clinical chemistry analyzer, carry out the parameter setting according to table 1 data.At first add sample; Add the R1 reagent (mixed liquor of antibody and R1 damping fluid) among the embodiment 2 again; Add R2 reagent (mixed liquor of G6PDH-6MMP conjugate and R2 damping fluid) at last, measure the OD340 light absorption value of different time points, calculate the absorbance rate of change of variable concentrations standard items; Obtain comparatively ideal reaction normal curve, the result is as shown in Figure 1.
The preparation of the ELISA testing reagent of embodiment 4 6-MMP
(1) contains the anti-6-MMP specific antibody that embodiment 1 prepares
(2) contain HRP-6MMP conjugate and substrate.
The preparation method of enzyme mark conjugate:
A) take by weighing 20mg HRP and be dissolved in 5ml 0.2M at ambient temperature, in the phosphate buffer of pH 8.5;
B) activation 6-MMP derivant: take by weighing 10mg 6-MMP derivant in small beaker; And add 350 μ L DMF, 350 μ L absolute ethyl alcohols, 700 μ L 10mM successively; The kaliumphosphate buffer of pH 5.0,40mg EDAC and 5mg Sulfo-NHS, stirring reaction 30min at ambient temperature;
C) subsequently the 6-MMP derivant of activation is added drop-wise in the HRP enzyme solutions, stirred overnight under 2-8 ℃ of condition, and the conjugate purifying of dialysing obtained the HRP-6-MMP conjugate.
The embodiment 5 6-MMP ELISA methods of inspection and calibration result
(1) 6-MMP ELISA method of inspection step
1) will resist the 6-MMP antibody dilution to become 1: 5000 final concentration solution with PBS, 100 μ L/ holes are coated on the 96 hole elisa plates, and 4 ℃ are spent the night;
2) with after the PBS washing 3 times, add 0.5% the BSA solution in 200 μ L/ holes, 4 ℃ of sealings are spent the night, PBS washing 3 times;
3) standard items in adding 20 μ L/ holes;
4) the HRP-6-MMP conjugate of adding 100 μ L/ hole working concentrations;
5) hatch 30min under the room temperature, PBS washes plate 5 times;
6) every hole adds 100 μ L tmb substrates, incubated at room 30min.
7) every hole adds 100 μ L stop buffers (2M sulfuric acid).
8) light absorption value of mensuration 450nm.
(2) the calibration result is as shown in Figure 2.
The purpose of this recovery experiment is to confirm that described 6-MMP homogeneous enzyme immunoassay detection method can be used for the detection of whole blood sample 6-MMP.
1. the calibration curve of the check of the homogeneous enzyme immunoassay through 6-MMP, replication is blank, basic, normal, high concentration serum (is dissolved in the 6-MMP standard items in the blank whole blood, is respectively 0 to final concentration; 0.6; 2.0,4.0 μ g/mL) and 3 times, the recovery of results sample high (＞95%).
2. assay method: the homogeneous enzyme immunoassay method like the 6-MMP among the embodiment 3 is said, and the result sees table 2.
The homogeneous enzyme immunoassay check recovery experiment of table 2 6-MMP
| Blood serum sample | Blank | Low | In | High |
| Sample concentration (μ g/ml) | 0.0 | 0.60 | 2.0 | 4.0 |
| |
0.00 | 0.62 | 2.10 | 4.10 |
| |
0.00 | 0.58 | 1.90 | 4.07 |
| |
0.00 | 0.63 | 2.06 | 4.05 |
| Mean value (μ g/ml) | 0.00 | 0.61 | 2.02 | 4.07 |
| Standard deviation (μ g/ml) | 0.00 | 0.02 | 0.09 | 0.02 |
| CV(％) | - | 3.5 | 4.3 | 0.5 |
| The recovery (%) | - | 102 | 101 | 102 |
This experimental result shows: the 6-MMP recovery in the sample of variable concentrations is high, all＞90%, explain that described 6-MMP homogeneous enzyme immunoassay detection method can be used for the detection of whole blood sample 6-MMP, and the result is accurate, and is credible.
The purpose of this recovery experiment is to confirm that described 6-MMP ELISA detection method can be used for the detection of whole blood sample 6-MMP.
1. the calibration curve of the check of the ELISA through 6-MMP, replication is blank, basic, normal, high concentration serum (is dissolved in the 6-MMP standard items in the blank whole blood, is respectively 0 to final concentration; 0.6; 2.0,4.0 μ g/mL) and 3 times, the recovery of results sample high (＞90%).
2. assay method: the ELISA method like the 6-MMP among the embodiment 4 is said, and the result sees table 3.
The ELISA of table 3 6-MMP detects recovery experiment
| Blood serum sample | Blank | Low | In | High |
| Sample concentration (μ g/ml) | 0.0 | 0.60 | 2.0 | 4.0 |
| |
0.02 | 0.68 | 1.92 | 4.13 |
| |
0.10 | 0.61 | 2.27 | 3.84 |
| |
0.03 | 0.65 | 2.15 | 3.87 |
| Mean value (μ g/ml) | 0.05 | 0.65 | 2.11 | 3.95 |
| The recovery (%) | - | 107 | 106 | 99 |
This experimental result shows: the 6-MMP recovery in the sample of variable concentrations is high, all＞90%, explain that described 6-MMP ELISA detection method can be used for the detection of whole blood sample 6-MMP, and the result is accurate, and is credible.
List of references:
[1]Mardini?et?al.Utility?of?Measuring?6-Methylmercaptopurine?and?6-Thioguanine?Nucleotide?Levels?in?Managing?Inflammatory?Bowel?Disease?Patients?Treated?With?6-Mercaptopurine?in?a?Clinical?Practice?Setting.Journal?of?Clinical?Gastroenterology.2003，36(5)：390-395.
[2]Hermanson.Bioconjugate?techniques.2
nd?edition，215-221.
[3]Wong，Jameson.Chemisty?of?protein?and?nucleic?acid?cross-linking?and?conjugation.2
nd?edition，368-369。
Claims (6)
1. the homogeneous enzyme immunoassay detection method (Homogeneous Immunoassay) of a 6-(first sulfydryl) purine; Use the homogeneous enzyme immunoassay testing reagent of 6-(first sulfydryl) purine; This detectable is made up of anti-6-(first sulfydryl) purine (6-MMP) specific antibody and 6-(first sulfydryl) purinase mark conjugate, comprises following operation steps:
(1) adopts automatic clinical chemistry analyzer, sample to be tested and 6-(first sulfydryl) purine detectable are put into sample storehouse and reagent storehouse respectively;
(2) carry out the setting of test item parameter according to following table;
(3) light absorption value of mensuration OD340nm;
(4) the production standard curve comes 6-(first sulfydryl) the purine concentration in the quantitative sample;
Aforesaid operations step (2) can also realize through following preferred version.
Said anti-6-MMP specific antibody is produced after by 6-MMP immunogen immune animal and is obtained,
Described 6-MMP immunogene, its structural formula is suc as formula shown in (I):
In the formula, R is a linking group, and carrier has immunogenicity.
R is-(CH
2)
n-COO-,-O-(CH
2)
n-COO-,-S-(CH
2)
n-COO-or-NH-(CH
2)
n-COO-etc., n are the integers between 1 to 20.Preferred R is-(CH
2)
n-COO-, the n value is 1 to 10.More preferably R is-(CH
2)
5-COO-; Said carrier is often selected protein for use for having immunogenic material.Be preferably haemocyanin, hemocyanin and thyroglobulin, bovine serum albumin more preferably,
The preparation method of 6-MMP enzyme mark conjugate is following:
(1) enzyme solutions preparation: take by weighing enzyme; Be selected from beta galactosidase (β-galactosidase) or glucose-6-phosphate dehydrogenase (G6PD) (and Glucose-6-phosphate Dehydrogenase, G6PDH), preferred G6PDH; Be dissolved at ambient temperature in the phosphate buffer, final concentration is 2-6mg/mL;
(2) with the 6-MMP derivant of organic solvent dissolution link R linking group; Making its final concentration is 10-50mg/mL, carries out activation through the tri-n-butylamine method, and carries out cross-linking reaction with enzyme solutions; Obtain 6-MMP enzyme mark conjugate after purified and the dialysis; Described organic solvent is selected from DMF, DMSO, methyl alcohol or ethanol, and preferably, organic solvent is DMF.
2. the homogeneous enzyme immunoassay detection method of 6-according to claim 1 (first sulfydryl) purine is characterized in that described detectable preparation method comprises following steps:
(1) will resist 6-(first sulfydryl) purine specific antibody with 1: 1000-1: 10000 volume ratio is diluted in the homogeneous phase R1 damping fluid; Homogeneous phase R1 damping fluid contains 50mM Tris; 0.25% bovine serum albumin, 50mM G-6-P and 50mM nicotine adenine-dinucleotide;
(2) 6-(first sulfydryl) purinase is marked conjugate with 1: 1000-1: 10000 volume ratio is diluted in the R2 damping fluid, and the R2 damping fluid contains 100mM Tris, 0.25% bovine serum albumin;
The volume ratio of described anti-6-(first sulfydryl) purine specific antibody and R1 damping fluid is preferably 1: 1000-1: 3000;
The volume ratio of described 6-(first sulfydryl) purinase mark conjugate and R2 damping fluid is preferably 1: 1000-1: 3000.
3. the ELISA detection method of a 6-(first sulfydryl) purine is used 6-(first sulfydryl) purine ELISA testing reagent, and this detectable contains: (1) anti-6-(first sulfydryl) purine (6-MMP) specific antibody; (2) enzyme mark conjugate and substrate comprise following operation steps:
(1) will resist the 6-MMP specific antibody to be diluted to 1 with PBS: 1000-1: 20000 final concentration solution, 100 μ L/ holes are coated on the 96 hole elisa plates, and 4 ℃ are spent the night;
(2) with after the PBS washing 3 times, add 0.5% the BSA solution in 200 μ L/ holes, 4 ℃ of sealings are spent the night, PBS washing 3 times;
(3) standard items in adding 20 μ L/ holes;
(4) 6-(first sulfydryl) purinase that adds 100 μ L/ hole working concentrations is marked conjugate;
(5) hatch 30min under the room temperature, PBS washes plate 5 times;
(6) every hole adds 100 μ L substrates, preferred TMB, incubated at room 30min;
(7) every hole adds 100 μ L stop buffers (2M sulfuric acid);
(8) light absorption value of mensuration 450nm;
Aforesaid operations step (1) is preferably: will resist 6-(first sulfydryl) purine specific antibody to be diluted to 1 with PBS: 2000-1: 10000 final concentration solution, and 100 μ L/ holes are coated on the 96 hole elisa plates, and 4 ℃ are spent the night; Said anti-6-MMP specific antibody is produced after by 6-MMP immunogen immune animal and is obtained,
Described 6-MMP immunogene, its structural formula is suc as formula shown in (I):
In the formula, R is a linking group, and carrier has immunogenicity.
R is-(CH
2)
n-COO-,-O-(CH
2)
n-COO-,-S-(CH
2)
n-COO-or-NH-(CH
2)
n-COO-etc., n are the integers between 1 to 20.Preferred R is-(CH
2)
n-COO-, the n value is 1 to 10.More preferably R is-(CH
2)
5-COO-; Said carrier is often selected protein for use for having immunogenic material.Be preferably haemocyanin, hemocyanin and thyroglobulin, bovine serum albumin more preferably,
The enzyme of enzyme mark conjugate is horseradish peroxidase (HRP), alkaline phosphatase, is preferably HRP, and substrate is the substrate of corresponding enzyme; Be preferably 3,3 ', 5; 5 '-tetramethyl benzidine (3,3 ' 5,5 '-Tetramethylbenzidine; TMB), described enzyme mark conjugate is obtained by following preparation method:
A) take by weighing enzyme and be dissolved at ambient temperature in the phosphate buffer, final concentration is 2-6mg/ml;
B) be connected to the 6-MMP derivant of R linking group with the organic solvent dissolution chain; Final concentration is 10-50mg/ml, carries out activation through the method for EDAC, and carries out cross-linking reaction with enzyme solutions; Behind the dialysis purifying, obtain 6-MMP enzyme mark conjugate; Described organic solvent is selected from DMF, DMSO, methyl alcohol or ethanol, and preferably, organic solvent is DMF.
4. according to claim 1 or 3 described detection methods, wherein said antibody is polyclonal antibody or monoclonal antibody, is preferably polyclonal antibody; Described animal is selected from rabbit, goat, and mouse, sheep, cavy or Malaysia and China a kind of is preferably rabbit.
5. detection method according to claim 4, the preparation method of wherein said anti-6-(first sulfydryl) purine specific antibody comprises following steps:
(1) with phosphate buffer synthetic 6-(first sulfydryl) purine immunogene is diluted to 0.5-5.0mg/mL;
(2) through conventional Freund method animal is injected, the animal specific corrosioning anteserum is extracted in the injection back, obtains effective antibody.
6. according to the detection method described in the claim 5, the immunogenic preparation method of wherein said 6-(first sulfydryl) purine comprises following steps:
(1) with the 6-MMP of 10-100ml organic solvent A dissolving 1.0-10.0g and the carbonate of 1.0-5.0g, obtains solution 1; The 6-bromocaproic acid second fat of 1.0-5.0g is joined in the organic solvent A of 5-50ml; Obtain solution 2; Solution 1 and 2 merging are carried out stirring reaction; Reacted solution is neutralized with inorganic acid solution, and purifying, drying after the organic solvent B extract and separate obtain white solid 6-(first sulfydryl) purine derivative;
(2) will have the 0.2M that immunogenic protein 100-300mg is dissolved in 10-100ml, in pH 8.5 phosphate buffers;
(3) with 6-(first sulfydryl) purine derivative of the organic solvent A of 1.0-5.0ml dissolving 50-500mg; Carry out activation and carry out cross-linking reaction through 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, behind the dialysis purifying, obtain having immunogenic 6-(first sulfydryl) purine immunogene with carrier solution;
Described organic solvent A is selected from a kind of in dimethyl sulfoxide (DMSO), dimethyl formamide, methyl alcohol or the ethanol, is preferably dimethyl formamide;
Described organic solvent B is selected from ethyl acetate, and a kind of in ether or the chloroform is preferably ethyl acetate,
Described carbonate is Na
2CO
3Or K
2CO
3, be preferably Na
2CO
3
Described inorganic acid solution is hydrochloric acid solution or sulfuric acid solution, is preferably hydrochloric acid solution.
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| CN110950820A (en) * | 2019-11-06 | 2020-04-03 | 苏州博源医疗科技有限公司 | Chlorpromazine derivative, preparation method thereof and chlorpromazine detection reagent |
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| CN110950820B (en) * | 2019-11-06 | 2023-05-02 | 苏州博源医疗科技有限公司 | Chlorpromazine derivative, preparation method thereof and chlorpromazine detection reagent |
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Effective date of registration: 20190516 Address after: 050000 Shijiazhuang Development Zone, Taishan Street, Shijiazhuang High-tech Zone, Shijiazhuang City, Hebei Province Patentee after: Shijiazhuang Lidekang Pharmaceutical Technology Co., Ltd. Address before: 215000 No. 78 Keling Road, Suzhou High-tech Industrial Development Zone, Suzhou City, Jiangsu Province Patentee before: Suzhou Evermed Co., Ltd. |