CN102621323B - 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-(first sulfydryl) purine (6-Methylmercaptopurine, 6-MMP), its structural formula is suc as formula shown in (I).
6-MMP is the metabolin of 6-(sulfydryl) purine (6-Mercaptopurine, 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]suggestion decides the dosage of 6-MP in the time using 6-MP by measuring the concentration of its metabolin 6-MMP in patient blood, document result shows, in the time of 6-MMP concentration < 0.6 μ g/mL in blood, dosage does not reach relevant curative effect; And in the time of 6-MMP concentration > 5.0 μ g/mL, can cause toxicity to liver.Therefore, within generally the concentration of metabolin 6-MMP need to being controlled to 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 to make up these shortcomings.Classic method need to be carried out complicated pre-treatment to sample, and the time is long, and expense is high; Detection method of the present invention, can determine in sample to be tested whether have 6-MMP, can carry out quantitative measurement to the content of 6-MMP equally.Can pass through assaying reaction product 6-MMP with the immunity inspection of anti-6-MMP specific antibody development and directly instruct clinical reasonable standard medication.Compare with HPLC method with Genotyping, immunologic detection method provided by the invention has the advantages such as easy and simple to handle, quick, 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 the defect of the detection 6-MMP method complexity in order to overcome prior art existence, adopts the immunity inspection reagent of 6-MMP specific antibody development to make up.The invention provides the method for testing with immunity inspection reagent of the present invention.
One of object of the present 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 6-MMP detection method is easy to operate, result is accurate, highly sensitive, overcome the defect of the detection 6-MMP method complexity that prior art exists.The present invention is achieved by the following technical solutions:
A homogeneous enzyme immunoassay method of inspection of 6-MMP, the homogeneous enzyme immunoassay testing reagent of use 6-MMP, this detection reagent is made up of anti-6-MMP specific antibody and 6-MMP enzyme mark conjugate, comprises following operation steps:
(1) adopt automatic clinical chemistry analyzer, sample to be tested and 6-MMP testing reagent are put into respectively to sample storehouse and agent bin;
(2) carry out the setting of test item parameter according to table 1;
Table 1 6-MMP homogeneous enzyme immunoassay inspection parameter arranges table
(3) light absorption value of mensuration OD340nm;
(4) production standard curve carrys out the 6-MMP concentration in quantitative sample.
More than operation (2) step can also be undertaken by following preferred version:
6-MMP homogeneous enzyme immunoassay inspection parameter arranges table
Described 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 formula, R is 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 is the integer between 1 to 20.Preferably R is-(CH
2)
n-COO-, n value is 1 to 10.More preferably R is-(CH
2)
5-COO-.
Above-mentioned carrier, for having immunogenic material, is often selected protein.Be preferably haemocyanin, hemocyanin and thyroglobulin.More preferably bovine serum albumin.
When R is-(CH
2)
5when-COO-, the immunogenic route of synthesis of this 6-MMP and method are as follows:
(1) 6-MMP derivant is synthetic:
Dissolve the 6-MMP of 1.0-10.0g and the carbonate of 1.0-5.0g by 10-100ml organic solvent A, 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 and carries out stirring reaction.Reacted solution is neutralized with inorganic acid solution, and after organic solvent B extract and separate, purifying, dry, obtains white powder 6-MMP derivant.
(2) preparation of carrier solution: will there is immunogenic protein 100-300mg and be dissolved in the 0.2M of 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 by the organic solvent A of 1.0-5.0ml, by the method for 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (1-Ethyl-3-(3-dimethylaminopropyl) Carbodiimide, EDAC)
[2]activate and carry out cross-linking reaction with carrier solution, after dialysis purifying, obtaining having immunogenic 6-MMP immunogene.
Organic solvent A described in said method is dimethyl sulfoxide (DMSO) (Dimethyl Sulfoxide, DMSO), dimethyl formamide (N, N-Dimethylformamide, DMF), methyl alcohol or ethanol, preferably DMF.Described organic solvent B is: ethyl acetate, ether or chloroform, ethyl acetate; Described carbonate is Na
2cO
3or K
2cO
3, preferably Na
2cO
3; Described inorganic acid solution is hydrochloric acid solution or sulfuric acid solution, preferred salt acid solution.
When R is-O-(CH
2)
n-COO-,-S-(CH
2)
n-COO-or-NH-(CH
2)
nwhen-COO-etc., the immunogenic route of synthesis of 6-MMP and R are-(CH
2)
nbasic identical when-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's adjuvant method, animal is injected and obtains antibody, after injection, extract animal specific antisera, obtain effective antibody.
In said method, preferably with phosphate buffer, 6-MMP immunogene is diluted to 1.0-2.0mg/mL.
In the present invention, " antibody " of indication not only refers to complete antibody molecule, also comprises the antibody fragment or the derivant that retain complete antibody specific binding capacity.Antibody of the present invention can be for polyclonal antibody can be also monoclonal antibody, is preferably polyclonal antibody.
Antibody of the present invention can prepare by prior art.The typical method that obtains polyclonal antibody is to use single immunogene, adding or do not add after 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 rabbit.Animal timing blood sampling obtains appropriate specific corrosioning anteserum, and antiserum can purifying.Monoclonal antibody can be made by 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 6-MMP testing reagent used is as follows:
(3) enzyme solutions preparation: take enzyme, be selected from beta galactosidase (β-galactosidase) or glucose-6-phosphate dehydrogenase (G6PD) (Glucose-6-phosphate Dehydrogenase, G6PDH), preferably G6PDH, be dissolved at ambient temperature in 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, by tri-n-butylamine method
[3](Wong, Jameson.Chemisty of protein and nucleic acid cross-linking and conjugation.2
ndedition, the method for recording in 368-369) activate, and carry out cross-linking reaction with enzyme solutions, after purified and dialysis, obtain 6-MMP enzyme mark conjugate.
Organic solvent described in 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 as follows:
(1) preparation of R1 reagent: above-mentioned anti-6-MMP specific antibody is diluted in homogeneous phase R1 damping fluid, homogeneous phase R1 damping fluid is containing 50mM Tris, 0.25%BSA, 50mM G-6-P and 50mM nicotine adenine-dinucleotide, the volume ratio of described 6-MMP specific antibody and R1 damping fluid is 1: 1000-1: 10000, and 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 described 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, by automatic clinical chemistry analyzer, adds sample, then adds R1 reagent when detection, finally adds R2 reagent, measures the OD340 light absorption value of different time points, calculates the reaction rate of variable concentrations standard items.In actual mechanical process, need constantly to adjust the ratio of R1 reagent and R2 reagent, obtain comparatively ideal reaction normal curve.By the reaction rate value of sample, and obtain the concentration value of sample according to the relational expression of reaction rate in typical curve and standard items concentration.
This detection method is a kind of competitive reaction, the 6-MMP of being combined with antibody in reaction system does not need to separate by solid phase with free 6-MMP, and its ultimate principle is: 6-MMP free in liquid sample is at war with to the binding site of specific antibody with the 6-MMP derivant being coupled on G6PDH.The 6-MMP enzyme conjugates that the emulative replacement of 6-MMP in liquid sample is combined with antibody, and its binding site from antibody is discharged, thus make enzyme activity recovery.Therefore, in liquid sample, the content of 6-MMP is more, and free 6-MMP derivant-G6PDH enzyme conjugates is just more, thereby can obtain stronger signal.Therefore can carry out the 6-MMP concentration in quantitative sample by 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 detection reagent by: anti-6-MMP specific antibody, 6-MMP enzyme mark conjugate and substrate form, and comprise following operation steps:
(1) will resist 6-MMP antibody dilution to become 1 with PBS: 1000-1: 20000 final concentration solution, 100 μ L/ holes are coated on 96 hole elisa plates, and 4 ℃ are spent the night;
(2) with after 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) add the standard items in 20 μ L/ holes;
(4) add the 6-MMP enzyme mark conjugate of 100 μ L/ hole working concentrations;
(5) under room temperature, hatch 30min, PBS washes plate 5 times;
(6) every hole adds 100 μ L substrates, preferably 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.
In above operation steps (1), preferably will resist 6-MMP antibody dilution to become 1 with PBS: 2000-1: 10000 final concentration solution, 100 μ L/ holes are coated on 96 hole elisa plates, and 4 ℃ are spent the night;
6-MMP ELISA testing reagent of the present invention, contains:
(1) anti-6-MMP specific antibody
(2) enzyme mark conjugate and substrate: the enzyme of enzyme mark conjugate can be horseradish peroxidase (HRP), alkaline phosphatase (Alkaline phosphatase, AP) etc., be preferably HRP, 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 enzyme and be dissolved at ambient temperature in 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, method by EDAC activates, and carries out cross-linking reaction with enzyme solutions, after dialysis purifying, obtains 6-MMP enzyme mark conjugate.
Organic solvent described in 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.In liquid sample, the content of 6-MMP is more, and 6-MMP derivant-enzyme conjugates that the antibody on solid support is combined is just fewer, develops the color more shallow.Therefore can carry out the 6-MMP concentration in quantitative sample by production standard curve.
The ultimate principle of this ELISA method of inspection is: in liquid sample, free 6-MMP and the 6-MMP derivant of HRP mark are at war with to the binding site that is coated on the specific antibody on solid support, remove unconjugated molecule by washing, then add the substrate of HRP to carry out chromogenic reaction.In liquid sample, the content of 6-MMP is more, and 6-MMP derivant-HRP enzyme conjugates that the antibody on solid support is combined is just fewer, develops the color more shallow.Therefore can carry out the 6-MMP concentration in quantitative sample by production standard curve.
Accompanying drawing explanation
Fig. 1 is 6-MMP homogeneous enzyme immunoassay detection reaction curve
Fig. 2 is the ELISA detection reaction curve of 6-MMP
Embodiment
The preparation of the anti-6-MMP specific antibody of embodiment 1
Synthesizing of 1.6-MMP derivant, its chemical constitution is suc as formula shown in (II).
Route of synthesis and the method for this 6-MMP derivant are as follows:
(1) dissolve 5.0g 6-MMP and 4.45g K with 30ml DMF
2cO
3, add 10ml to contain the DMF solution of 4.06g 6-bromocaproic acid second fat, stirring reaction 30min under 40 ℃ of conditions.
(2) will react subsequently the neutralization of rear solution 1N HCl solution, and be extracted with ethyl acetate.Organic phase drying, filtration, vacuum drying and purification on normal-phase silica gel purifying.With 25ml methyl alcohol dissolving 1.5g purified product, add subsequently the aqueous solution containing 600mg LiOH.H2O, stirring at room temperature reaction 4 hours.Product is dilute with water, ether washing after concentrated, and aqueous phase solution is adjusted pH to 5.0 with 1N HCl solution.Finally use dichloromethane extraction, organic phase is through Na
2sO
4after being dried, filtering and concentrating, obtain white solid 6-MMP derivant.LCMS result shows: purity is 99.7%; Molecular weight is 281; Molion is 282 (M+1).
2.6-MMP is immunogenic synthetic
6-MMP immunogene is by haemocyanin, pass through-(CH of hemocyanin or thyroglobulin and 6-MMP
2)
5-COOH group is formed by connecting, take bovine serum albumin (Bovine Serum Albumin, BSA) as the concrete synthetic method of example as follows:
(1) 200mg BSA is dissolved in to 50ml 0.2M, in the phosphate buffer of pH 8.5;
(2) following chemicals is joined to the synthetic 6-MMP derivant of stirring and dissolving in small beaker: 200mg, 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), by its stirring and dissolving, at room temperature react 30min;
(3) solution having dissolved is dropped in BSA solution, and stir and spend the night at 2～8 ℃, obtain antigen; Synthetic antigen, through dialysis purifying, is obtained to 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, then mixes with Freund's complete adjuvant with antigenic solution, rabbit is injected;
After (2) 2～3 weeks, then after mixing with incomplete Freund's adjuvant with the identical antigenic solution of 1.0ml, rabbit is injected once, afterwards every surrounding 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 R1 damping fluid, homogeneous phase R1 damping fluid is containing 50mM Tris, 0.25%BSA, 50Mm G-6-P and 50mM NAD by the antibody dilution preparing.The volume ratio of antibody and R1 damping fluid is 1: 1000.
(2) preparation of R2 reagent
1) preparation of G6PDH-6MMP
A) take 15mg G6PDH, be dissolved in 12ml, in 0.05M Tris damping fluid, add successively the DMF of 100mg NADH, 0.5ml carbitol and 1ml to mix;
B) the 6-MMP derivant of 10mg is dissolved in 420 μ l dimethyl sulfoxide (DMSO)s and 180 μ l DMF, add 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) under 2～8 ℃ of conditions, stir and spend the night subsequently, and the G6PDH-6MMP obtaining is carried out to purifying.
2) G6PDH-6MMP preparing is diluted in R2 damping fluid.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
By automatic clinical chemistry analyzer, carry out parameter setting according to table 1 data.First add sample, add again the R1 reagent (mixed liquor of antibody and R1 damping fluid) in embodiment 2, finally add R2 reagent (mixed liquor of G6PDH-6MMP conjugate and R2 damping fluid), 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, result as shown in Figure 1.
The preparation of the ELISA testing reagent of embodiment 4 6-MMP
(1) contain anti-6-MMP specific antibody prepared by embodiment 1
(2) contain HRP-6MMP conjugate and substrate.
The preparation method of enzyme mark conjugate:
A) take 20mg HRP and be dissolved at ambient temperature 5ml 0.2M, in the phosphate buffer of pH 8.5;
B) activation 6-MMP derivant: take 10mg 6-MMP derivant in small beaker, and add successively 350 μ L DMF, 350 μ L absolute ethyl alcohols, 700 μ L 10mM, kaliumphosphate buffer, 40mg EDAC and the 5mg Sulfo-NHS of pH 5.0, stirring reaction 30min at ambient temperature;
C) subsequently the 6-MMP derivant of activation is added drop-wise in HRP enzyme solutions, under 2-8 ℃ of condition, stirs and spend the night, and the conjugate purifying of dialysing is obtained to HRP-6-MMP conjugate.
The embodiment 5 6-MMP ELISA method of inspection and the calibration results
(1) 6-MMP ELISA method of inspection step
1) will resist 6-MMP antibody dilution to become the final concentration solution of 1: 5000 with PBS, 100 μ L/ holes are coated on 96 hole elisa plates, and 4 ℃ are spent the night;
2) with after 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) add the standard items in 20 μ L/ holes;
4) add the HRP-6-MMP conjugate of 100 μ L/ hole working concentrations;
5) under room temperature, hatch 30min, 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 results as shown in Figure 2.
The object of this recovery experiment is to determine that described 6-MMP homogeneous enzyme immunoassay detection method can be for the detection of 6-MMP in whole blood sample.
1. the calibration curve of checking by the homogeneous enzyme immunoassay of 6-MMP, replication blank, basic, normal, high concentration serum (are dissolved in 6-MMP standard items in blank whole blood, be respectively 0 to final concentration, 0.6,2.0,4.0 μ g/mL) 3 times, the recovery high (> 95%) of results sample.
2. assay method: as described in the homogeneous enzyme immunoassay method of the 6-MMP in embodiment 3, the results are shown in 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%, and the 6-MMP homogeneous enzyme immunoassay detection method described in illustrate can be for the detection of 6-MMP in whole blood sample, and result is accurate, credible.
Embodiment 7 applies 6-MMPELISA and detects the recovery test that carries out 6-MMP in serum
The object of this recovery experiment is to determine that described 6-MMP ELISA detection method can be for the detection of 6-MMP in whole blood sample.
1. the calibration curve of checking by the ELISA of 6-MMP, replication blank, basic, normal, high concentration serum (are dissolved in 6-MMP standard items in blank whole blood, be respectively 0 to final concentration, 0.6,2.0,4.0 μ g/mL) 3 times, the recovery high (> 90%) of results sample.
2. assay method: as described in the ELISA method of the 6-MMP in embodiment 4, the results are shown in 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%, and the 6-MMP ELISA detection method described in illustrate can be for the detection of 6-MMP in whole blood sample, and result is accurate, 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 (7)
1. the homogeneous enzyme immunoassay detection method of 6-(first sulfydryl) purine (6-MMP), use the homogeneous enzyme immunoassay testing reagent of 6-(first sulfydryl) purine, this detection reagent is made up of R1 reagent and R2 reagent, comprises following operation steps:
(1) adopt automatic clinical chemistry analyzer, sample to be tested and 6-(first sulfydryl) purine detection reagent are put into respectively to sample storehouse and agent bin;
(2) carry out the setting of test item parameter according to following table;
(3) light absorption value of mensuration OD340nm;
(4) production standard curve carrys out 6-(first sulfydryl) the purine concentration in quantitative sample;
Wherein:
The preparation of R1 reagent: will resist 6-MMP specific antibody to be diluted in homogeneous phase R1 damping fluid, homogeneous phase R1 damping fluid is containing 50mM Tris, 0.25%BSA, 50mM G-6-P and 50mM nicotine adenine-dinucleotide, the volume ratio of described 6-MMP specific antibody and R1 damping fluid is 1: 1000-1: 3000;
The preparation of R2 reagent: enzyme-6MMP conjugate is diluted in R2 damping fluid, and R2 damping fluid is containing 100mM Tris and 0.25% bovine serum albumin, and the volume ratio of described enzyme-6MMP conjugate and R2 damping fluid is 1: 1000-1: 3000;
Being prepared as follows of described enzyme-6MMP conjugate:
A. enzyme solutions preparation: take enzyme, it is selected from beta galactosidase or glucose-6-phosphate dehydrogenase (G6PD), is dissolved at ambient temperature in phosphate buffer, and final concentration is 2-6mg/mL;
B.6-MMP the activation of derivant and conjugate is synthetic: the 6-MMP derivant that dissolves structural formula (II) with DMF, making its final concentration is 10-50mg/mL, activate by tri-n-butylamine method, and carry out cross-linking reaction with enzyme solutions, purified and dialysis after obtain 6-MMP enzyme mark conjugate;
Described 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):
Formula (I)
In formula, R is-(CH
2)
5-COO-, carrier is bovine serum albumin,
Described 6-MMP immunogen synthesis method is as follows:
(1) 6-MMP derivant is synthetic, and its chemical constitution is suc as formula shown in (II):
Formula (II)
1) dissolve 5.0g6-MMP and 4.45g K with 30ml DMF
2cO
3, add 10ml to contain the DMF solution of 4.06g6-bromocaproic acid second fat, stirring reaction 30min under 40 ℃ of conditions;
2) will react subsequently the neutralization of rear solution 1N HCl solution, and be extracted with ethyl acetate, organic phase drying, filtration, vacuum drying and purification on normal-phase silica gel purifying, with 25ml methyl alcohol dissolving 1.5g purified product, add the LiOH.H containing 600mg subsequently
2the aqueous solution of O, stirring at room temperature reaction 4 hours, product is dilute with water, ether washing after concentrated, and aqueous phase solution is adjusted pH to 5.0 with 1N HCl solution, finally uses dichloromethane extraction, and organic phase is through Na
2sO
4after being dried, filtering and concentrating, obtain white solid 6-MMP derivant;
(2) 6-MMP is immunogenic synthetic
1) 200mg bovine serum albumin (BSA) is dissolved in to 50ml 0.2M, in the phosphate buffer of pH8.5;
2) following chemicals is joined to the synthetic 6-MMP derivant of stirring and dissolving in small beaker: 200mg, 3.5ml DMF, 3.5ml ethanol, 7.0ml 10mM, the kaliumphosphate buffer of pH5.0,200mg EDAC, 50mg N-hydroxy-succinamide (N-hydroxysuccinimide, Sulfo-NHS), by its stirring and dissolving, at room temperature react 30min;
3) solution having dissolved is dropped in BSA solution, and stir and spend the night at 2～8 ℃, obtain antigen; Synthetic antigen, through dialysis purifying, is obtained to 6-MMP immunogene.
2. the homogeneous enzyme immunoassay detection method of 6-according to claim 1 (first sulfydryl) purine, wherein step (2) can also realize by following scheme:
。
3. the ELISA detection method of a 6-(first sulfydryl) purine, use 6-(first sulfydryl) purine ELISA testing reagent, this detection reagent contains: (1) anti-6-(first sulfydryl) purine (6-MMP) specific antibody; (2) enzyme mark conjugate and substrate, comprises following operation steps:
1) will resist 6-MMP specific antibody to be diluted to 1 with PBS: 2000-1: 10000 final concentration solution, 100u L/ hole is coated on 96 hole elisa plates, and 4 ℃ are spent the night;
2) with after 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) add the standard items in 20 μ L/ holes;
4) add 6-(first sulfydryl) the purinase mark conjugate of 100 μ L/ hole working concentrations;
5) under room temperature, hatch 30min, PBS washes plate 5 times;
6) every hole adds 100 μ L TMB, incubated at room 30min;
7) every hole adds 100 μ L2M sulfuric acid;
8) light absorption value of mensuration 450nm;
Described 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):
Formula (I)
In formula, R is-(CH
2)
5-COO-, carrier is bovine serum albumin;
Described 6-MMP immunogen synthesis method is as follows:
(1) 6-MMP derivant is synthetic, and its chemical constitution is suc as formula shown in (II):
Formula (II)
A). with 30ml DMF dissolving 5.0g6-MMP and 4.45g K
2cO
3, add 10ml to contain the DMF solution of 4.06g6-bromocaproic acid second fat, stirring reaction 30min under 40 ℃ of conditions;
B). 1N HCl solution neutralization for solution after reacting subsequently, and be extracted with ethyl acetate, organic phase drying, filtration, vacuum drying and purification on normal-phase silica gel purifying, with 25ml methyl alcohol dissolving 1.5g purified product, add the LiOH.H containing 600mg subsequently
2the aqueous solution of O, stirring at room temperature reaction 4 hours, product is dilute with water, ether washing after concentrated, and aqueous phase solution is adjusted pH to 5.0 with 1N HCl solution, finally uses dichloromethane extraction, and organic phase is through Na
2sO
4after being dried, filtering and concentrating, obtain white solid 6-MMP derivant;
(2) 6-MMP is immunogenic synthetic
A). 200mg bovine serum albumin (BSA) is dissolved in to 50ml 0.2M, in the phosphate buffer of pH8.5;
B). following chemicals is joined to the synthetic 6-MMP derivant of stirring and dissolving in small beaker: 200mg, 3.5ml DMF, 3.5ml ethanol, 7.0ml 10mM, the kaliumphosphate buffer of pH5.0,200mg EDAC, 50mg N-hydroxy-succinamide (N-hydroxysuccinimide, Sulfo-NHS), by its stirring and dissolving, at room temperature react 30min;
C). the solution having dissolved is dropped in BSA solution, and stir and spend the night at 2～8 ℃, obtain antigen; Synthetic antigen, through dialysis purifying, is obtained to 6-MMP immunogene;
The enzyme of described enzyme mark conjugate is that horseradish peroxidase (HRP), substrate are TMB (TMB), and described enzyme mark conjugate is obtained by following preparation method:
A) take enzyme and be dissolved at ambient temperature in phosphate buffer, final concentration is 2-6mg/ml;
B) be connected to the 6-MMP derivant of R linking group with organic solvent dissolution chain, final concentration is 10-50mg/ml, activates, and carry out cross-linking reaction with enzyme solutions by the method for EDAC, after dialysis purifying, obtain 6-MMP enzyme mark conjugate, described organic solvent is DMF.
4. according to the detection method described in claim 2 or 3, wherein said antibody is polyclonal antibody or monoclonal antibody, and described animal is selected from rabbit, goat, mouse, sheep, the one of cavy or Malaysia and China.
5. detection method according to claim 4, described animal is rabbit.
6. according to the detection method described in claim 2 or 3, the preparation method of wherein said anti-6-(first sulfydryl) purine specific antibody comprises the following steps:
(1) with phosphate buffer, synthetic 6-(first sulfydryl) purine immunogene is diluted to 0.5-5.0mg/mL;
(2) through conventional Freund's adjuvant method, animal is injected, after injection, extract animal specific corrosioning anteserum, obtain effective antibody.
7. according to the detection method described in claim 2 or 3, it can be used for the detection of 6-MMP in whole blood sample.
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