CN104569373A - Methotrexate homogenous enzyme immunoassay reagent as well as preparation method and detection method thereof - Google Patents
Methotrexate homogenous enzyme immunoassay reagent as well as preparation method and detection method thereof Download PDFInfo
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Abstract
The invention relates to a methotrexate detection reagent as well as a preparation method and a detection method thereof, and specifically relates to a methotrexate homogenous enzyme immunoassay reagent as well as a preparation method and a detection method thereof. The methotrexate homogenous enzyme immunoassay reagent comprises an anti-methotrexate specific antibody, and an indication reagent for detecting an anti-methotrexate specific antibody-methotrexate compound, wherein the anti-methotrexate specific antibody is obtained from immune animals with methotrexate immunogen. The methotrexate homogenous enzyme immunoassay reagent disclosed by the invention has the following beneficial effects: the methotrexate immunogen is high in specificity and immunogenicity, and the prepared anti-methotrexate specific antibody is high in specificity and valence, and free from any cross reaction with 62 common medicines; the homogenous enzyme immunoassay reagent containing the anti-methotrexate specific antibody is capable of conveniently, rapidly and accurately determining the content of methotrexate in a sample and measuring a plurality of samples on a fully-automatic biochemical analyser to realize high-flux rapid measurement for methotrexate, is high in accuracy and high in specificity, and is capable of greatly improving the accuracy and the detection efficiency.
Description
Technical field
The present invention relates to a kind of methotrexate (MTX) and detect reagent and preparation and determination methods method thereof, be specifically related to a kind of methotrexate (MTX) homogeneous enzyme immunoassay and detect reagent and preparation and determination methods method thereof.
Background technology
Methotrexate (MTX) (Methotrexate) structural formula is such as formula shown in (III):
Methotrexate (MTX) is a kind of folic acid reductase inhibitor, is anti-folic acid series antineoplastic medicament, mainly through hindering the synthesis of DNA of tumor cell to the inhibiting effect of dihyrofolate reductase, thus the Growth and reproduction of inhibition tumor cell.It optionally acts on the S phase, belongs to CCSA.In acute leukemia (especially acute lymphatic leukemia), chorioepithelioma and chorioadenoma etc., result for the treatment of is better clinically.All have certain curative effect to head and neck neoplasm, breast cancer, lung cancer and tumor of pelvis, also can with other drug therapeutic alliance Burkitts lymthoma, lymphosarcoma in late period (III and IV phase, PeterShi stage system) and late period mycosis fungoides.Methotrexate (MTX) safe range is narrow, and the individuation difference of body metabolism and excretion after taking medicine is large, cannot grow by Tumor suppression, then can cause the spinoffs such as gastrointestinal reaction, cirrhosis, kidney damage higher than its effective blood drug concentration lower than its effective blood drug concentration.This medicine cytotoxicity is comparatively large, and bad reaction is more, should closely monitor patient's blood concentration, accomplish individual administration.Therefore, therapeutic drug monitoring is carried out to the patient taking methotrexate (MTX), to reducing bad reaction and instructing clinical individual safe medication significant.
At present, the method measuring methotrexate (MTX) mainly contains high performance liquid chromatography (HPLC), fluorescence polarization immunoassay (FPIA) etc., and HPLC method complex operation, is mainly used in lab analysis, FPIA method needs to be equipped with expensive instrumentation, and cost is higher.Therefore, these methods all have certain defective in clinical practice.Although the existing methotrexate (MTX) that can be applicable to Biochemical Analyzer of external producer measures kit listing, product quantity far can not meet clinical demand.Still deficient in stability methotrexate (MTX) that is good, highly sensitive, high specificity detects reagent, the especially measured Automated inspection reagent of matter in the market.Therefore, development & production quality reaches clinical requirement, practical, cost performance is high, and the methotrexate (MTX) that can be applicable to automatic clinical chemistry analyzer measures the focus that reagent has become domestic and international external diagnosis reagent industry.Methotrexate (MTX) homogeneous enzyme immunoassay of the present invention detects reagent and can be implemented on automatic clinical chemistry analyzer the high flux of methotrexate (MTX), rapid detection, and there is the advantages such as easy and simple to handle, highly sensitive, high specificity, result are accurate, can also effectively reduce methotrexate (MTX) testing cost, be conducive to clinical expansion and use.
Summary of the invention
For solving the deficiencies in the prior art, the object of the present invention is to provide a kind of not only safe but also quick, efficient, sensitive, accurately can detect that the methotrexate (MTX) homogeneous enzyme immunoassay of methotrexate (MTX) content in sample to be tested detects reagent and preparation method thereof, and can with various types of automatic biochemistry analyzer coupling, to testing staff's less demanding methotrexate (MTX) homogeneous enzyme immunoassay detection method.
Another object of the present invention contributes to preparing for providing a kind of the methotrexate derivatives that the methotrexate (MTX) homogeneous enzyme immunoassay measuring methotrexate (MTX) content rapidly and accurately detects reagent.
In order to realize above-mentioned target, the technical solution used in the present invention is:
A kind of methotrexate (MTX) homogeneous enzyme immunoassay detects reagent, it is characterized in that, comprising: anti-methotrexate (MTX) specific antibody, for detecting the indicator of anti-methotrexate (MTX) specific antibody-methotrexate (MTX) compound; Above-mentioned anti-methotrexate (MTX) specific antibody is obtained by methotrexate (MTX) immunogen immune animal, and the immunogenic structural formula of methotrexate (MTX) is such as formula shown in (I):
In formula, carrier, for having immunogenic protein, is preferably haemocyanin, hemocyanin or thyroglobulin; Above-mentioned indicator is selected from enzyme reagent, radioactive isotope reagent, fluorescent reagent or chemical illuminating reagent.
Aforesaid methotrexate (MTX) homogeneous enzyme immunoassay detects reagent, and above-mentioned indicator is selected from enzyme reagent, comprising: the substrate of enzyme mark conjugate and enzyme; Above-mentioned enzyme mark conjugate comprises glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate; The substrate of above-mentioned enzyme is G-6-P.
Aforesaid methotrexate (MTX) homogeneous enzyme immunoassay detects reagent, above-mentioned glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate is reacted by glucose-6-phosphate dehydrogenase (G6PD) and methotrexate derivatives and is formed, and the structural formula of above-mentioned methotrexate derivatives is such as formula shown in (II):
Methotrexate derivatives, structural formula is such as formula shown in (II):
Its synthetic route is:
Methotrexate (MTX) homogeneous enzyme immunoassay detects a preparation method for reagent, it is characterized in that, comprises the steps:
(1) synthesis of methotrexate derivatives and purifying, and carry out Structural Identification;
(2) the immunogenic synthesis of methotrexate (MTX): make the terminal carboxyl group of methotrexate (MTX) and there is immunogenic protein carrier be connected;
(3) with methotrexate (MTX) immunogen immune animal, preparation is the anti-methotrexate (MTX) specific antibody of purifying also;
(4) preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing: prepare glucose-6-phosphate dehydrogenase (G6PD) solution, prepare methotrexate derivatives solution, glucose-6-phosphate dehydrogenase (G6PD) is connected with the terminal carboxyl group of methotrexate (MTX), and purifying connects product;
(5) methotrexate (MTX) homogeneous enzyme immunoassay detects the preparation of reagent:
The preparation of reagent A: mixed by anti-methotrexate (MTX) specific antibody and homogeneous phase zymolyte; The preparation of reagent B: mixed by glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing and Tris damping fluid.
Aforesaid a kind of methotrexate (MTX) homogeneous enzyme immunoassay detects the preparation method of reagent, and in described step (2), protein carrier is BSA, and concrete synthesis step is as follows:
A. take 2.72g potassium dihydrogen phosphate, 4.26g sodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, be jointly dissolved in 1L deionized water, regulate pH to 7.4, make buffer solution A;
B. take 3mg BSA, be dissolved under room temperature in the above-mentioned buffer solution A of 3mL, make BSA solution;
C. take 3mg methotrexate derivatives, be dissolved in the above-mentioned buffer solution A of 300ul, make methotrexate derivatives solution;
D., when above-mentioned methotrexate derivatives solution has just become clarification, it is dropwise added in above-mentioned BSA solution, then this mixed solution is stirred 1 hour at 2-8 DEG C;
E. dialysed by the above-mentioned buffer solution A of reacted above-mentioned mixed solution, after dialysis, gained solution is methotrexate (MTX) immunogen solution, adds the NaN of massfraction 0.1% in methotrexate (MTX) immunogen solution
3, store at-20 DEG C.
Aforesaid a kind of methotrexate (MTX) homogeneous enzyme immunoassay detects the preparation method of reagent, and described step (4) detailed process is:
A. take 1.09g potassium dihydrogen phosphate, 1.70g sodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, be jointly dissolved in 1L deionized water, regulate pH to 7.4, make buffer solution B;
B. take 3mg glucose-6-phosphate dehydrogenase (G6PD), be dissolved under room temperature in the above-mentioned buffer solution B of 3mL, make glucose-6-phosphate dehydrogenase (G6PD) solution;
C. take 3mg methotrexate derivatives, be dissolved in the above-mentioned buffer solution B of 300ul, make methotrexate derivatives solution;
D., when above-mentioned methotrexate derivatives solution has just become clarification, it is dropwise added in above-mentioned glucose-6-phosphate dehydrogenase (G6PD) solution, then this mixed solution is stirred 1 hour at 2-8 DEG C;
E. the above-mentioned buffer solution B of reacted above-mentioned mixed solution is dialysed, after dialysis, gained solution is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution, adds the BSA of the massfraction 0.5% and NaN of massfraction 0.1% in glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution
3, store at 2-8 DEG C.
Aforesaid a kind of methotrexate (MTX) homogeneous enzyme immunoassay detects the preparation method of reagent, and the detailed process of step (5) is as follows:
The preparation of reagent A: 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 are made homogeneous phase zymolyte; Be added in above-mentioned homogeneous phase zymolyte by the anti-methotrexate (MTX) specific antibody of preparation, the volume ratio of antibody and homogeneous phase zymolyte is 1:100 ~ 1:10000;
The preparation of reagent B: be added in the Tris damping fluid of 120mM, pH=8.2 by the glucose-6-phosphate dehydrogenase (G6PD) of preparation-hapten conjugation thing, the volume ratio of above-mentioned conjugate and Tris damping fluid is 1:100 ~ 1:10000.
Utilize methotrexate (MTX) homogeneous enzyme immunoassay to detect the detection method of reagent, it is characterized in that, comprise the following steps:
1) sample to be tested is contacted with anti-methotrexate (MTX) specific antibody;
2) according to methotrexate (MTX) in sample to be tested and anti-methotrexate (MTX) specific antibody in conjunction with situation, utilize the content of methotrexate (MTX) in indicator judgement sample;
Described sample to be tested is serum, blood plasma, saliva or urine.
Above-mentioned enzyme reagent comprises: the substrate of enzyme mark conjugate and enzyme; Above-mentioned enzyme mark conjugate comprises glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate; The substrate of above-mentioned enzyme is G-6-P;
Above-mentioned glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate is reacted by glucose-6-phosphate dehydrogenase (G6PD) and methotrexate derivatives and is formed, and the structural formula of above-mentioned methotrexate derivatives is such as formula shown in (II):
Usefulness of the present invention is: methotrexate (MTX) immunogens of the present invention is strong, immunogenicity is high, the anti-methotrexate (MTX) specific antibody high specificity prepared, height of tiring, and with common 62 kinds of medicines without any cross reaction; Homogeneous enzyme immunoassay detection reagent containing above-mentioned anti-methotrexate (MTX) specific antibody can determine the methotrexate (MTX) content in sample easily and fast, exactly, and can on automatic clinical chemistry analyzer the multiple sample of Simultaneously test, realize the rapid mensuration of high flux of methotrexate (MTX), accuracy is high, high specificity, degree of accuracy is all enhanced before comparing with detection efficiency, achieve the full-automation of testing process simultaneously, less demanding to testing staff, is easy to realize and promote the use of.
Accompanying drawing explanation
Fig. 1 is methotrexate (MTX) homogeneous enzyme immunoassay reaction normal curve map;
Fig. 2 is methotrexate (MTX) homogeneous enzyme immunoassay correlation analysis figure.
Embodiment
The technical solution used in the present invention is:
Methotrexate (MTX) immunogene, its structural formula is such as formula shown in (I):
In formula, carrier has immunogenicity, and preferably, carrier is for having immunogenic protein.Also can, as carrier, select protein as carrier under normal circumstances although what other were enough large possesses immunogenic material.The most frequently used immunogenic carrier comprises haemocyanin, hemocyanin (KLH) and thyroglobulin.Carrier in the present invention is preferably haemocyanin.
A kind of anti-methotrexate (MTX) specific antibody, is obtained by the methotrexate (MTX) immunogen immune animal shown in formula (I).
In the present invention, " antibody " of indication not only refers to complete antibody molecule, also comprises the antibody fragment or derivant that retain complete antibody specific binding capacity.Antibody of the present invention can be polyclonal antibody also can be monoclonal antibody, is preferably polyclonal antibody.
The method obtaining polyclonal antibody is the methotrexate (MTX) immunogene shown in use formula (I), and after adding or not adding adjuvant, carry out immunity at one or more position of animal, host animal comprises: rabbit, goat, mouse, sheep, cavy or horse.Persistent immunological carries out always, until antibody titer reaches the highest.Animal timing blood sampling obtains appropriate specific corrosioning anteserum, and antiserum can purifying.
Monoclonal antibody makes by somatocyte hybriding technology.
A kind of methotrexate (MTX) homogeneous enzyme immunoassay detects reagent, comprising: above-mentioned anti-methotrexate (MTX) specific antibody, for detecting the indicator of anti-methotrexate (MTX) specific antibody-methotrexate (MTX) compound.Indicator is selected from enzyme reagent, radioactive isotope reagent, fluorescent reagent and chemical illuminating reagent.Preferably, indicator is enzyme reagent, comprising: the substrate of enzyme mark conjugate and enzyme.Wherein, enzyme mark conjugate comprises glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate, and it obtains by chemical synthesis process.
Above-mentioned methotrexate (MTX) homogeneous enzyme immunoassay detects the using method of reagent, comprises the following steps:
1) sample to be tested is contacted with above-mentioned anti-methotrexate (MTX) specific antibody;
2) according to methotrexate (MTX) in sample to be tested and above-mentioned anti-methotrexate (MTX) specific antibody in conjunction with situation, utilize the content of methotrexate (MTX) in indicator judgement sample.
Sample to be tested is various physiology samples, such as serum, blood plasma, urine, saliva etc.Preferably, sample to be tested is serum 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 examples is such as formula shown in (II):
The synthetic route of this methotrexate derivatives is as follows:
Concrete synthesis step is as follows:
The synthesis of compound 2
Take 5.0g compound 1, be dissolved in the MeOH of 50mL, at 0 DEG C, dropwise add 4.0g (34.1mmol) SOCl
2, then this reaction mixture is stirred at 70 DEG C and spends the night, by decompression method, solvent is evaporated, by the NaHCO of residue by DCM and 200mL of 200mL
3saturated aqueous solution is separated, and is rinsed by organic phase bittern, then passes through Na
2sO
4carry out drying, and carry out concentrated obtaining 5.1g compound as white solid 2, productive rate 96%.
The synthesis of compound 3
Take 5.1g compound 2, be dissolved in the MeOH of 100mL, at 0 DEG C, add 1.3g (33.3mmol) NaBH several times
4then at room temperature stirred by this reaction mixture and spend the night, concentrated by reacted potpourri, the residue obtained after concentrated carries out purifying by silica dehydrator post (DCM:MeOH=10:1), obtain the compound 3 of 4.0g colorless oil, productive rate 92%.
The synthesis of methotrexate derivatives
Take 2.5g compound 3, be dissolved in the THF of 40mL, under 0 DEG C and nitrogen protection condition, add 1.2g (12.6mmol) maleimide, 3.8g (14.56mmol) PPh
3and 2.9g (14.56mmol) DIAD, then this reaction mixture is stirred at 70 DEG C and spend the night, reacted potpourri is concentrated, the residue obtained after concentrated carries out purifying by silica dehydrator post (DCM:MeOH=50:1), finally obtain 500mg tan solid Compound 4, productive rate 15%.This compound 4 is the methotrexate derivatives shown in formula (II).
The immunogenic synthesis of embodiment two: BSA-methotrexate (MTX)
BSA-methotrexate (MTX) immunogene is formed by connecting by the terminal carboxyl group of the methotrexate (MTX) shown in bovine serum albumin(BSA) (BSA) Yu formula (III), and this immunogenic concrete synthesis step is as follows:
A. take 2.72g potassium dihydrogen phosphate, 4.26g sodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, be jointly dissolved in 1L deionized water, regulate pH to 7.4, make buffer solution A;
B. take 3mg BSA, be dissolved under room temperature in the above-mentioned buffer solution A of 3mL, make BSA solution;
C. take 3mg methotrexate derivatives, be dissolved in the above-mentioned buffer solution A of 300ul, make methotrexate derivatives solution;
D., when above-mentioned methotrexate derivatives solution has just become clarification, it is dropwise added in above-mentioned BSA solution, then this mixed solution is stirred 1 hour at 2-8 DEG C;
E. dialysed by the above-mentioned buffer solution A of reacted above-mentioned mixed solution, after dialysis, gained solution is methotrexate (MTX) immunogen solution, adds the NaN of 0.1% in methotrexate (MTX) immunogen solution
3, store at-20 DEG C.The NaN of 0.1%
3refer to that addition accounts for the mass percent of the immunogen solution of final gained, concrete addition is determined according to the concrete quality of the immunogen solution of gained after dialysis.
Embodiment three: the preparation of anti-methotrexate (MTX) specific antibody
Above-mentioned obtained BSA-methotrexate (MTX) immunogene is adopted conventional method inoculation experiments animal rabbit, get antiserum after booster immunization, concrete steps are as follows:
With PBS, the BSA-methotrexate (MTX) immunogene of above-mentioned synthesis is diluted to 1.0mg/ml, obtains antigenic solution, then mix with Freund's complete adjuvant with 1.0ml antigenic solution, experimental animal rabbit is injected.
After 2 ~ 3 weeks, then with the identical antigenic solution of 1.0ml and incomplete Freund's adjuvant, above-mentioned experimental animal rabbit is injected once, afterwards every surrounding injection once, amount to injection 4 times.
Blood is got to above-mentioned experimental animal rabbit, separation and purification obtain tiring be 1: 30000-1: 50000 anti-methotrexate (MTX) specific antibody.
Embodiment four: the preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing
A. take 1.09g potassium dihydrogen phosphate, 1.70g sodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, be jointly dissolved in 1L deionized water, regulate pH to 7.4, make buffer solution B;
B. take 3mg glucose-6-phosphate dehydrogenase (G6PD), be dissolved under room temperature in the above-mentioned buffer solution B of 3mL, make glucose-6-phosphate dehydrogenase (G6PD) solution;
C. take 3mg methotrexate derivatives, be dissolved in the above-mentioned buffer solution B of 300ul, make methotrexate derivatives solution;
D., when above-mentioned methotrexate derivatives solution has just become clarification, it is dropwise added in above-mentioned glucose-6-phosphate dehydrogenase (G6PD) solution, then this mixed solution is stirred 1 hour at 2-8 DEG C; E. the above-mentioned buffer solution B of reacted above-mentioned mixed solution is dialysed, after dialysis, gained solution is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution, in glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution, add the BSA of the 0.5% and NaN of 0.1%
3, store at 2-8 DEG C.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 addition is determined according to the concrete quality of the conjugate solution of gained after dialysis.
Embodiment five: methotrexate (MTX) homogeneous enzyme immunoassay detects the preparation of reagent
Methotrexate (MTX) homogeneous enzyme immunoassay detects reagent, comprising: above-mentioned anti-methotrexate (MTX) specific antibody, for detecting the indicator of anti-methotrexate (MTX) specific antibody-methotrexate (MTX) compound.Indicator is selected from enzyme reagent, radioactive isotope reagent, fluorescent reagent and chemical illuminating reagent.Preferably, indicator is enzyme reagent, comprising: the substrate of enzyme mark conjugate and enzyme.Wherein, enzyme mark conjugate comprises glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate, and it is obtained by above-mentioned chemical synthesis process.
Methotrexate (MTX) homogeneous enzyme immunoassay detects reagent before the use, in order to avoid the substrate of the enzyme mark conjugate in indicator and enzyme reacts, the substrate of enzyme mark conjugate and enzyme is separated, does not mix, so the substrate of enzyme and above-mentioned anti-methotrexate (MTX) specific antibody are mixed.That is, methotrexate (MTX) homogeneous enzyme immunoassay detects reagent and comprises two kinds of reagent be provided separately, specific as follows:
1. the preparation of reagent A: the nicotinamide adenine dinucleotide (NAD) of 4.036g (11.25mM) oxidation state, 1.711g (11.25mM) G-6-P (G-6-P) are placed in beaker D, make homogeneous phase zymolyte with the Tris buffer solution of 1L 55mM, pH=8.0; Be added in above-mentioned homogeneous phase zymolyte by the anti-methotrexate (MTX) specific antibody of above-mentioned preparation, the volume ratio of antibody and homogeneous phase zymolyte can be 1:100 ~ 1:10000, and ratio concrete is in the present embodiment 1:400.
2. the preparation of reagent B: the glucose-6-phosphate dehydrogenase (G6PD) of above-mentioned preparation-hapten conjugation thing is added in the Tris damping fluid of 120mM, pH=8.2, the volume ratio of above-mentioned conjugate and Tris damping fluid can be 1:100 ~ 1:10000, and ratio concrete is in the present embodiment 1:1500.
Above-mentioned methotrexate (MTX) homogeneous enzyme immunoassay detects the using method of reagent, comprises the following steps:
1) sample to be tested is contacted with above-mentioned anti-methotrexate (MTX) specific antibody;
2) according to methotrexate (MTX) in sample to be tested and above-mentioned anti-methotrexate (MTX) specific antibody in conjunction with situation, utilize indicator to judge the content of methotrexate (MTX) in sample to be tested.
Concrete, during detection, be added to by sample to be tested in reagent A, the anti-methotrexate (MTX) specific antibody generation specific binding in the methotrexate (MTX) in sample to be tested and reagent A, generates anti-methotrexate (MTX) specific antibody-methotrexate (MTX) compound; Add reagent B again, glucose-6-phosphate dehydrogenase (G6PD) now in reagent B-hapten conjugation thing mixes with the substrate of the enzyme in reagent A, contacts, there is enzymatic reaction, form and detect the indicator of anti-methotrexate (MTX) specific antibody-methotrexate (MTX) compound, indicator is according to the content judging methotrexate (MTX) in sample to be tested in conjunction with situation of methotrexate (MTX) in sample to be tested and above-mentioned anti-methotrexate (MTX) specific antibody.
Due to the anti-methotrexate (MTX) specific antibody of the methotrexate (MTX) competitive binding in glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing and sample to be tested, so, in sample to be tested, the amount of methotrexate (MTX) is more, the amount of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing free in homogeneous phase enzyme solutions is more, enzymatic reaction is faster, causes OD
340rise.
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 (MTX) homogeneous enzyme immunoassay is checked
1, typical curve is obtained: arrange auspicious BS200 automatic clinical chemistry analyzer response parameter (see table 1) advanced in years, operating process is: first reagent adding A, then adds standard items, finally adds reagent B.After adding reagent B, measure the OD of different time points
340light absorption value, calculates reaction rate during various criterion product concentration, needs the volume ratio constantly adjusting reagent A and reagent B in actual mechanical process, adjusts light-metering point simultaneously, finally draws comparatively ideal reaction normal curve map, as shown in Figure 1.
Table 1 steps auspicious BS200 automatic clinical chemistry analyzer response parameter
By the typical curve that homogeneous enzyme immunoassay detection reagent of the present invention obtains, replication basic, normal, high concentration Quality Control sample 10 times, above-mentioned Quality Control sample is: be dissolved in human serum by methotrexate (MTX) standard items, is respectively 0.25,1.00,2.00 μm of ol/L to concentration.Detection data and data analysis are in table 2.
Table 2 sample determination and precision and recovery assessment
| 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 |
| Mean value (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 |
| Recovery % | 100.0 | 101.0 | 100.0 |
Testing result: the accuracy that homogeneous enzyme immunoassay of the present invention detects reagent mensuration is high, and the recovery reaches 95%-105%, and precision is high, and CV is all lower than 5%.
Embodiment seven: interfering effects of drug is tested
Choose 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 are specifically see 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 (MTX) is all less than 0.1 μm of ol/L.Visible, antibody of the present invention is the specific antibody of anti-methotrexate (MTX).
Embodiment eight: correlation analysis
Use the methotrexate (MTX) of De Ling diagnostic products (Shanghai) Co., Ltd. detection reagent (adopting enzyme to amplify immunoassay) and homogeneous enzyme immunoassay reagent of the present invention to carry out correlation analysis respectively to 100 routine clinical samples, the data of mensuration are see table 4.
Table 4 clinical sample measured value
Map to above-mentioned data, see Fig. 2, the linear equation obtained is: y=0.9953x+0.0095, coefficient R
2=0.9970, show that the accuracy of detection reagent of the present invention mensuration methotrexate (MTX) clinical samples is high.
Because testing process of the present invention is completed by instrument full-automation, so less demanding to testing staff, be easy to realize and promote the use of.
It should be noted that; the foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize instructions of the present invention and accompanying drawing content to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other correlative technology fields, be all in like manner included in scope of patent protection of the present invention.
Claims (10)
1. methotrexate (MTX) homogeneous enzyme immunoassay detects a reagent, it is characterized in that, comprising: anti-methotrexate (MTX) specific antibody, for detecting the indicator of anti-methotrexate (MTX) specific antibody-methotrexate (MTX) compound; Above-mentioned anti-methotrexate (MTX) specific antibody is obtained by methotrexate (MTX) immunogen immune animal, and the immunogenic structural formula of methotrexate (MTX) is such as formula shown in (I):
In formula, carrier is for having immunogenic protein; Above-mentioned indicator is selected from enzyme reagent, radioactive isotope reagent, fluorescent reagent or chemical illuminating reagent.
2. methotrexate (MTX) homogeneous enzyme immunoassay according to claim 1 detects reagent, and it is characterized in that, above-mentioned indicator is selected from enzyme reagent, comprising: the substrate of enzyme mark conjugate and enzyme; Above-mentioned enzyme mark conjugate comprises glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate; The substrate of above-mentioned enzyme is G-6-P; Described glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate is reacted by glucose-6-phosphate dehydrogenase (G6PD) and methotrexate derivatives and is formed.
3. methotrexate (MTX) homogeneous enzyme immunoassay according to claim 2 detects reagent, and it is characterized in that, the structural formula of above-mentioned methotrexate derivatives is such as formula shown in (II):
4. methotrexate derivatives, structural formula is such as formula shown in (II):
5. methotrexate derivatives according to claim 4, is characterized in that, its synthetic route is:
6. methotrexate (MTX) homogeneous enzyme immunoassay detects a preparation method for reagent, it is characterized in that, comprises the steps:
(1) synthesis of the methotrexate derivatives described in claim 4 or 5 and purifying, and carry out Structural Identification;
(2) the immunogenic synthesis of methotrexate (MTX): make the terminal carboxyl group of methotrexate derivatives and there is immunogenic protein carrier be connected;
(3) with methotrexate (MTX) immunogen immune animal, preparation is the anti-methotrexate (MTX) specific antibody of purifying also;
(4) preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing: prepare glucose-6-phosphate dehydrogenase (G6PD) solution, prepare methotrexate derivatives solution, glucose-6-phosphate dehydrogenase (G6PD) is connected with the terminal carboxyl group of methotrexate (MTX), and purifying connects product;
(5) methotrexate (MTX) homogeneous enzyme immunoassay detects the preparation of reagent:
The preparation of reagent A: mixed by anti-methotrexate (MTX) specific antibody and homogeneous phase zymolyte; The preparation of reagent B: mixed by glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing and Tris damping fluid.
7. a kind of methotrexate (MTX) homogeneous enzyme immunoassay according to claim 6 detects the preparation method of reagent, and it is characterized in that, in described step (2), protein carrier is BSA, and concrete synthesis step is as follows:
A. take 2.72g potassium dihydrogen phosphate, 4.26g sodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, be jointly dissolved in 1L deionized water, regulate pH to 7.4, make buffer solution A;
B. take 3mg BSA, be dissolved under room temperature in the above-mentioned buffer solution A of 3mL, make BSA solution;
C. take 3mg methotrexate derivatives, be dissolved in the above-mentioned buffer solution A of 300ul, make methotrexate derivatives solution;
D., when above-mentioned methotrexate derivatives solution has just become clarification, it is dropwise added in above-mentioned BSA solution, then this mixed solution is stirred 1 hour at 2-8 DEG C;
E. dialysed by the above-mentioned buffer solution A of reacted above-mentioned mixed solution, after dialysis, gained solution is methotrexate (MTX) immunogen solution, adds the NaN of massfraction 0.1% in methotrexate (MTX) immunogen solution
3, store at-20 DEG C.
8. a kind of methotrexate (MTX) homogeneous enzyme immunoassay according to claim 6 detects the preparation method of reagent, and it is characterized in that, described step (4) detailed process is:
A. take 1.09g potassium dihydrogen phosphate, 1.70g sodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, be jointly dissolved in 1L deionized water, regulate pH to 7.4, make buffer solution B;
B. take 3mg glucose-6-phosphate dehydrogenase (G6PD), be dissolved under room temperature in the above-mentioned buffer solution B of 3mL, make glucose-6-phosphate dehydrogenase (G6PD) solution;
C. take 3mg methotrexate derivatives, be dissolved in the above-mentioned buffer solution B of 300ul, make methotrexate derivatives solution;
D., when above-mentioned methotrexate derivatives solution has just become clarification, it is dropwise added in above-mentioned glucose-6-phosphate dehydrogenase (G6PD) solution, then this mixed solution is stirred 1 hour at 2-8 DEG C;
E. the above-mentioned buffer solution B of reacted above-mentioned mixed solution is dialysed, after dialysis, gained solution is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution, adds the BSA of the massfraction 0.5% and NaN of massfraction 0.1% in glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution
3, store at 2-8 DEG C.
9. a kind of methotrexate (MTX) homogeneous enzyme immunoassay according to claim 6 detects the preparation method of reagent, and it is characterized in that, the detailed process of step (5) is as follows:
The preparation of reagent A: the Tris buffer solution of G-6-P 1L 55mM, pH=8.0 of the nicotinamide adenine dinucleotide of the oxidation state of 4.036g 11.25mM, 1.711g 11.25mM is made homogeneous phase zymolyte; Be added in above-mentioned homogeneous phase zymolyte by the anti-methotrexate (MTX) specific antibody of preparation, the volume ratio of antibody and homogeneous phase zymolyte is 1:100 ~ 1:10000;
The preparation of reagent B: be added in the Tris damping fluid of 120mM, pH=8.2 by the glucose-6-phosphate dehydrogenase (G6PD) of preparation-hapten conjugation thing, the volume ratio of above-mentioned conjugate and Tris damping fluid is 1:100 ~ 1:10000.
10. utilize the methotrexate (MTX) homogeneous enzyme immunoassay described in claim 1 to 3 any one to detect the detection method of reagent, it is characterized in that, comprise the following steps:
(1) sample to be tested is contacted with anti-methotrexate (MTX) specific antibody;
(2) according to methotrexate (MTX) in sample to be tested and anti-methotrexate (MTX) specific antibody in conjunction with situation, utilize the content of methotrexate (MTX) in indicator judgement sample;
Described sample to be tested is serum, blood plasma, saliva or urine;
Above-mentioned enzyme reagent comprises: the substrate of enzyme mark conjugate and enzyme; Above-mentioned enzyme mark conjugate comprises glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate; The substrate of above-mentioned enzyme is G-6-P;
Above-mentioned glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate is reacted by glucose-6-phosphate dehydrogenase (G6PD) and methotrexate derivatives and is formed, and the structural formula of above-mentioned methotrexate derivatives is such as formula shown in (II):
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