CN104569373B - A kind of methotrexate homogeneous enzyme immunoassay detectable and preparation thereof and detection method - Google Patents
A kind of methotrexate homogeneous enzyme immunoassay detectable and preparation thereof and detection method Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
Abstract
The present invention relates to a kind of methotrexate detectable and preparation thereof and detection method, it is specifically related to a kind of methotrexate homogeneous enzyme immunoassay detectable and preparation thereof and detection method, including: anti-methotrexate specific antibody, for detecting the indicator of anti-methotrexate specific antibody methotrexate complex；Above-mentioned anti-methotrexate specific antibody is obtained by methotrexate immunogen immune animal.The invention have benefit that: the methotrexate immunogens of the present invention is strong, immunogenicity is high, anti-methotrexate 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-methotrexate specific antibody can easily and fast, the methotrexate content that accurately determines in sample, and multiple sample can be measured on automatic clinical chemistry analyzer simultaneously, realize the rapid mensuration of high flux of methotrexate, accuracy is high, high specificity, degree of accuracy and detection efficiency are enhanced.
Description
Technical field
The present invention relates to a kind of methotrexate detectable and preparation thereof and detection method, be specifically related to a kind of methotrexate
Homogeneous enzyme immunoassay detectable and preparation thereof and detection method.
Background technology
Methotrexate (Methotrexate) structural formula is as shown in formula III:
Methotrexate is a kind of folic acid reductase inhibitor, for anti-folic acid series antineoplastic medicament, mainly by dihydro leaf
The inhibitory action of acid reductase hinders the synthesis of DNA of tumor cell, thus suppresses growth and the breeding of tumor cell.Its selectivity
Act on the S phase, belong to cell cycle specific agents.In acute leukemia, (especially acute lymphocytic is white clinically
Disorders of blood), the aspect therapeutic effect such as chorionic epithelioma and malignant mole preferable.To tumor of head and neck, breast carcinoma, pulmonary carcinoma and basin
Chamber tumor all has certain curative effect, it is possible to other drug therapeutic alliance Burkitts lymphoma, late period lymphosarcoma (III and IV
Phase, PeterShi stage system) and mycosis fungoides in late period.Methotrexate safety range is narrow, body metabolism and excretion after taking medicine
Individuation difference is big, cannot suppress tumor growth less than its effective blood drug concentration, then can cause stomach higher than its effective blood drug concentration
The side effect such as intestinal reaction, liver cirrhosis, kidney damage.This medicine cytotoxicity is relatively big, and untoward reaction is more, should closely monitor patient
Blood drug level, accomplishes individual administration.Therefore, the patient taking methotrexate is carried out therapeutic drug monitoring, bad to reducing
React and instruct clinical individual safe medication significant.
At present, the method measuring methotrexate mainly has high performance liquid chromatography (HPLC), fluorescence polarization immunoassay
(FPIA) etc., HPLC method complex operation, it is mainly used in lab analysis, FPIA method needs to be equipped with expensive instrumentation, cost
Higher.Therefore, these methods all have certain defective in clinical practice.Although external producer can be applicable to biochemistry
The methotrexate of analyser measures test kit listing, but product quantity is far from meeting clinical demand.The most still lack
Good stability, highly sensitive, the methotrexate detectable of high specificity, especially matter measured Automated inspection reagent.Cause
This, development & production quality reaches clinical requirement, practical, cost performance is high, can be applicable to the first ammonia of automatic clinical chemistry analyzer
Pterin measures reagent has become the focus of domestic and international external diagnosis reagent industry.The methotrexate homogeneous enzyme immunoassay detection of the present invention
Reagent can be implemented in high flux, the rapid detection on automatic clinical chemistry analyzer to methotrexate, and have easy and simple to handle,
The advantages such as highly sensitive, high specificity, result are accurate, moreover it is possible to effectively reduce methotrexate testing cost, beneficially clinical expansion
Use.
Summary of the invention
For solving the deficiencies in the prior art, it is an object of the invention to provide one not only safety but also can be quick, efficient, clever
Quick, methotrexate homogeneous enzyme immunoassay detectable accurately detecting methotrexate content in sample to be tested and preparation method thereof,
And can be combined with various types of automatic biochemistry analyzers, methotrexate homogeneous enzyme immunoassay less demanding to testing staff
Detection method.
Another object of the present invention is for providing a kind of first contributing to preparing mensuration methotrexate content rapidly and accurately
The methotrexate derivatives of aminopterin homogeneous enzyme immunoassay detectable.
In order to realize above-mentioned target, the technical solution used in the present invention is:
A kind of methotrexate homogeneous enzyme immunoassay detectable, it is characterised in that including: anti-methotrexate specific antibody,
For detecting the indicator of anti-methotrexate specific antibody-methotrexate complex；Above-mentioned anti-methotrexate specificity resists
Body is obtained by methotrexate immunogen immune animal, and the immunogenic structural formula of methotrexate is as shown in formula I:
In formula, carrier for having immunogenic protein, preferably serum albumin, hemocyanin or thyroid ball egg
In vain；Above-mentioned indicator is selected from enzyme reagent, radiosiotope reagent, fluorometric reagent or chemical illuminating reagent.
Aforesaid methotrexate homogeneous enzyme immunoassay detectable, above-mentioned indicator is selected from enzyme reagent, including: enzyme mark coupling
Thing and the substrate of enzyme；Above-mentioned enzyme mark conjugate includes glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark conjugate；Above-mentioned enzyme
Substrate is G-6-P.
Aforesaid methotrexate homogeneous enzyme immunoassay detectable, above-mentioned glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark is even
Connection thing is reacted with methotrexate derivatives by glucose-6-phosphate dehydrogenase (G6PD) and is formed, the structural formula of above-mentioned methotrexate derivatives
As shown in formula II:
Methotrexate derivatives, structural formula is as shown in formula II:
Its synthetic route is:
The preparation method of a kind of methotrexate homogeneous enzyme immunoassay detectable, it is characterised in that comprise the steps:
(1) synthesis of methotrexate derivatives and purification, and carry out Structural Identification；
(2) the immunogenic synthesis of methotrexate: make the terminal carboxyl group of methotrexate carry with having immunogenic protein
Body connects；
(3) with methotrexate immunogen immune animal, preparation purification anti-methotrexate specific antibody；
(4) preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing: prepare glucose-6-phosphate dehydrogenase (G6PD) molten
Liquid, prepares methotrexate derivatives solution, makes glucose-6-phosphate dehydrogenase (G6PD) be connected with the terminal carboxyl group of methotrexate, purification
Connect product；
(5) preparation of methotrexate homogeneous enzyme immunoassay detectable:
The preparation of reagent A: mixed by anti-methotrexate specific antibody and homogeneous zymolyte；The preparation of reagent B: by
Glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing mixes with Tris buffer.
The preparation method of aforesaid a kind of methotrexate homogeneous enzyme immunoassay detectable, in described step (2), protein
Carrier is BSA, and concrete synthesis step is as follows:
A. weigh 2.72g potassium dihydrogen phosphate, 4.26g disodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, jointly dissolve
In 1L deionized water, regulate pH to 7.4, make buffer solution A；
B. weigh and be dissolved under 3mg BSA, room temperature in 3mL above-mentioned buffer solution A, make BSA solution；
C. weigh 3mg methotrexate derivatives, be dissolved in 300ul above-mentioned buffer solution A, make methotrexate derivatives
Solution；
D. when above-mentioned methotrexate derivatives solution just becomes clarification, it is added dropwise in above-mentioned BSA solution, then will
This mixed solution stirs 1 hour at 2-8 DEG C；
E. being dialysed with above-mentioned buffer solution A by reacted above-mentioned mixed solution, after dialysis, gained solution is first
Aminopterin immunogen solution, adds the NaN of mass fraction 0.1% in methotrexate immunogen solution3, store at-20 DEG C.
The preparation method of aforesaid a kind of methotrexate homogeneous enzyme immunoassay detectable, described step (4) detailed process
For:
A. weigh 1.09g potassium dihydrogen phosphate, 1.70g disodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, jointly dissolve
In 1L deionized water, regulate pH to 7.4, make buffer solution B；
B. weigh and be dissolved under 3mg glucose-6-phosphate dehydrogenase (G6PD), room temperature in 3mL above-mentioned buffer solution B, make Fructus Vitis viniferae
Sugar-6-phosphate dehydrogenase enzymatic solution；
C. weigh 3mg methotrexate derivatives, be dissolved in 300ul above-mentioned buffer solution B, make methotrexate derivatives
Solution；
D., when above-mentioned methotrexate derivatives solution just becomes clarification, it is added dropwise over above-mentioned G-6-P and is taken off
In hydrogen enzymatic solution, then this mixed solution is stirred 1 hour at 2-8 DEG C；
E. being dialysed with above-mentioned buffer solution B by reacted above-mentioned mixed solution, after dialysis, gained solution is Portugal
Glucose-6-phosphate dehydrogenase-hapten conjugation thing solution, adds in glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution
Enter the BSA and the NaN of mass fraction 0.1% of mass fraction 0.5%3, store at 2-8 DEG C.
The preparation method of aforesaid a kind of methotrexate homogeneous enzyme immunoassay detectable, the detailed process of step (5) is as follows:
The preparation of reagent A: by the nicotinamide adenine dinucleotide (NAD) of 4.036g (11.25mM) oxidation state, 1.711g
(11.25mM) G-6-P (G-6-P) the Tris buffer solution of 1L 55mM, pH=8.0 makes homogeneous zymolyte；
The anti-methotrexate specific antibody of preparation being added in above-mentioned homogeneous zymolyte, antibody is 1 with the volume ratio of homogeneous zymolyte:
100～1:10000；
The preparation of reagent B: the glucose-6-phosphate dehydrogenase (G6PD) of preparation-hapten conjugation thing is added to 120mM, pH=8.2
Tris buffer in, the volume ratio of above-mentioned conjugate and Tris buffer is 1:100～1:10000.
Utilize the detection method of methotrexate homogeneous enzyme immunoassay detectable, it is characterised in that comprise the following steps:
1) sample to be tested is contacted with anti-methotrexate specific antibody；
2) according to the combination situation of methotrexate in sample to be tested Yu anti-methotrexate specific antibody, indicator is utilized
The content of methotrexate in judgment sample；
Described sample to be tested is serum, blood plasma, saliva or urine.
Above-mentioned enzyme reagent includes: enzyme mark conjugate and the substrate of enzyme；Above-mentioned enzyme mark conjugate includes that G-6-P takes off
Hydrogen enzyme-hapten enzyme mark conjugate；The substrate of above-mentioned enzyme is G-6-P；
Above-mentioned glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark conjugate is by glucose-6-phosphate dehydrogenase (G6PD) and first ammonia butterfly
Purine derivatives reaction is formed, and the structural formula of above-mentioned methotrexate derivatives is as shown in formula II:
The invention have benefit that: the methotrexate immunogens of the present invention is strong, immunogenicity is high, prepares
Anti-methotrexate specific antibody high specificity, titer high, and with 62 kinds of common medicines without any cross reaction；Contain
The homogeneous enzyme immunoassay detectable of above-mentioned anti-methotrexate specific antibody can easily and fast, accurately determine in sample
Methotrexate content, and multiple sample can be measured on automatic clinical chemistry analyzer simultaneously, it is achieved the high pass of methotrexate
Measuring rapid mensuration, accuracy is high, and high specificity, degree of accuracy is all enhanced before comparing with detection efficiency, simultaneously
Achieve the full-automation of detection process, less demanding to testing staff, it is easy to accomplish and promote the use of.
Accompanying drawing explanation
Fig. 1 is methotrexate homogeneous enzyme immunoassay reaction normal curve chart；
Fig. 2 is methotrexate homogeneous enzyme immunoassay correlation analysis figure.
Detailed description of the invention
The technical solution used in the present invention is:
Methotrexate immunogen, its structural formula is as shown in formula I:
In formula, carrier has immunogenicity, it is preferred that carrier is for having immunogenic protein.Although other are enough
Big possess immunogenic material and as carrier, but protein can also be selected under normal circumstances as carrier.The most frequently used
Immunogenic carrier includes serum albumin, hemocyanin (KLH) and Elityran.Carrier in the present invention is preferably serum
Albumen.
A kind of anti-methotrexate specific antibody, is obtained by the methotrexate immunogen immune animal shown in formula I.
In the present invention, " antibody " of indication refers not only to complete antibody molecule, also includes retaining complete antibody specificity knot
The antibody fragment of conjunction ability or derivant.The antibody of the present invention can be polyclonal antibody can also be monoclonal antibody, excellent
Elect polyclonal antibody as.
The method obtaining polyclonal antibody is to use the methotrexate immunogen shown in formula I, is adding or is being not added with adjuvant
After, carrying out immunity at one or more position of animal, host animal includes: rabbit, goat, mice, sheep, Cavia porcellus or horse.
Persistent immunological is carried out always, until antibody titer reaches the highest.Animal timing blood sampling obtains appropriate specific corrosioning anteserum, antiserum
Can be with purification.
Monoclonal antibody can be made by somatocyte hybriding technology.
A kind of methotrexate homogeneous enzyme immunoassay detectable, including: above-mentioned anti-methotrexate specific antibody, is used for detecting
The indicator of anti-methotrexate specific antibody-methotrexate complex.Indicator is selected from enzyme reagent, radiosiotope
Reagent, fluorometric reagent and chemical illuminating reagent.Preferably, indicator is enzyme reagent, including: enzyme mark conjugate and the end of enzyme
Thing.Wherein, enzyme mark conjugate includes glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark conjugate, and it can pass through chemosynthesis side
Method obtains.
The using method of above-mentioned methotrexate homogeneous enzyme immunoassay detectable, comprises the following steps:
1) sample to be tested is contacted with above-mentioned anti-methotrexate specific antibody；
2) according to the combination situation of methotrexate in sample to be tested Yu above-mentioned anti-methotrexate specific antibody, instruction is utilized
The content of methotrexate in reagent judgment sample.
Sample to be tested is various physiology samples, such as serum, blood plasma, urine, saliva etc..Preferably, sample to be tested is blood
Clear or blood plasma.
Below in conjunction with specific embodiment, further illustrate the present invention.
Embodiment one: the synthesis of methotrexate derivatives and structural confirmation thereof
The methotrexate derivatives chemical constitution used in following example is as shown in formula II:
The synthetic route of this methotrexate derivatives is as follows:
Concrete synthesis step is as follows:
The synthesis of compound 2
Weigh 5.0g compound 1, be dissolved in the MeOH of 50mL, at 0 DEG C, be added dropwise over 4.0g (34.1mmol) SOCl2,
Then this reaction mixture is stirred overnight at 70 DEG C, makes solvent evaporate by decompression method, by residue by 200mL's
The NaHCO of DCM and 200mL3Saturated aqueous solution separates, and is rinsed by organic facies salt, then passes through Na2SO4Do
Dry, and carry out being concentrated to give 5.1g compound as white solid 2, productivity 96%.
The synthesis of compound 3
Weigh 5.1g compound 2, be dissolved in the MeOH of 100mL, at 0 DEG C, add 1.3g (33.3mmol) several times
NaBH4, then this reaction mixture is stirred at room temperature overnight, reacted mixture is concentrated, obtains after concentration
Residue be purified by silica dehydrator post (DCM:MeOH=10:1), obtain the compound 3 of 4.0g colorless oil, productivity
92%.
The synthesis of methotrexate derivatives
Weigh 2.5g compound 3, be dissolved in the THF of 40mL, under 0 DEG C and nitrogen protective condition, add 1.2g
(12.6mmol) maleimide, 3.8g (14.56mmol) PPh3And 2.9g (14.56mmol) DIAD, then by mixed for this reaction
Closing liquid to be stirred overnight at 70 DEG C, concentrated by reacted mixture, the residue obtained after concentration passes through silica dehydrator post
(DCM:MeOH=50:1) it is purified, finally gives 500mg tan solid Compound 4, productivity 15%.This compound 4 is i.e.
For the methotrexate derivatives shown in formula II.
The immunogenic synthesis of embodiment two: BSA-methotrexate
BSA-methotrexate immunogen is by the terminal carboxyl group of bovine serum albumin (BSA) with the methotrexate shown in formula III
Being formed by connecting, this immunogenic concrete synthesis step is as follows:
A. weigh 2.72g potassium dihydrogen phosphate, 4.26g disodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, jointly dissolve
In 1L deionized water, regulate pH to 7.4, make buffer solution A；
B. weigh and be dissolved under 3mg BSA, room temperature in 3mL above-mentioned buffer solution A, make BSA solution；
C. weigh 3mg methotrexate derivatives, be dissolved in 300ul above-mentioned buffer solution A, make methotrexate derivatives
Solution；
D. when above-mentioned methotrexate derivatives solution just becomes clarification, it is added dropwise in above-mentioned BSA solution, then will
This mixed solution stirs 1 hour at 2-8 DEG C；
E. being dialysed with above-mentioned buffer solution A by reacted above-mentioned mixed solution, after dialysis, gained solution is first
Aminopterin immunogen solution, adds the NaN of 0.1% in methotrexate immunogen solution3, store at-20 DEG C.0.1%
NaN3Referring to that addition accounts for the mass percent of the immunogen solution of final gained, concrete addition is according to gained after dialysis
Depending on the concrete quality of immunogen solution.
Embodiment three: the preparation of anti-methotrexate specific antibody
Above-mentioned prepared BSA-methotrexate immunogen is used conventional method inoculation experiments animal rabbit, takes after booster immunization
Antiserum, specifically comprises the following steps that
With PBS, the BSA-methotrexate immunogen of above-mentioned synthesis is diluted to 1.0mg/ml, obtains antigenic solution, then use
1.0ml antigenic solution mixes with Freund's complete adjuvant, injects experimental animal rabbit.
After 2～3 weeks, more above-mentioned experimental animal rabbit is injected with incomplete Freund's adjuvant with antigenic solution identical for 1.0ml
Once, inject once every surrounding afterwards, amount to injection 4 times.
Above-mentioned experimental animal rabbit takes blood, and isolated and purified to obtain the anti-methotrexate that titer is 1: 30000-1: 50000 special
Heterogenetic antibody.
Embodiment four: the preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing
A. weigh 1.09g potassium dihydrogen phosphate, 1.70g disodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, jointly dissolve
In 1L deionized water, regulate pH to 7.4, make buffer solution B；
B. weigh and be dissolved under 3mg glucose-6-phosphate dehydrogenase (G6PD), room temperature in 3mL above-mentioned buffer solution B, make Fructus Vitis viniferae
Sugar-6-phosphate dehydrogenase enzymatic solution；
C. weigh 3mg methotrexate derivatives, be dissolved in 300ul above-mentioned buffer solution B, make methotrexate derivatives
Solution；
D., when above-mentioned methotrexate derivatives solution just becomes clarification, it is added dropwise over above-mentioned G-6-P and is taken off
In hydrogen enzymatic solution, then this mixed solution is stirred 1 hour at 2-8 DEG C；E. by reacted above-mentioned mixed solution with above-mentioned
Buffer solution B dialyses, and after dialysis, gained solution is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution, in Portugal
Glucose-6-phosphate dehydrogenase-hapten conjugation thing solution adds the NaN of the BSA and 0.1% of 0.5%3, store up at 2-8 DEG C
Deposit.The BSA of the 0.5% and NaN of 0.1%3Refer to that addition accounts for the mass percent of the conjugate solution of final gained, concrete
Depending on addition is according to the concrete quality of the conjugate solution of gained after dialysis.
Embodiment five: the preparation of methotrexate homogeneous enzyme immunoassay detectable
Methotrexate homogeneous enzyme immunoassay detectable, including: above-mentioned anti-methotrexate specific antibody, it is used for detecting anti-first
The indicator of aminopterin specific antibody-methotrexate complex.Indicator is selected from enzyme reagent, radiosiotope examination
Agent, fluorometric reagent and chemical illuminating reagent.Preferably, indicator is enzyme reagent, including: enzyme mark conjugate and the substrate of enzyme.
Wherein, enzyme mark conjugate includes glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark conjugate, and it is by above-mentioned chemosynthesis side
Method obtains.
Methotrexate homogeneous enzyme immunoassay detectable before the use, in order to avoid the enzyme mark conjugate in indicator and
The substrate of enzyme reacts, and the substrate of enzyme mark conjugate and enzyme is separated, does not mixes, so by the substrate of enzyme with above-mentioned
Anti-methotrexate specific antibody mixes.It is to say, methotrexate homogeneous enzyme immunoassay detectable includes two kinds points
Offer the reagent put, specific as follows:
1. the preparation of reagent A: by the nicotinamide adenine dinucleotide (NAD) of 4.036g (11.25mM) oxidation state,
1.711g (11.25mM) G-6-P (G-6-P) is placed in beaker D, with the Tris buffer of 1L 55mM, pH=8.0
Homogeneous zymolyte is made in dissolving；The anti-methotrexate specific antibody of above-mentioned preparation is added in above-mentioned homogeneous zymolyte, antibody
Can be 1:100～1:10000 with the volume ratio of homogeneous zymolyte, the most concrete ratio be 1:400.
2. the preparation of reagent B: the glucose-6-phosphate dehydrogenase (G6PD) of above-mentioned preparation-hapten conjugation thing is added to 120mM,
In the Tris buffer of pH=8.2, above-mentioned conjugate can be 1:100～1:10000 with the volume ratio of Tris buffer, at this
Ratio concrete in embodiment is 1:1500.
The using method of above-mentioned methotrexate homogeneous enzyme immunoassay detectable, comprises the following steps:
1) sample to be tested is contacted with above-mentioned anti-methotrexate specific antibody；
2) according to the combination situation of methotrexate in sample to be tested Yu above-mentioned anti-methotrexate specific antibody, instruction is utilized
Reagent judges the content of methotrexate in sample to be tested.
Concrete, during detection, sample to be tested is added in reagent A, the methotrexate in sample to be tested and resisting in reagent A
Methotrexate specific antibody occurs specific binding, generates anti-methotrexate specific antibody-methotrexate complex；Add again
Enter reagent B, now the glucose-6-phosphate dehydrogenase (G6PD) in reagent B-hapten conjugation thing mix with the substrate of the enzyme in reagent A,
, there is enzymatic reaction in contact, constitutes the indicator detecting anti-methotrexate specific antibody-methotrexate complex, instruction
Reagent judges in sample to be tested according to the combination situation of methotrexate in sample to be tested Yu above-mentioned anti-methotrexate specific antibody
The content of methotrexate.
Due to glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing and the methotrexate competitive binding in sample to be tested
Anti-methotrexate specific antibody, so, in sample to be tested, the amount of methotrexate is the most, Fructus Vitis viniferae free in homogeneous enzymatic solution
The amount of sugar-6-phosphate dehydrogenase-hapten conjugation thing is the most, and enzymatic reaction is the fastest, causes OD340Rise.
Above-mentioned sample to be tested is physiology sample, such as serum, blood plasma, urine, saliva etc..
As the preferred scheme of one, above-mentioned sample to be tested is serum or blood plasma.
Embodiment six: methotrexate homogeneous enzyme immunoassay is checked
1, obtain standard curve: auspicious BS200 automatic clinical chemistry analyzer response parameter (being shown in Table 1) advanced in years, operating process are set
For: first reagent adding A, add standard substance, be eventually adding reagent B.After adding reagent B, measure the OD of different time points340Extinction
Value, calculates reaction rate during various criterion product concentration, needs constantly to adjust reagent A and the volume of reagent B in actual mechanical process
Ratio, adjusts light-metering point simultaneously, finally draws comparatively ideal reaction normal curve chart, as shown in Figure 1.
Table 1 steps auspicious BS200 automatic clinical chemistry analyzer response parameter
The standard curve obtained by the homogeneous enzyme immunoassay detectable of the present invention, replication basic, normal, high concentration Quality Control
Sample 10 times, above-mentioned Quality Control sample is: be dissolved in human serum by methotrexate standard substance, to concentration be respectively 0.25,1.00,
2.00μmol/L.Detection data and data analysis are shown in Table 2.
Table 2 sample determination and precision and the response rate are assessed
| Blood sample | Low | In | High |
| Sample concentration (μm ol/L) | 0.25 | 1.00 | 2.00 |
| 1 | 0.24 | 1.06 | 1.90 |
| 2 | 0.26 | 1.05 | 2.05 |
| 3 | 0.25 | 0.98 | 2.11 |
| 4 | 0.25 | 0.93 | 1.99 |
| 5 | 0.26 | 1.03 | 2.07 |
| 6 | 0.24 | 0.97 | 1.92 |
| 7 | 0.24 | 1.06 | 2.01 |
| 8 | 0.26 | 1.00 | 1.88 |
| 9 | 0.23 | 1.03 | 2.13 |
| 10 | 0.25 | 0.96 | 1.91 |
| Meansigma methods (μm ol/L) | 0.25 | 1.01 | 2.00 |
| Standard deviation (SD) | 0.0103 | 0.0457 | 0.0915 |
| Precision (CV%) | 4.12 | 4.52 | 4.58 |
| Response rate % | 100.0 | 101.0 | 100.0 |
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 5%.
Embodiment seven: interfering effects of drug is tested
Choosing 62 kinds of Common drugs, adjustment concentration, to 10.0 μm ol/L, carries out interference test mensuration.62 kinds of common medicines
And measurement result is referring specifically to table 3.
Table 3 common interference medicine and measurement result
Measurement result: the concentration that above-mentioned 62 kinds of Common drugs are equivalent to methotrexate is respectively less than 0.1 μm ol/L.Visible, this
The antibody of invention is the specific antibody of anti-methotrexate.
Embodiment eight: correlation analysis
100 example clinical samples are used respectively the methotrexate detectable of De Ling diagnostic products (Shanghai) Co., Ltd.
(using enzyme to amplify immunoassay) and the homogeneous enzyme immunoassay reagent of the present invention carry out correlation analysis, and the data of mensuration see table
4。
Table 4 clinical sample measured value
Mapping above-mentioned data, see Fig. 2, the linear equation obtained is: y=0.9953x+0.0095, coefficient R2
=0.9970, show that the detectable of the present invention measures the accuracy height of methotrexate clinical samples.
Owing to the detection process of the present invention is to be completed by instrument full-automation, so less demanding, easily to testing staff
In realizing and promoting the use of.
It should be noted that the foregoing is only embodiments of the invention, not thereby limit the scope of the claims of the present invention,
Every equivalent structure utilizing description of the invention and accompanying drawing content to be done or equivalence flow process conversion, or be directly or indirectly used in
Other correlative technology fields, are the most in like manner included in the scope of patent protection of the present invention.
Claims (7)
1. a methotrexate homogeneous enzyme immunoassay detectable, it is characterised in that including: anti-methotrexate specific antibody, use
In the indicator detecting anti-methotrexate specific antibody-methotrexate complex；Above-mentioned anti-methotrexate specific antibody
Being obtained by methotrexate immunogen immune animal, the immunogenic structural formula of methotrexate is as shown in formula I:
Formula I
In formula, carrier is for having immunogenic protein；
Above-mentioned indicator is selected from enzyme reagent, including: enzyme mark conjugate and the substrate of enzyme；Above-mentioned enzyme mark conjugate includes Fructus Vitis viniferae
Sugar-6-phosphate dehydrogenase-hapten enzyme mark conjugate；The substrate of above-mentioned enzyme is G-6-P；Described glucose-6-phosphorus
Acidohydrogenase-hapten enzyme mark conjugate is reacted with methotrexate derivatives by glucose-6-phosphate dehydrogenase (G6PD) and is formed；Above-mentioned first
The structural formula of aminopterin derivant is as shown in formula II:
Formula II.
2. the synthetic method of methotrexate derivatives, it is characterised in that methotrexate derivatives structural formula is as shown in formula II:
Formula II
Its synthetic route is:
。
3. the preparation method of a methotrexate homogeneous enzyme immunoassay detectable, it is characterised in that comprise the steps:
(1) synthesis of the methotrexate derivatives described in claim 1 and purification, and carry out Structural Identification；
(2) the immunogenic synthesis of methotrexate: make the terminal carboxyl group of methotrexate derivatives and there is immunogenic protein
Carrier connects；
(3) with methotrexate immunogen immune animal, preparation purification anti-methotrexate specific antibody；
(4) preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing: prepare glucose-6-phosphate dehydrogenase (G6PD) solution, system
Standby methotrexate derivatives solution, makes glucose-6-phosphate dehydrogenase (G6PD) be connected with the terminal carboxyl group of methotrexate, and purification connects product
Thing；
(5) preparation of methotrexate homogeneous enzyme immunoassay detectable:
The preparation of reagent A: mixed by anti-methotrexate specific antibody and homogeneous zymolyte；The preparation of reagent B: by Fructus Vitis viniferae
Sugar-6-phosphate dehydrogenase-hapten conjugation thing mixes with Tris buffer.
The preparation method of a kind of methotrexate homogeneous enzyme immunoassay detectable the most according to claim 3, it is characterised in that
In described step (2), protein carrier is BSA, and concrete synthesis step is as follows:
A. weigh 2.72g potassium dihydrogen phosphate, 4.26g disodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, be jointly dissolved in 1L
In deionized water, regulate pH to 7.4, make buffer solution A；
B. weigh and be dissolved under 3mg BSA, room temperature in 3mL above-mentioned buffer solution A, make BSA solution；
C. weigh 3mg methotrexate derivatives, be dissolved in 300ul above-mentioned buffer solution A, make methotrexate derivatives molten
Liquid；
D., when above-mentioned methotrexate derivatives solution just becomes clarification, it is added dropwise in above-mentioned BSA solution, then this is mixed
Close solution to stir 1 hour at 2-8 DEG C；
E. being dialysed with above-mentioned buffer solution A by reacted above-mentioned mixed solution, after dialysis, gained solution is first ammonia butterfly
Purine immunogen solution, adds the NaN of mass fraction 0.1% in methotrexate immunogen solution3, store at-20 DEG C.
The preparation method of a kind of methotrexate homogeneous enzyme immunoassay detectable the most according to claim 3, it is characterised in that
Described step (4) detailed process is:
A. weigh 1.09g potassium dihydrogen phosphate, 1.70g disodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, be jointly dissolved in 1L
In deionized water, regulate pH to 7.4, make buffer solution B；
B. weigh and be dissolved under 3mg glucose-6-phosphate dehydrogenase (G6PD), room temperature in 3mL above-mentioned buffer solution B, make glucose-6-
Phosphate dehydrogenase enzymatic solution；
C. weigh 3mg methotrexate derivatives, be dissolved in 300ul above-mentioned buffer solution B, make methotrexate derivatives molten
Liquid；
D., when above-mentioned methotrexate derivatives solution just becomes clarification, it is added dropwise over above-mentioned glucose-6-phosphate dehydrogenase (G6PD)
In solution, then this mixed solution is stirred 1 hour at 2-8 DEG C；
E. reacted above-mentioned mixed solution is dialysed with above-mentioned buffer solution B, after dialysis gained solution be glucose-
6-phosphate dehydrogenase-hapten conjugation thing solution, adds matter in glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution
The BSA and the NaN of mass fraction 0.1% of amount mark 0.5%3, store at 2-8 DEG C.
The preparation method of a kind of methotrexate homogeneous enzyme immunoassay detectable the most according to claim 3, it is characterised in that
The detailed process of step (5) is as follows:
The preparation of reagent A: by the nicotinamide adenine dinucleotide of oxidation state of 4.036g 11.25mM, 1.711g 11.25mM
The Tris buffer solution of G-6-P 1L 55mM, pH=8.0 make homogeneous zymolyte；Anti-first ammonia by preparation
Pterin specific antibody is added in above-mentioned homogeneous zymolyte, and antibody is 1:100～1:10000 with the volume ratio of homogeneous zymolyte；
The preparation of reagent B: the glucose-6-phosphate dehydrogenase (G6PD) of preparation-hapten conjugation thing is added to 120mM, pH=8.2
In Tris buffer, above-mentioned conjugate is 1:100～1:10000 with the volume ratio of Tris buffer.
7. utilize the detection method of methotrexate homogeneous enzyme immunoassay detectable described in claim 1, it is characterised in that include
Following steps:
(1) sample to be tested is contacted with anti-methotrexate specific antibody；
(2) according to the combination situation of methotrexate in sample to be tested Yu anti-methotrexate specific antibody, indicator is utilized to sentence
The content of methotrexate in disconnected sample；
Described sample to be tested is serum, blood plasma, saliva or urine；
Above-mentioned enzyme reagent includes: enzyme mark conjugate and the substrate of enzyme；Above-mentioned enzyme mark conjugate includes G-6-P dehydrogenation
Enzyme-hapten enzyme mark conjugate；The substrate of above-mentioned enzyme is G-6-P；
Above-mentioned glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark conjugate is spread out with methotrexate by glucose-6-phosphate dehydrogenase (G6PD)
Biological respinse is formed, and the structural formula of above-mentioned methotrexate derivatives is as shown in formula II:
Formula II.
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| CN108614107A (en) * | 2016-12-12 | 2018-10-02 | 上海复星长征医学科学有限公司 | A kind of methotrexate (MTX) latex immunoturbidimetry assay kit and preparation method thereof |
| CN107219357B (en) * | 2017-04-21 | 2020-07-07 | 中国人民解放军第三军医大学第一附属医院 | ELISA kit for detecting methotrexate and application thereof |
| CN108593908A (en) * | 2017-12-22 | 2018-09-28 | 太原瑞盛生物科技有限公司 | A kind of methotrexate (MTX) immunologic function test reagent and its preparation and detection method |
| CN110357886B (en) * | 2018-04-09 | 2022-06-24 | 浙江准策生物技术有限公司 | Methotrexate hapten and complete antigen as well as preparation method and application thereof |
| CN110174363A (en) * | 2019-01-09 | 2019-08-27 | 北京九强生物技术股份有限公司 | Glucose-6-phosphate dehydrogenase mutant and its purposes in preparation detection reagent |
| KR20230035332A (en) * | 2020-07-06 | 2023-03-13 | 비온디스 비.브이. | Antifolate Linker-Drug and Antibody-Drug Conjugates |
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