CN112763702B - Latex-enhanced immunonephelometry kit for detecting imatinib blood concentration and preparation method thereof - Google Patents
Latex-enhanced immunonephelometry kit for detecting imatinib blood concentration and preparation method thereof Download PDFInfo
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- 239000005517 L01XE01 - Imatinib Substances 0.000 title claims abstract description 53
- 229960002411 imatinib Drugs 0.000 title claims abstract description 53
- KTUFNOKKBVMGRW-UHFFFAOYSA-N imatinib Chemical compound C1CN(C)CCN1CC1=CC=C(C(=O)NC=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)C=C1 KTUFNOKKBVMGRW-UHFFFAOYSA-N 0.000 title claims abstract description 52
- 239000004816 latex Substances 0.000 title claims abstract description 43
- 229920000126 latex Polymers 0.000 title claims abstract description 43
- 210000004369 blood Anatomy 0.000 title claims abstract description 20
- 239000008280 blood Substances 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 55
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims abstract description 28
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 28
- 238000003018 immunoassay Methods 0.000 claims abstract description 23
- 239000007853 buffer solution Substances 0.000 claims abstract description 18
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 14
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 14
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 14
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- 238000001514 detection method Methods 0.000 claims abstract description 10
- 229920002307 Dextran Polymers 0.000 claims abstract description 7
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 30
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 19
- 239000007995 HEPES buffer Substances 0.000 claims description 19
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- 238000006243 chemical reaction Methods 0.000 claims description 10
- 102000014914 Carrier Proteins Human genes 0.000 claims description 9
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- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 9
- 230000002163 immunogen Effects 0.000 claims description 9
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
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- GVJXGCIPWAVXJP-UHFFFAOYSA-N 2,5-dioxo-1-oxoniopyrrolidine-3-sulfonate Chemical compound ON1C(=O)CC(S(O)(=O)=O)C1=O GVJXGCIPWAVXJP-UHFFFAOYSA-N 0.000 claims description 3
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 claims description 3
- 239000007993 MOPS buffer Substances 0.000 claims description 3
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- 238000000502 dialysis Methods 0.000 claims description 3
- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 claims description 3
- 239000012452 mother liquor Substances 0.000 claims description 3
- 238000011587 new zealand white rabbit Methods 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 claims description 3
- 239000012460 protein solution Substances 0.000 claims description 3
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- 238000001647 drug administration Methods 0.000 description 2
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- QAWFLJGZSZIZHO-UHFFFAOYSA-N methyl 4-bromobutanoate Chemical compound COC(=O)CCCBr QAWFLJGZSZIZHO-UHFFFAOYSA-N 0.000 description 1
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Classifications
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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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
-
- 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
- G01N33/531—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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a latex enhanced turbidimetric immunoassay kit for detecting imatinib blood concentration and a preparation method thereof, comprising the following steps: reagent R1 buffer solution, reagent R2 buffer solution and calibrator; the reagent R1 buffer solution comprises 25mM MES, 0.1wt% bovine serum albumin, 0.9 wt% NaCl, 1wt% D-trehalose, 2.75wt% dextran, 0.1wt% sodium azide, 1 mug/ml imatinib-bovine serum albumin complex and detection antigen; the reagent R2 buffer included 50mM HEPES-HCl, 0.5vol%Blockmaster DB1130, 0.9% wt NaCl, 1% wt D-trehalose, 0.1% wt sodium azide, 0.02% T-20, 0.1% wt bovine serum albumin, and 0.11% wt anti-imatinib antibody conjugated polystyrene latex microspheres and imatinib antibody. The invention has the advantages of simple and quick operation and high sensitivity, and can be applied to an automatic biochemical analyzer.
Description
Technical Field
The invention relates to the technical field of reagent detection, in particular to a latex enhanced immunonephelometry kit for detecting imatinib blood concentration and a preparation method thereof.
Background
Imatinib, a tyrosine kinase inhibitor, is a small molecule protein kinase inhibitor that has the effect of blocking one or more protein kinases. Clinically, the composition is used for treating chronic myelogenous leukemia and malignant gastrointestinal stromal tumors, in particular to treating patients with chronic myelogenous leukemia in embryonic crisis stage, accelerated stage or chronic stage of failure of interferon-alpha treatment. Because of the large degree of pharmacokinetic variability of imatinib and its salts in and among individuals, and due to a number of factors, including: organ function, genetic modulation, disease state, age, drug interactions, drug intake time, compliance, etc., equal doses of the same drug in different individuals can result in significantly different clinical effects, the effectiveness of the same dose of imatinib or a salt thereof varies significantly depending on individual drug clearance and final serum drug concentration in the patient. The monitoring of the drug concentration in blood can enable a clinician to know the individual differences of patients in the drug administration process in time, and to conduct individual adjustment on the drug administration amounts of different patients, so that the treatment effect can be effectively improved, the toxic and side effects of the drug can be obviously reduced, and the method has important significance for clinically reasonable medication.
At present, the analysis methods for detecting imatinib at home and abroad mainly comprise HPLC-UV, LC-MS-MS and the like, but the methods are not suitable for clinical large-scale application and cannot meet the increasing clinical detection demands. At present, imatinib detection reagents with good stability, high sensitivity and strong specificity, in particular to automatic detection reagents with good quality are lacking in the market.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide the latex-enhanced turbidimetric immunoassay kit for detecting the imatinib blood concentration and the preparation method thereof, and the kit has the advantages of simplicity and rapidness in operation and high sensitivity, and can be applied to an automatic biochemical analyzer. To achieve the above objects and other advantages and in accordance with the purpose of the invention, there is provided a latex-enhanced turbidimetric immunoassay kit for detecting an imatinib blood concentration, comprising:
reagent R1 buffer solution, reagent R2 buffer solution and calibrator;
the reagent R1 buffer comprises 25mM MES, 0.1wt% bovine serum albumin, 0.9 wt% NaCl, 1wt% D-trehalose, 2.75wt% dextran, 0.1wt% sodium azide;
the reagent R2 buffer included 50mM HEPES-HCl, 0.5vol% Blockmaster DB1130, 0.9 wt% NaCl, 1wt% D-trehalose, 0.1wt% sodium azide, 0.02% T-20,0.1wt% bovine serum albumin.
The preparation method of the latex enhanced turbidimetric immunoassay kit for detecting the imatinib blood concentration comprises the following steps:
s1, preparing an imatinib derivative, wherein the structural formula of the imatinib derivative is as follows:
wherein R is a bondRadical- (CH) 2 ) n -COO-, n is an integer between 1 and 20;
s2, preparing imatinib immune antigen;
s3, coupling the imatinib derivative with Bovine Serum Albumin (BSA) to obtain an imatinib detection antigen;
s4, using male New Zealand white rabbits as immune animals, and using a conjugate of imatinib and blue carrier protein as an imatinib antibody;
s5, preparing imatinib sensitization latex microspheres;
s6, preparing a latex enhanced turbidimetric immunoassay reagent;
s7, adding imatinib standard substances into serum of normal people according to the requirement to obtain imatinib calibrator with different concentrations.
Preferably, in the step S1, when n=2, the synthesis steps are as follows:
preferably, the step S2 further includes:
s21, dissolving 100mg of blue carrier protein (CCH) in 20mL of 0.1M phosphate buffer with pH of 8.5;
s22, weighing 100mg of the imatinib derivative, 2mL of dimethylformamide, 2mL of ethanol, 4.0mL 10mM,pH 5.0 of phosphate buffer, 80mg of 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide and 20mg of N-hydroxysulfosuccinimide, and stirring and dissolving the chemicals at room temperature for 30min;
s23, dripping the dissolved solution into a carrier protein solution, and stirring overnight at 2-8 ℃ to obtain an antigen;
s24, purifying the synthesized antigen through dialysis to obtain the imatinib immunogen.
Preferably, the step S4 further includes:
s41, adopting a trace long-range immunization method, wherein the first immunization is carried out by mixing an immunogen solution and an adjuvant solution in equal volume, and the immunization dose of each rabbit is about 1mg of immunogen;
s42, three booster immunizations were performed with freund' S incomplete adjuvant.
Preferably, the step S5 further includes:
s51, adding 900 mu LMES buffer (containing 0.2% sodium chloride) and 100 mu L of microspheres (10%) into a centrifuge tube, and uniformly mixing;
s52, adding 1mg of EDC and 10mg of EDC for reaction, and oscillating at room temperature for 30min;
s53, ultracentrifugation for 40min, dumping the supernatant, adding 1.00mL HEPES buffer (pH 7.6) into each centrifuge tube, blowing off, and performing ultrasonic treatment for 10 times;
s54, repeating the steps for cleaning once;
s55, adding 20-100 mg of bridging arm reagent, and reacting for 30min;
s56, ultracentrifugation for 40min, dumping the supernatant, adding 1.00mL HEPES buffer (pH 7.6) into each centrifuge tube, blowing off, and performing ultrasonic treatment for 10 times;
s57, adding 1mg of EDC and 10mg of EDC again for reaction, and oscillating at room temperature for 30min;
s58, ultracentrifugation for 40min, dumping the supernatant, adding 1.00mL HEPES buffer (pH 7.6) into each centrifuge tube, blowing off, and performing ultrasonic treatment for 10 times;
s59, crosslinking: adding 0.1-0.5 mg of polyclonal antibody, immediately mixing, and performing ultrasonic treatment for 10 times; oscillating at room temperature for 90min;
s59, sealing: adding 40-80 mu L of sealing solution into 1mL of latex solution, uniformly mixing, carrying out ultrasonic treatment for 10 times, and carrying out oscillation reaction at room temperature for 30min;
s510, washing: centrifuging for 2 times: ultracentrifugation for 40min, decanting, adding HEPES buffer (pH 7.6) into the centrifuge tube, pipetting, ultracentrifugation, decanting the supernatant. The precipitated latex was blown off with 4ml HEPES; storing at 4deg.C to obtain crosslinked mother liquor with particle content of 0.25%.
Preferably, the latex-enhanced turbidimetric immunoassay in step S6 comprises two formulations, wherein the first formulation comprises: 25mM MES, 0.9% NaCl, 0.02% Tween-20, 0.05% NaN3, 1% trehalose, 2.75% dextran, 1000ng/mL IMA-CCH (pH 6.1); the second formulation comprises: microsphere content 0.125%, 50mM HEPES, 0.9% NaCl, 0.5%Blockmaster DB1130, 1% trehalose, 0.02% T-20 and 0.1% NaN3 (pH 7.6).
Preferably, in the preparation of the sensitized latex microsphere according to step S55, a bridging arm is coupled to the surface of the latex microsphere and then coupled to the antibody, wherein the bridging arm is NH2- (CH 2) n-COOH, n=6-20, or-NH 2- (CH 2 CHO) n-COOH n=6-20.
Preferably, the buffer in the buffer solution of the reagent R1 and the reagent R2 is selected from a buffer pair consisting of any one of the following reagents and a conjugate acid or a conjugate base thereof: 2- (N-morpholino) ethanesulfonic acid (MES), tris (hydroxymethyl) aminomethane (Tris), 3- (N-morphism) propanesulfonic acid (MOPS), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES)
Preferably, the pH of the buffer solutions of the reagent R1 and the reagent R2 is 5.0-9.0, preferably 5.5-7.5, and the concentration of the buffer solutions of the reagent R1 and the reagent R2 is 15-100mM.
Compared with the prior art, the invention has the beneficial effects that: the latex enhanced turbidimetric immunoassay kit for detecting the imatinib blood concentration and the preparation method thereof have the advantages that the variation coefficient is lower than 10.0%, the recovery rate is 93.0% -103.7%, the kit has good repeatability and accuracy, the reagent has good sample correlation with HPLC measured values, a relatively accurate reference value can be provided for clinic, the latex enhanced turbidimetric immunoassay kit which is easy and convenient to operate and low in price is provided, and the preparation method thereof can meet the increasing clinical monitoring requirement of imatinib, and compared with the traditional method, when in latex coupling, a bridging arm NH2- (CH 2) n-COOH, n=6-20, or-NH 2- (CH 2 CHO) n-COOH n=6-20 is coupled with an antibody, so that the antibody is subjected to relatively small steric hindrance by the microsphere, the situation that the antibody falls down on the surface of the microsphere is avoided, the antigen determinant of the antibody is ensured to be fully exposed, and the antigen binding with small molecular weight can be realized. Thereby ensuring the sensitivity and the specificity of the kit.
Drawings
FIG. 1 is a schematic diagram of a calibration curve obtained by performing multi-point calibration on a standard substance of a latex-enhanced turbidimetric immunoassay kit for detecting imatinib blood concentration and a preparation method thereof according to the present invention and calculating the calibration curve by using a spline function.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1, a latex-enhanced turbidimetric immunoassay kit for detecting imatinib blood concentration, comprising; reagent R1 buffer solution, reagent R2 buffer solution and calibrator;
the reagent R1 buffer comprises 25mM MES, 0.1wt% bovine serum albumin, 0.9 wt% NaCl, 1wt% D-trehalose, 2.75wt% dextran, 0.1wt% sodium azide;
the reagent R2 buffer included 50mM HEPES-HCl, 0.5vol% Blockmaster DB1130, 0.9 wt% NaCl, 1wt% D-trehalose, 0.1wt% sodium azide, 0.02% T-20,0.1wt% bovine serum albumin.
The preparation method of the latex enhanced turbidimetric immunoassay kit for detecting the imatinib blood concentration comprises the following steps: s1, preparing an imatinib derivative, wherein the structural formula of the imatinib derivative is as follows:
wherein R is a linking group- (CH) 2 ) n -COO-, n is an integer between 1 and 20;
s2, preparing imatinib immune antigen;
s3, coupling the imatinib derivative with Bovine Serum Albumin (BSA) to obtain an imatinib detection antigen;
s4, using male New Zealand white rabbits as immune animals, and using a conjugate of imatinib and blue carrier protein as an imatinib antibody;
s5, preparing imatinib sensitization latex microspheres;
s6, preparing a latex enhanced turbidimetric immunoassay reagent;
s7, adding imatinib standard substances into serum of normal people according to the requirement to obtain imatinib calibrator with different concentrations.
Further, in the step S1, when n=2, the synthesis steps are as follows:
when n is an integer other than 2, the synthesis steps of the imatinib derivative differ from the above synthesis steps only in that: in the second step, the starting methyl 4-bromobutyrate employed was replaced with its analogue.
Further, the step S2 further includes:
s21, dissolving 100mg of blue carrier protein (CCH) in 20mL of 0.1M phosphate buffer with pH of 8.5;
s22, weighing 100mg of the imatinib derivative, 2mL of dimethylformamide, 2mL of ethanol, 4.0mL 10mM,pH 5.0 of phosphate buffer, 80mg of 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide and 20mg of N-hydroxysulfosuccinimide, and stirring and dissolving the chemicals at room temperature for 30min;
s23, dripping the dissolved solution into a carrier protein solution, and stirring overnight at 2-8 ℃ to obtain an antigen;
s24, purifying the synthesized antigen through dialysis to obtain the imatinib immunogen.
Further, the step S4 further includes:
s41, adopting a trace long-range immunization method, wherein the first immunization is carried out by mixing an immunogen solution and an adjuvant solution in equal volume, and the immunization dose of each rabbit is about 1mg of immunogen;
s42, three booster immunizations were performed with freund' S incomplete adjuvant.
Further, the step S5 further includes:
s51, adding 900 mu LMES buffer (containing 0.2% sodium chloride) and 100 mu L of microspheres (10%) into a centrifuge tube, and uniformly mixing;
s52, adding 1mg of EDC and 10mg of EDC for reaction, and oscillating at room temperature for 30min;
s53, ultracentrifugation for 40min, dumping the supernatant, adding 1.00mL HEPES buffer (pH 7.6) into each centrifuge tube, blowing off, and performing ultrasonic treatment for 10 times;
s54, repeating the steps for cleaning once;
s55, adding 20-100 mg of bridging arm reagent, and reacting for 30min;
s56, ultracentrifugation for 40min, dumping the supernatant, adding 1.00mL HEPES buffer (pH 7.6) into each centrifuge tube, blowing off, and performing ultrasonic treatment for 10 times;
s57, adding 1mg of EDC and 10mg of EDC again for reaction, and oscillating at room temperature for 30min;
s58, ultracentrifugation for 40min, dumping the supernatant, adding 1.00mL HEPES buffer (pH 7.6) into each centrifuge tube, blowing off, and performing ultrasonic treatment for 10 times;
s59, crosslinking: adding 0.1-0.5 mg of polyclonal antibody, immediately mixing, and performing ultrasonic treatment for 10 times; oscillating at room temperature for 90min;
s59, sealing: adding 40-80 mu L of sealing solution into 1mL of latex solution, uniformly mixing, carrying out ultrasonic treatment for 10 times, and carrying out oscillation reaction at room temperature for 30min;
s510, washing: centrifuging for 2 times: ultracentrifugation for 40min, decanting, adding HEPES buffer (pH 7.6) into the centrifuge tube, pipetting, ultracentrifugation, decanting the supernatant. The precipitated latex was blown off with 4ml HEPES; storing at 4deg.C to obtain crosslinked mother liquor with particle content of 0.25%.
Further, the latex enhanced turbidimetric immunoassay in step S6 includes two formulations, wherein the first formulation includes: 25mM MES, 0.9% NaCl, 0.02% Tween-20, 0.05% NaN3, 1% trehalose, 2.75% dextran, 1000ng/mL IMA-CCH (pH 6.1); the second formulation comprises: microsphere content 0.125%, 50mM HEPES, 0.9% NaCl, 0.5%Blockmaster DB1130, 1% trehalose, 0.02% T-20 and 0.1% NaN3 (pH 7.6).
The latex enhanced turbidimetric immunoassay in step S6 comprises two formulations, the first formulation comprising: 25mM MES, 0.9% NaCl, 0.02% Tween-20, 0.05% NaN3, 1% trehalose, 2.75% dextran, 1000ng/mL IMA-CCH (pH 6.1); the second formulation comprises: microsphere content 0.125%, 50mM HEPES, 0.9% NaCl, 0.5%Blockmaster DB1130, 1% trehalose, 0.02% T-20 and 0.1% NaN3 (pH 7.6).
Further, in the preparation of the sensitized latex microsphere according to step S55, a bridging arm is coupled to the surface of the latex microsphere and then coupled to the antibody, wherein the bridging arm is NH2- (CH 2) n-COOH, n=6-20, or-NH 2- (CH 2 CHO) n-COOH n=6-20.
Further, the buffer in the buffer solution of the reagent R1 and the reagent R2 is selected from a buffer pair consisting of any one of the following reagents and conjugate acid or conjugate base thereof: 2- (N-morpholino) ethanesulfonic acid (MES), tris (hydroxymethyl) aminomethane (Tris), 3- (N-morphism) propanesulfonic acid (MOPS), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES)
Further, the pH of the buffer solutions of the reagent R1 and the reagent R2 is 5.0-9.0, preferably 5.5-7.5, and the concentration of the buffer solutions of the reagent R1 and the reagent R2 is 15-100mM.
During the experimental test results:
pure imatinib was added to the blank serum. The recovery and coefficient of variation for each concentration were calculated from 5 replicates of 3 days, and the recovery was quantitatively calculated according to the plotted linear equation of the working curve, with the results shown in the following table:
TABLE 1 Imatinib detection values
From the measurement results, the variation coefficient is lower than 10.0%, and the recovery rate is 93.0% -103.7%, which shows that the kit has good repeatability and accuracy.
2 clinical sample alignment
Testing the reagent prepared by the proportion by using a Hitachi 7180 full-automatic biochemical analyzer, wherein the test wavelength is 546nm, taking a sample or calibrator 3uL, adding 120uL of reagent R1, keeping the temperature at 37 ℃ for 5min, adding 60uL of reagent R2, reading absorbance A1 after 20s, and reading absorbance A2 after incubation for 4 minutes and 40 seconds at 37 ℃, so as to obtain reaction absorbance delta A=A2-A1; firstly, performing multi-point calibration by using a standard substance, and calculating by using a spline function to obtain a calibration curve, as shown in figure 1. The sample is subjected to clinical sample measurement through the change of absorbance, and the detection result is compared with the HPLC measurement result, so that the verification result is as follows:
the sample comparison result shows that the kit prepared by the invention has better sample correlation with the HPLC measurement value, and can provide more accurate reference value for clinic.
The number of devices and the scale of processing described herein are intended to simplify the description of the invention, and applications, modifications and variations of the invention will be apparent to those skilled in the art. Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
Claims (8)
1. The preparation method of the latex enhanced turbidimetric immunoassay reagent for detecting the blood concentration of imatinib is characterized by comprising the following steps of:
s1, preparing an imatinib derivative, wherein the structural formula of the imatinib derivative is as follows:
wherein R is a linking group- (CH) 2 ) n -COO-, n is an integer between 1 and 20;
s2, preparing imatinib immune antigen;
s3, coupling the imatinib derivative with Bovine Serum Albumin (BSA) to obtain an imatinib detection antigen;
s4, using male New Zealand white rabbits as immune animals, and using a conjugate of imatinib and blue carrier protein as an imatinib antibody;
s5, preparing imatinib sensitization latex microspheres;
s6, adding imatinib standard substances into serum of normal people according to the requirement to obtain imatinib calibrator with different concentrations;
the kit also comprises a latex enhanced turbidimetric immunoassay kit, wherein the kit comprises a reagent R1 buffer solution, a reagent R2 buffer solution and a calibrator;
the reagent R1 buffer comprises 25mM MES, 0.1wt% bovine serum albumin, 0.9 wt% NaCl, 1wt% D-trehalose, 2.75wt% dextran, 0.1wt% sodium azide;
the reagent R2 buffer included 50mM HEPES-HCl, 0.5vol%Blockmaster DB1130, 0.9% wt NaCl, 1% wt D-trehalose, 0.1% wt sodium azide, 0.02% T-20, 0.1% wt bovine serum albumin.
2. The method for preparing the latex-enhanced turbidimetric immunoassay reagent for detecting the blood concentration of imatinib according to claim 1, wherein in the step S1, when n=2, the synthesis steps are as follows:
3. the method for preparing a latex-enhanced turbidimetric immunoassay reagent for detecting imatinib blood concentration according to claim 1, wherein the step S2 further comprises:
s21, dissolving 100mg of blue carrier protein (CCH) in 20mL of 0.1M phosphate buffer with pH of 8.5;
s22, weighing 100mg of the imatinib derivative, 2mL of dimethylformamide, 2mL of ethanol, 4.0mL 10mM,pH 5.0 of phosphate buffer, 80mg of 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide and 20mg of N-hydroxysulfosuccinimide, and stirring and dissolving the chemicals at room temperature for 30min;
s23, dripping the dissolved solution into a carrier protein solution, and stirring overnight at 2-8 ℃ to obtain an antigen;
s24, purifying the synthesized antigen through dialysis to obtain the imatinib immunogen.
4. The method for preparing a latex-enhanced turbidimetric immunoassay for detecting imatinib blood concentration according to claim 1, wherein the step S4 further comprises:
s41, adopting a trace long-range immunization method, wherein the first immunization is carried out by mixing an immunogen solution and an adjuvant solution in equal volume, and the immunization dose of each rabbit is about 1mg of immunogen;
s42, three booster immunizations were performed with freund' S incomplete adjuvant.
5. The method for preparing a latex-enhanced turbidimetric immunoassay for detecting imatinib blood concentration according to claim 1, wherein the step S5 further comprises:
s51, adding 900 mu LMES buffer (containing 0.2% sodium chloride) and 100 mu L of microspheres (10%) into a centrifuge tube, and uniformly mixing;
s52, adding 1mg of EDC and 10mg of EDC for reaction, and oscillating at room temperature for 30min;
s53, ultracentrifugation for 40min, dumping the supernatant, adding 1.00mL HEPES buffer (pH 7.6) into each centrifuge tube, blowing off, and performing ultrasonic treatment for 10 times;
s54, repeating the steps for cleaning once;
s55, adding 20-100 mg of bridging arm reagent, and reacting for 30min;
s56, ultracentrifugation for 40min, dumping the supernatant, adding 1.00mL HEPES buffer (pH 7.6) into each centrifuge tube, blowing off, and performing ultrasonic treatment for 10 times;
s57, adding 1mg of EDC and 10mg of EDC again for reaction, and oscillating at room temperature for 30min;
s58, ultracentrifugation for 40min, dumping the supernatant, adding 1.00mL HEPES buffer (pH 7.6) into each centrifuge tube, blowing off, and performing ultrasonic treatment for 10 times;
s59, crosslinking: adding 0.1-0.5 mg of polyclonal antibody, immediately mixing, and performing ultrasonic treatment for 10 times; oscillating at room temperature for 90min;
s59, sealing: adding 40-80 mu L of sealing solution into 1mL of latex solution, uniformly mixing, carrying out ultrasonic treatment for 10 times, and carrying out oscillation reaction at room temperature for 30min;
s510, washing: centrifuging for 2 times: ultracentrifugation for 40min, decanting, adding HEPES buffer (pH 7.6) into the centrifuge tube, blowing off, ultracentrifugation again, decanting the supernatant, and blowing off the precipitated latex with 4 mLHEPES; storing at 4deg.C to obtain crosslinked mother liquor with particle content of 0.25%.
6. The method for preparing the latex-enhanced turbidimetric immunoassay reagent for detecting the blood concentration of imatinib according to claim 5, wherein in the step S55, a bridging arm is coupled to the surface of the latex microsphere before the latex microsphere is coupled to the antibody, and the bridging arm is NH2- (CH 2) n-COOH, n=6-20, or-NH 2- (CH 2 CHO) n-COOH n=6-20.
7. The method for preparing the latex-enhanced turbidimetric immunoassay reagent for detecting the blood concentration of imatinib according to claim 1, wherein the buffer in the buffer solution of the reagent R1 and the reagent R2 is selected from a buffer pair consisting of any one of the following reagents and conjugate acid or conjugate base thereof: 2- (N-morpholino) ethanesulfonic acid (MES), tris (hydroxymethyl) aminomethane (Tris), 3- (N-morphino) propanesulfonic acid (MOPS), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES).
8. The method for preparing a latex-enhanced turbidimetric immunoassay reagent for detecting the blood concentration of imatinib according to claim 7, wherein the pH of the buffer solutions of the reagent R1 and the reagent R2 is 5.0 to 9.0 or the pH is 5.5 to 7.5, and the concentration of the buffer solutions of the reagent R1 and the reagent R2 is 15 mM to 100mM.
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| CN110736835A (en) * | 2018-07-19 | 2020-01-31 | 上海云泽生物科技有限公司 | reagent kit for determining immunosuppressant cyclosporin A in whole blood by high sensitivity latex enhanced immunoturbidimetry |
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| CN110736835A (en) * | 2018-07-19 | 2020-01-31 | 上海云泽生物科技有限公司 | reagent kit for determining immunosuppressant cyclosporin A in whole blood by high sensitivity latex enhanced immunoturbidimetry |
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| Saita, T 等.An ultra-specific and sensitive sandwich ELISA for imatinib using two anti-imatinib antibodies.《Analytica Chimica Acta》.2017,第969卷72-78. * |
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