CN108614107A - A kind of methotrexate (MTX) latex immunoturbidimetry assay kit and preparation method thereof - Google Patents
A kind of methotrexate (MTX) latex immunoturbidimetry assay kit and preparation method thereof Download PDFInfo
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Abstract
A kind of methotrexate (MTX) latex immunoturbidimetry assay kit and preparation method thereof.The present invention provides the kit for measuring blood medicine methotrexate (MTX) content in human serum：Reagent R1 and reagent R2 are with 1:1 composition.Its weight percent proportioning is R1：0.01%~4% emulsifier, 0.01%~4% methotrexate (MTX) conjugate, 0.1%~2% sodium chloride, 0.05%~2% sensitizer, 0.02% preservative, 30~100mmol/L buffer solutions.R2：0.1%~0.5% is combined with anti-methotrexate (MTX) antibody latex particle, 0.1%~1.0% bovine serum albumin(BSA), 0.02%~2% polysorbas20,0.02%~2% stabilizer 2,0.02%~2% protein protective agent 2,0.02% preservative, 20~50mmol/L buffer solutions.Kit sensitivity of the present invention and stability are high, have preferable potential applicability in clinical practice, the present invention provides preparation methods.
Description
Technical field
The present invention relates to biological reagents, and in particular to detection kit more particularly to a kind of high sensitivity, high specificity
Latex immunoturbidimetry measures the kit and preparation method thereof of human serum methotrexate (MTX) content.
Background technology
Methotrexate (MTX) (Methotrexate, MTX) is a kind of folic acid reductase inhibitor, for anti-folic acid class antineoplastic
Object, mainly by hindering the inhibiting effect of dihyrofolate reductase the synthesis of DNA of tumor cell, to inhibit tumour cell
Growth and breeding.Its antitumor machanism is：Methotrexate (MTX) is as similar to folic acid structure, belongs to pteridine derivatives, therefore MTX energy
It is enough attached on the active position of dihyrofolate reductase, blocks the formation of tetrahydrofolic acid.It is selectively applied to S phase (DNA
The synthesis phase), belong to cell cycle specific drugs.Clinically in acute leukemia (the especially white blood of acute lymphocytic
Disease), the therapeutic effect of chorioepithelioma and chorioadenoma etc. it is preferable.To head and neck neoplasm, breast cancer, lung cancer and basin
Chamber tumour has certain curative effect, also can be with other drugs combination therapy Burkitts lymthomas, late period lymphosarcoma (III and IV
Phase, PeterShi stage systems) and late period mycosis fungoides.
Because methotrexate (MTX) safe range is narrow, body metabolism and the individuation difference of excretion are big after medication, are less than its effective blood
Concentration can not then inhibit tumour growth, can cause gastrointestinal reaction, hepatic sclerosis, kidney damage etc. higher than its effective blood drug concentration
Side effect.The cytotoxicity of the medicine is larger, and adverse reaction is more, should monitor patient's blood concentration closely, accomplish individual administration.
Therefore, therapeutic drug monitoring is carried out to the patient for taking methotrexate (MTX), reduce adverse reaction and clinical individualization is instructed to use safely
Medical instrument is significant.
Currently, the method for measuring methotrexate (MTX) mainly has high performance liquid chromatography (HPLC), fluorescence polarization immunoassay
(FPIA) etc..HPLC methods are cumbersome, are mainly used for lab analysis, and FPIA methods need to be equipped with expensive special instrument, cost
It is higher.Thus, these methods all have certain limitation in clinical application.Although the existing Biochemical Analyzer of can be applied to
Methotrexate (MTX) assay kit lists, but far from meeting clinical demand.Still deficient in stability is good currently on the market, sensitivity
High, high specificity methotrexate (MTX) detection reagent, especially high-quality Automated inspection reagent.Therefore, development ＆ production quality
Reach clinical requirement, highly practical, cost-effective, the methotrexate (MTX) measure reagent that can be applied to automatic clinical chemistry analyzer has become
For the hot spot of domestic and international external diagnosis reagent industry.
The methotrexate (MTX) latex immunoturbidimetry measure reagent of the present invention may be implemented on automatic clinical chemistry analyzer to first
High-throughput, the rapid detection of aminopterin, and have many advantages, such as that easy to operate, high sensitivity, high specificity, result are accurate, have
Effect reduces methotrexate (MTX) testing cost, is conducive to clinical promotion and application.
Invention content
Technical problem to be solved by the present invention lies in above-mentioned shortcoming is overcome, research and design one kind is in full-automatic biochemical
To quick, sensitive, the measure reagent and preparation method thereof that accurately detects of methotrexate (MTX) on analyzer.
The present invention provides a kind of methotrexate (MTX) latex immunoturbidimetry assay kits.
Kit of the present invention is made of reagent R1 and reagent R2.
The volume ratio of the reagent R1 and reagent R2 is 1:1.
The group that reagent R1 is matched by following weight percent described in kit of the present invention is grouped as：
0.01%~4% emulsifier, 0.01%~4% methotrexate (MTX) conjugate, 0.1%~2% sodium chloride,
0.05%~2% sensitizer, 0.02% preservative, 88.00%~99.80% 30~100mmol/L buffer solutions.
The reagent R1 is the conjugation competitive reaction liquid combined with anti-methotrexate (MTX) specific antibody.
Emulsifier described in the reagent R1 is selected from polysorbas20, Tween 80 or Triton X-100.
The sensitizer is selected from Macrogol 4000, Macrogol 6000, PEG 8000 or PEG 20000,
Preferably PEG 20000.Sensitizer mainly acts on the reaction rate of antibody antigen.
The buffer solution is selected from phosphate buffer (PBS), TRIS buffer (Tris), 3- (N-
Quinoline base) propane sulfonic acid buffer solution (Mops), 2- (N- morpholines) ethanesulfonic acid buffer (MES), borate buffer solution, glycine buffer
Or it is one or more in hydroxyethyl piperazine second sulphur acid buffer；The pH of cushioning fluid is 6~9.
Reagent R2 described in kit of the present invention is grouped as by the group of following per distribution ratio：
0.1%~0.5% to be combined with the present latex particulate of anti-methotrexate (MTX) antibody, 0.1%~1.0% (w/w) ox blood pure
Albumen, 0.02%~2% (w/w) polysorbas20,0.02%~2% (w/w) stabilizer 2,0.02%~2% (w/w) protein protection
Agent 2,0.02% (w/w) preservative, 92.50%~97.70% 20~50mmol/L buffer solutions.
The present latex particulate for being combined with anti-methotrexate (MTX) antibody is：Select the carboxylic polystyrene latex of different-grain diameter
The compound that particle is formed with anti-methotrexate (MTX) antibody；A diameter of 50~300nm of the nano rubber latex particle of the different-grain diameter.
The anti-methotrexate (MTX) antibody is selected from rabbit-anti polyclonal antibody, goat-anti clonal antibody or the anti-monoclonal antibody of mouse.
The stabilizer and protein protective agent of the reagent R2, can improve the stability of reagent.
The stabilizer is polyoxyethylene resin class, selected from Tween-80, polyoxyethylene
Sugar alcohol acid anhydride monopalmitate or ethoxylated dodecyl alcohol sulfuric ester sodium salt, protein protective agent are trehalose.
It is horizontal that the buffer solution of the reagent R2 is selected from phosphate buffer, TRIS buffer, 2- morpholines second
It is one or more in acid buffer, borate buffer solution, glycine buffer or hydroxyethyl piperazine second sulphur acid buffer；Institute
It is 6~9 to state pH of cushioning fluid.
The preservative of the reagent R2 is selected from sodium azide, N- methyl-isothiazols ketone, N'- methylene-bis- [N'- (3- hydroxyls
Methyl -2,5- diketone -4- imidazolidinyls)] it is one or more in urea or ProClin 300.Wherein ProClin 300 is commodity
Name, ProClin 300 are molten for the mixing of 2-methyl-4-isothiazolin-3-one and 5-Chloro-2-methyl-4-isothiazolin-3-one
Liquid.
The principle of the present invention is：The present invention is using the present latex particulate conjugate for being combined with antibody in R2, with the first in R1
Aminopterin conjugate forms aggregation after immune response occurs, and changes the turbidity of system, and methotrexate (MTX) and being total in R1 in sample
Yoke object specifically competes the present latex particulate conjugate for being combined with antibody in R2, reduces the variation of turbidity.In certain wavelength
Under, the variation of front and back turbidity is reacted by measuring, you can measure the content of methotrexate (MTX) in sample.
It is a further object of the present invention to provide the preparation sides of the kit for measuring human serum methotrexate (MTX) content
Method.
This method includes the following steps：
I, reagent preparation R1：Take 1L water, then sequentially add sodium chloride, sensitizer, emulsifier, methotrexate (MTX) conjugate,
Preservative, the needs that add materials every time are stirred to being completely dissolved, and are adjusted pH value to 6.0~9.0, preferably 7.4, are then crossed 0.22 μm
Filter membrane obtains reagent R1；
The methotrexate (MTX) conjugate is the conjugate for competing anti-methotrexate (MTX) antibody in detection with methotrexate (MTX).
Methotrexate (MTX) conjugate described in reagent R1 is prepared by methotrexate (MTX) and chitosan reaction, is included the following steps：
(1) in the 2- of 50mmol/L (N- morpholines) ethanesulfonic acid (MES) buffer solution, 1.0g methotrexate (MTX)s (MTX) with
50mgN- HOSu NHSs (NHS) are in coupling agent 30mg 1- (3- dimethyl propyls) -3- ethyl-carbodiimide hydrochlorides
(EDCHCl) it under acting on, is reacted 30 minutes under room temperature (20-25 DEG C), forms the intermediate fat of MTX-NHS activation；
(2) 5.0g chitosans are added, that is, press 1：10 mass ratio (MTX：Chitosan), it is small that 2 are reacted under room temperature (20-25 DEG C)
When, condensation reaction forms the conjugate of methotrexate (MTX)-chitosan；
(3) inactivation of the intermediate fat of MTX-NHS activation, is directly added into 0.5ml ethanol amines and is inactivated to intermediate fat body, room temperature
Reaction 30 minutes；
(4) reaction solution after inactivating is in bag filter, and in the phosphate buffer of 20mmol/L, 2-8 DEG C of dialysis 12 is small
When, freeze-drying obtains the sterling of methotrexate (MTX)-chitosan conjugate, as methotrexate (MTX) conjugate.
II, reagent preparation R2：
Reagent R2 methotrexate (MTX)s specific antibody is prepared by methotrexate (MTX) immunogen immune animal, is included the following steps：
(1) methotrexate (MTX) immunogene is synthesized：The amino on the carboxyl and carrier protein of methotrexate (MTX) end is set to connect, carrier
For the protein with immunogenicity, preferably hemocyanin (KLH), haemocyanin (BSA) or thyroglobulin；
(2) antibody starting material used in R2 is prepared：With methotrexate (MTX) immunogen immune animal, prepares and purify anti-methotrexate (MTX)
Specific antibody；Monoclonal antibody is prepared with somatocyte hybriding technology.
The preparation process of reagent R2 is as follows：
(1) activation of polystyrene latex microparticles：1- ethyls -3- is added in the polystyrene latex microparticles with carboxyl
(3- DimethylAminopropyls) carbodiimide hydrochloride (EDAC), after mixing (20 DEG C -25 DEG C) of room temperature activation 30min；
(2) coupling of anti-methotrexate (MTX) antibody and polystyrene latex microparticles：The polystyrene colloidal that will be activated in step (1)
After lactoconium and the mixing of anti-methotrexate (MTX) antibody, covalent coupling is carried out in (20 DEG C -25 DEG C) reaction 2h of room temperature；
(3) it is combined with the washing of the present latex particulate compound of anti-methotrexate (MTX) antibody：Anti- methotrexate (MTX) antibody will be combined with
The reaction solution of present latex particulate be filtered washing in tangential flow filtration system, remove free antibodies, small molecular weight impurity；It is described
Cleaning solution is 50~100mM buffer solutions (pH7.0~8.0), and the latex for being combined with anti-methotrexate (MTX) antibody after being washed is micro-
Grain；
(4) closing and preservation of the present latex particulate of anti-methotrexate (MTX) antibody are combined with：After the washing that step (5) is obtained
It is combined in the present latex particulate of anti-methotrexate (MTX) antibody and polyoxyethylene sorbitan monopalmitate and trehalose is added, closing
Unreacted radical and other adsorption sites on polystyrene latex microparticles obtain initial action liquid after closing 1h；
(6) 3-6 times is diluted to above-mentioned initial action liquid using buffer solution and obtains final reagent R2；
III, kit is formed：By the reagent R1 of above-mentioned preparation and reagent R2 R1 by volume：R2=1:1 is distributed into a bottle group
At kit.
Beneficial effects of the present invention：
(1) the methotrexate (MTX) immunogens of kit of the present invention are strong, and the anti-methotrexate (MTX) specific antibody of preparation is special
It is anisotropic strong, and with 40 kinds of common drug no cross reactions.
(2) polystyrene latex microparticles used in the present invention and antibody coupling compound, can be significantly increased first
The sensitivity for analysis of aminopterin measure reagent.Meanwhile reagent of the present invention is not limited to monospecific antibody to the application method of antibody, together
Sample is suitable for the method that Multiple Antibodies are used in mixed way, and it is polyclonal to be related to the anti-methotrexate (MTX) monoclonal antibody of mouse, rabbit-anti methotrexate (MTX)
Antibody, goat-anti methotrexate (MTX) polyclonal antibody etc..
(3) methotrexate (MTX) conjugate of the invention, good water solubility and high sensitivity；With the glue for being coupled anti-methotrexate (MTX) antibody
The reaction sensitivity of lactoconium is high, is conjugated the mechanism of competitive reaction and can accurately, delicately measure containing for methotrexate (MTX) in serum
Amount.
Kit high sensitivity, high specificity and stability of the present invention are good, there is preferable potential applicability in clinical practice.The present invention tries
Agent box is prepared simply, is suitable for industrialized production, there is larger application value.
Description of the drawings
Fig. 1 is calibration curve of the reagent of the preparation of embodiment 4 on 7100 type automatic clinical chemistry analyzer of Hitachi, and X-axis is
Concentration of Methotrexate, Y-axis are absorbance change value.
Fig. 2 is that reagent prepared by embodiment 4 is carried out with moral spirit reagent on 7100 type automatic clinical chemistry analyzer of Hitachi
Sample compares correlation results.
Specific implementation mode
The present invention is further described in detail with reference to embodiments, but is not limited solely to following embodiment.
Embodiment is using raw material by being commercially available.
Embodiment 1
The synthesis of KLH- methotrexate (MTX) immunogenes
KLH- methotrexate (MTX) immunogenes are the antigen used in reagent R2 in the preparation process of antibody starting material, by blood indigo plant egg
(KLH) and the terminal carboxyl group of methotrexate (MTX) are formed by connecting in vain, and the specific synthesis step of the immunogene is as follows：
(1) 2.13g 2- (N- morpholines) ethanesulfonic acid, 8.5g sodium chloride, 0.95g magnesium chlorides are weighed, 1L deionizations are dissolved in
In water, pH to 6.1 is adjusted, buffer solution A is made；
(2) 3mg KLH are weighed, is dissolved at room temperature in the above-mentioned buffer solution A of 3mL, KLH solution is made；
(3) 3mg methotrexate (MTX)s are weighed, is dissolved in the above-mentioned buffer solution A of 300ul, methotrexate (MTX) solution is made；
(4) 5mg NHS and 1mg EDCHCl are added in methotrexate (MTX) solution, after reacting 30 minutes, by above-mentioned KLH solution
It is added dropwise in methotrexate (MTX) solution, then stirs this mixed solution 2 hours at 2-8 DEG C, obtained after the closing of 2ml ethanol amines is added
To mixed solution；
(5) the above-mentioned mixed solution after reaction is dialysed with above-mentioned buffer solution A, acquired solution is first after dialysis
0.1% NaN is added in aminopterin immunogen solution in methotrexate (MTX) immunogen solution3, stored at -20 DEG C.0.1%
NaN3Refer to the mass percent for the immunogen solution that addition accounts for final gained, specific addition is according to gained after dialysis
Depending on the specific quality of immunogen solution.
Embodiment 2
The preparation of anti-methotrexate (MTX) specific antibody
Anti- methotrexate (MTX) specific antibody is specific antibody used in reagent R2.By KLH- first ammonia obtained above
Pterin immunogene uses conventional method inoculation experiments animal rabbit, takes antiserum after booster immunization, is as follows：
(1) the KLH- methotrexate (MTX) immunogenes of above-mentioned synthesis are diluted to 1.0mg/ml with PBS, obtain antigenic solution, so
It is mixed afterwards with Freund's complete adjuvant with 1.0ml antigenic solutions, experimental animal rabbit is injected.
After (2) 3 weeks, then with the identical antigenic solutions of 1.0ml and incomplete Freund's adjuvant above-mentioned experimental animal rabbit is injected
Once, primary every surrounding injection later, amount to injection 4 times.
(3) blood is taken to above-mentioned experimental animal rabbit, isolates and purifies to obtain the anti-methotrexate (MTX) that potency is 1: 30000-1: 50000
Specific antibody.
Embodiment 3
The preparation of methotrexate (MTX) conjugate
(1) 2.13g 2- (N- morpholines) ethanesulfonic acid, 8.5g sodium chloride are weighed, is dissolved in 1L deionized waters, pH is adjusted
To 6.1, buffer solution A is made
(2) 1.0g methotrexate (MTX)s (MTX) are dissolved in buffer solution A, and 50mgN- HOSu NHSs (NHS) and idol is added
Join agent 30mg 1- (3- dimethyl propyls) -3- ethyl-carbodiimide hydrochlorides (EDCHCl), reaction forms MTX- after 30 minutes
The intermediate fat of NHS activation；
(2) 50mg chitosans are added in above-mentioned solution, react 2 hours at room temperature, methotrexate (MTX) and chitosan condensation are anti-
The conjugate of methotrexate (MTX)-chitosan should be formed；
(3) 0.5ml ethanol amines are added to inactivate intermediate fat body；
(4) reaction solution is dialysed in bag filter, and in the phosphate buffer of 20mmol/L, 2-8 DEG C is dialysed 12 hours, cold
It is lyophilized dry, obtains the sterling of methotrexate (MTX)-chitosan conjugate, as methotrexate (MTX) conjugate.
Embodiment 4
It is prepared by the kit that the present invention measures human serum methotrexate (MTX) content
The preparation of reagent R1：Take 995g water, then sequentially add 2.4g sodium dihydrogen phosphates, 11.4g disodium hydrogen phosphates,
10.0g PEG 20000s, 50mg methotrexate (MTX)s conjugate (embodiment 3 is made), 9.0gNaCl, 0.5g polysorbas20,5.0g oxen
Seralbumin (w/w) and 0.2gProclin 300, the needs that add materials every time are stirred to being completely dissolved, and adjust pH to 7.4；
With 0.22 μm of membrane filtration, the reagent R1 of 1L is made.
The preparation of reagent R2：The polyphenyl second for being combined with methotrexate (MTX) antibody containing 0.3% in the Mops buffer solutions of 50mmol/L
Alkene present latex particulate (w/w), 1%NaCl (w/w), 0.05% polysorbas20 (w/w), 1%BSA (w/w), 0.02%Proclin 300
(w/w).It is as follows：
1. taking 4.5ml (100mg/ml) latex (a diameter of 200nm) respectively, respectively use 0.1M (mol/L), the MES of pH6.5 molten
Liquid (2-morpholine ethane sulfonic acid buffer solution) washs 3 times (each 4.5ml), dispersion；
2. 1- ethyls -3- (3- DimethylAminopropyls) carbon of the MES solution Fresh of 0.3ml 0.1M, pH6.5 is added
Change diimmonium salt hydrochlorate (EDAC) (20mg/ml) and mixes complete, (20~24 DEG C) mixing 30min of room temperature；
3. using 50mM, the phosphate buffer solution of pH7.0 dilutes 10 times after washing 1 time, and it is spare to obtain 45ml latex solutions；
4. 4.5ml rabbit-anti methotrexate (MTX) polyclonal antibodies (8mg/ml) is taken to be added to the 60nm latex activated in step 3
In solution；Separately 1.5ml rabbit-anti methotrexate (MTX) polyclonal antibodies (8mg/ml) is taken to be added to the 200nm latex activated in step 3
In solution；Covalent coupling is carried out respectively at (25-30 DEG C) reaction 2h of room temperature.
5. the reaction solution for two kinds of different-grain diameters that step 4 is obtained is in tangential flow filtration system (KrosFlo MiniKros
Piloti (KPMi)) in be filtered washing, the filter membrane in tangential flow filtration column be hollow-fibre membrane (aperture 500kD), washing
Liquid is 50mM, the Mops buffer solutions of pH7.4, removes free antibodies, small molecular weight impurity, is prepared in 4-6 times of step 4 with filtering
The amount of liquor of reaction solution quality is washing terminal, the present latex particulate for being combined with anti-methotrexate (MTX) antibody after being washed；
5. 10% aqueous trehalose of polyoxyethylene sorbitan monopalmitate and 0.45ml of 0.9ml 10% is added
It is closed, 25 DEG C of -30 DEG C of closing 1h of room temperature；
6. with liquid (1.0%BSA, 0.05% polysorbas20,0.05% trehalose, 0.02%Proclin 300 is preserved
50mM, pH7.4Mops buffer solution) it step 5 is prepared is diluted to latex final concentration of 0.3%, the as examination of 200nm grain sizes
Agent R2.
The preparation of calibration object：1L water is taken, buffer, sodium chloride, sodium ethylene diamine tetracetate (EDTA), ox are then sequentially added
Seralbumin (BSA), methotrexate (MTX), Sodium azide, the needs that add materials every time are stirred to being completely dissolved, and adjust pH value to 7.0
Then~8.0, preferably 7.4 cross 0.22 μm of filter membrane and obtain calibration object solution.Calibration object is carried out using commercially available moral spirit kit
Assignment.
Form kit：By the R1 reagents of above-mentioned preparation, R2 reagents R1 by volume：R2=1:1 is distributed into bottle composition examination
The packing specification of agent box, kit is：R1 (every bottle of 2 × 45ml/), R2 (every bottle of 2 × 45ml/).
Embodiment 5
It is prepared by the kit that the present invention measures human serum methotrexate (MTX) content
The preparation of reagent R1：Take 995g water, then sequentially add 2.4g sodium dihydrogen phosphates, 11.4g disodium hydrogen phosphates,
15.0g PEG 20000s, 50mg methotrexate (MTX)s conjugate, 9.0gNaCl, 0.8g polysorbas20,5.0g bovine serum albumin(BSA)s (w/
W) it is stirred with 0.2gProclin 300, the needs that add materials every time to being completely dissolved, adjusts pH to 7.2；With 0.22 μm of filter membrane mistake
Filter, is made the reagent R1 of 1L.
The preparation of reagent R2：The polyphenyl second for being combined with methotrexate (MTX) antibody containing 0.3% in the Mops buffer solutions of 50mmol/L
Alkene present latex particulate (w/w), 1%NaCl (w/w), 0.05% polysorbas20 (w/w), 1%BSA (w/w), 0.02%Proclin 300
(w/w).Wherein, the present latex particulate of small particle is coupled the anti-methotrexate (MTX) monoclonal antibody of mouse, and large-sized present latex particulate is coupled rabbit
Anti- methotrexate (MTX) polyclonal antibody.It is as follows：
1. 6.0ml (100mg/ml) latex (a diameter of 250nm) is taken, with 0.1M (mol/L), the MES solution (2- of pH6.5
Morpholino b acid buffer solution) it washs 3 times, dispersion；
2. EDAC (20mg/ml) mixing that the MES solution Fresh of 0.4ml 0.1M, pH6.5 is added is complete, room temperature
(20~24 DEG C) mixing 30min；
3. using 50mM, the PBS solution of pH7.0 dilutes 10 times after washing 1 time, and it is spare to obtain 60ml latex solutions；
4. the anti-methotrexate (MTX) monoclonal antibody (10mg/ml) of 1.0ml mouse is taken to be added to the 60nm latex activated in step 3
In solution, 2h is reacted at room temperature；
5. the reaction solution that step 4 is obtained is in tangential flow filtration system (KrosFlo MiniKros Piloti (KPMi))
In be filtered washing, the filter membrane in tangential flow filtration column be hollow-fibre membrane (aperture 500kD), cleaning solution 50mM, pH7.4
Mops buffer solutions, free antibodies, small molecular weight impurity are removed, to filter washing for the reaction solution quality prepared in 4-6 times of step 4
It is washing terminal, the present latex particulate for being combined with anti-methotrexate (MTX) antibody after being washed to wash liquid measure；
5. 10% aqueous trehalose of polyoxyethylene sorbitan monopalmitate and 0.60ml of 1.2ml 10% is added
It is closed, room temperature closes 1h；
6. with liquid is preserved (containing 1.0%BSA, 0.05% polysorbas20,0.05% trehalose, 0.02%Proclin 300
50mM, pH7.4Mops buffer solution) reaction solution after above-mentioned closing is diluted to latex final concentration of 0.3%, obtain 200ml
250nm reagents R2.
The preparation of calibration object：1L water is taken, buffer, sodium chloride, sodium ethylene diamine tetracetate (EDTA), ox are then sequentially added
Seralbumin (BSA), methotrexate (MTX), Sodium azide, the needs that add materials every time are stirred to being completely dissolved, and adjust pH value to 7.0
Then~8.0, preferably 7.4 cross 0.22 μm of filter membrane and obtain calibration object solution.Using commercially available moral spirit assay kit to calibration object
Carry out assignment.
Form kit：By the R1 reagents of above-mentioned preparation, R2 reagents, R1 by volume：R2=1:1 composition kit, examination
The packing specification of agent box is：R1 (every bottle of 2 × 45ml/), R2 (every bottle of 2 × 45ml/).
Embodiment 6
The present invention measures the correlation analysis analysis method of the kit (embodiment 4 is made) of methotrexate (MTX)：2 terminals
Method.Reagent R1 150ul are taken, sample 20ul is added, reagent R2 150ul are added after 37 DEG C of 5min, start to read, react 5min,
Read point again obtains absorbance difference (△ A).
Computational methods：Multiple spot is calibrated, and calibration curve is made according to the value of absorbance and reference serum, by standard curve in terms of
Calculation pattern, sample content can be calculated according to its absorbance on curve.
The range of linearity：0.02-10.00umol/L
Detecting instrument：7100 type automatic clinical chemistry analyzer of Hitachi.
Detection wavelength：600nm.
The clinical test results of 4 reagent of embodiment and the methotrexate (MTX) kit of De Ling diagnostic products (Shanghai) Co., Ltd.
Compare：
After 4 reagent of embodiment is calibrated respectively with moral spirit reagent, reagent calibration curve such as Fig. 1 of the present embodiment 4, detection 50
The clinical sample of example various concentration, testing result are as shown in table 1：
Table 1
According to measurement result above, this reagent and the clinical correlation of moral spirit methotrexate (MTX) assay kit are good, do not have
Significant difference.
Embodiment 7
Interfering effects of drug is tested
Take the normal healthy human blood's sample of physical examination (no apparent haemolysis, jaundice, chyle) that pooled serum (samples sources are made
In Zhongshan Hospital of Fudan University, Shanghai), precision weighs 40 kinds of Common drugs, is dissolved in the molten of above-mentioned similar human serum matrix
In liquid, the chaff interferent solution of 40 groups of 100umol/L is made.It is accurate respectively to measure high concentration chaff interferent solution and mix normal blood
Clearly, the pooled serum sample (interferent concentration 10.0umol/L) of 40 groups of disturbance objects, each sample replication 3 is made
It is secondary, take its average value.Common interference drug and measurement result
Table 2
Measurement result：The concentration that above-mentioned 40 kinds of Common drugs are equivalent to methotrexate (MTX) is respectively less than 0.1umol/L, it is seen then that this
The antibody of invention is the specific antibody of anti-methotrexate (MTX).
It should be noted that example the above is only the implementation of the present invention, is not intended to limit the scope of the invention,
It is every to be directly or indirectly used in other correlative technology fields using what description of the present invention and accompanying drawing content were done, similarly
It is included within the scope of the present invention.
Claims (10)
1. a kind of methotrexate (MTX) latex immunoturbidimetry assay kit, which is characterized in that the kit is by reagent R1 and reagent
R2 is formed；The volume ratio of the reagent R1 and reagent R2 is 1:1.
2. kit according to claim 1, which is characterized in that the reagent R1 is grouped by the group that following weight percent matches
At：
0.01%~4% emulsifier, 0.01%~4% methotrexate (MTX) conjugate, 0.1%~2% sodium chloride, 0.05%~
2% sensitizer, 0.02% preservative, 88.00%~99.80% 30~100mmol/L buffer solutions.
3. kit according to claim 2, which is characterized in that emulsifier described in reagent R1 be selected from polysorbas20, Tween 80 or
Triton X-100；The sensitizer is selected from Macrogol 4000, Macrogol 6000, PEG 8000 or poly- second
Glycol 20000, preferably PEG 20000；The buffer solution is selected from phosphate buffer, trishydroxymethylaminomethane buffers
Liquid, 3- (N- morpholinyls) propane sulfonic acid buffer solution, 2- (N- morpholines) ethanesulfonic acid buffer, borate buffer solution, glycine buffer
Or it is one or more in hydroxyethyl piperazine second sulphur acid buffer；The pH of cushioning fluid is 6~9.
4. kit according to claim 1, which is characterized in that the reagent R2 is grouped by the group that following weight percent matches
At：
0.1%~0.5% be combined with anti-methotrexate (MTX) antibody present latex particulate, 0.1%~1.0% bovine serum albumin(BSA),
0.02%~2% polysorbas20,0.02%~2% stabilizer, 0.02%~2% protein protective agent, 0.02% preservative,
92.50%~97.70% 20~50mmol/L buffer solutions.
5. kit according to claim 4, which is characterized in that the present latex particulate for being combined with anti-methotrexate (MTX) antibody
For：The compound for selecting the carboxylic polystyrene present latex particulate of different-grain diameter to be formed with anti-methotrexate (MTX) antibody；The difference
A diameter of 50~300nm of the nano rubber latex particle of grain size.
6. the kit according to claim 4 or 5, which is characterized in that the anti-methotrexate (MTX) antibody is selected from more grams of rabbit-anti
Grand antibody, goat-anti clonal antibody or the anti-monoclonal antibody of mouse.
7. kit according to claim 4, which is characterized in that the stabilizer is polyoxyethylene resin class, is selected from polyoxy
Ethylene sorbitan mono-oleic acid ester, polyoxyethylene sorbitan monopalmitate or ethoxylated dodecyl alcohol sodium sulfovinate
Salt, protein protective agent are trehalose；The buffer solution is selected from phosphate buffer, TRIS buffer, 2-
The horizontal acid buffer of quinoline second, borate buffer solution, glycine buffer or one kind in hydroxyethyl piperazine second sulphur acid buffer or
It is a variety of；The pH of cushioning fluid is 6~9；The preservative is selected from sodium azide, N- methyl-isothiazols ketone, N'- methylene-bis-
It is one or more in [N'- (3- methylol -2,5- diketone -4- imidazolidinyls)] urea or ProClin 300.
8. measuring the preparation method of the kit of human serum methotrexate (MTX) content as described in claim 1, which is characterized in that should
Method includes the following steps：
I, reagent preparation R1：1L water is taken, sodium chloride, sensitizer, emulsifier, methotrexate (MTX) conjugate, anti-corrosion are then sequentially added
Agent, the needs that add materials every time are stirred to being completely dissolved, and are adjusted pH value to 6.0~9.0, preferably 7.4, are then crossed 0.22 μm of filter membrane
Obtain reagent R1；
II, reagent preparation R2：
(1) methotrexate (MTX) immunogene is synthesized：The amino on the carboxyl and carrier protein of methotrexate (MTX) end is set to connect, carrier is tool
There are the protein of immunogenicity, preferably hemocyanin (KLH), haemocyanin (BSA) or thyroglobulin；
(2) antibody starting material used in R2 is prepared：With methotrexate (MTX) immunogen immune animal, prepares and to purify anti-methotrexate (MTX) special
Property antibody；Monoclonal antibody is prepared with somatocyte hybriding technology.
The preparation process of reagent R2 is as follows：
(1) activation of polystyrene latex microparticles：1- ethyl -3- (3- are added in the polystyrene latex microparticles with carboxyl
DimethylAminopropyl) carbodiimide hydrochloride, -25 DEG C of activation 30min of 20 DEG C of room temperature after mixing；
(2) coupling of anti-methotrexate (MTX) antibody and polystyrene latex microparticles：The polystyrene latex activated in step (1) is micro-
After grain and the mixing of anti-methotrexate (MTX) antibody, covalent coupling is carried out in 20 DEG C of -25 DEG C of reaction 2h of room temperature；
(3) it is combined with the washing of the present latex particulate compound of anti-methotrexate (MTX) antibody：The glue of anti-methotrexate (MTX) antibody will be combined with
The reaction solution of lactoconium is filtered washing in tangential flow filtration system, removes free antibodies, small molecular weight impurity；The washing
Liquid is 50~100mM pH of buffer 7.0~8.0, the present latex particulate for being combined with anti-methotrexate (MTX) antibody after being washed；
(4) closing and preservation of the present latex particulate of anti-methotrexate (MTX) antibody are combined with：Combination after the washing that step (5) is obtained
Have and polyoxyethylene sorbitan monopalmitate and trehalose are added in the present latex particulate of anti-methotrexate (MTX) antibody, closes polyphenyl
Unreacted radical and other adsorption sites on ethylene latex particle obtain initial action liquid after closing 1h；
(6) 3-6 times is diluted to above-mentioned initial action liquid using buffer solution and obtains final reagent R2；
III, kit is formed：By the reagent R1 of above-mentioned preparation and reagent R2 R1 by volume：R2=1:1 packing composition kit.
9. preparation method according to claim 8, which is characterized in that methotrexate (MTX) conjugate described in reagent R1 passes through first ammonia
Pterin prepares with chitosan reaction, includes the following steps：
(1) in the 2- of 50mmol/L (N- morpholines) ethanesulfonic acid buffer, 1.0g methotrexate (MTX)s and 50mgN- hydroxysuccinimidyl acyls
Imines is under the effect of coupling agent 30mg 1- (3- dimethyl propyls) -3- ethyl-carbodiimide hydrochlorides, at 20 DEG C -25 DEG C of room temperature
Reaction 30 minutes forms the intermediate fat of MTX-NHS activation；
(2) 5.0g chitosans, MTX is added：Chitosan is 1：10 mass ratioes react 2 hours at 20 DEG C -25 DEG C of room temperature, are condensed
Reaction forms the conjugate of methotrexate (MTX)-chitosan；
(3) inactivation of the intermediate fat of MTX-NHS activation, is directly added into 0.5ml ethanol amines and is inactivated to intermediate fat body, reacted at room temperature
30 minutes；
(4) reaction solution after inactivating is in bag filter, and in the phosphate buffer of 20mmol/L, 2-8 DEG C is dialysed 12 hours, cold
Freeze the sterling for being dried to obtain methotrexate (MTX)-chitosan conjugate, as methotrexate (MTX) conjugate.
10. preparation method according to claim 8, which is characterized in that reagent preparation R1 adjusts pH value to 7.4.
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| CN109541241A (en) * | 2019-01-25 | 2019-03-29 | 浙江夸克生物科技有限公司 | A kind of assay kit of lipoprotein (a) |
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