CN110736836B

CN110736836B - Kit for measuring immunosuppressant tacrolimus in whole blood by high-sensitivity latex-enhanced turbidimetric immunoassay

Info

Publication number: CN110736836B
Application number: CN201810798212.7A
Authority: CN
Inventors: 刘斌虎; 陈青青; 钱芸琦
Original assignee: Shanghai Inzex Biotechnology Co ltd
Current assignee: Shanghai Inzex Biotechnology Co ltd
Priority date: 2018-07-19
Filing date: 2018-07-19
Publication date: 2023-08-18
Anticipated expiration: 2038-07-19
Also published as: CN110736836A

Abstract

The invention relates to a kit for measuring an immunosuppressant tacrolimus in whole blood by a high-sensitivity latex-enhanced immunoturbidimetry, in particular to a tacrolimus detection kit which comprises a reagent R1, a reagent R2 and a calibrator, wherein the reagent R1 comprises tacrolimus protein complex, a preservative, a surfactant, a stabilizer and a buffer solution; reagent R2 comprises latex particles combined with anti-tac Mo Sishan clone antibody, preservative, surfactant, stabilizer and buffer solution; the calibrator comprises preservative, whole blood matrix, and tacks Mo Sichun. The method for reacting the monoclonal antibody coated latex particles with the protein complex ensures the high sensitivity and wide linear range of the kit, has the advantages of high accuracy, good repeatability, strong specificity, simple operation and the like, and can be used for a full-automatic biochemical analyzer commonly used in clinic.

Description

Kit for measuring immunosuppressant tacrolimus in whole blood by high-sensitivity latex-enhanced turbidimetric immunoassay

Technical Field

The invention belongs to the field of in-vitro diagnostic reagents, and particularly relates to a kit for determining tacrolimus in whole blood.

Background

Tacrolimus (Tacrolimus), also known as FK506, is a fermentation product isolated from Streptomyces (streptomyces tsukubaensis) and has a chemical structure that is a 23-membered macrolide antibiotic. Is a powerful novel immunosuppressant, and can completely inhibit T lymphocyte by inhibiting interleukin-2 (L-2) release, and is 100 times stronger than cyclosporin (CsA). In recent years, as a first-line drug for liver and kidney transplantation, it has been marketed in 14 countries such as japan and the united states. Clinical experiments show that the medicine has good curative effect when being applied to transplantation of heart, lung, intestine, marrow and the like. At the same time, FK506 plays a positive role in treating autoimmune diseases such as Atopic Dermatitis (AD), systemic Lupus Erythematosus (SLE), autoimmune eye diseases and the like.

Since different patients often show great individual differences in the absorption and metabolism of FK506, even with the same drug dosage, FK506 blood concentration of different patients often varies significantly; because of the hepatotoxicity and the nephrotoxicity of FK506 and the narrow therapeutic dosage range, the drug effect and the drug toxicity of FK506 are closely related to the blood drug concentration, so that accurate detection of the blood drug concentration of FK506 has important reference value for controlling the drug toxicity of FK506 and exerting the anti-rejection effect of FK 506.

Currently, methods for detecting FK506 plasma concentration mainly include high performance liquid chromatography-mass spectrometry (HPLC-MS), micro-particle enzyme immunoassay (MEIA), chemiluminescent micro-particle immunoassay (CMIA), enzyme-linked immunosorbent assay (ELISA), and the like. The HPLC-MS method is accurate and sensitive in measuring the blood concentration, but has the defects of complicated operation, long detection time, high detection cost and expensive equipment, and is mainly used in the scientific research field or used as a reference method. MEIA and CMIA are detection methods commonly used at present, and have high detection automation degree and accurate detection results, but the reagent is expensive and a matched instrument is needed. The ELISA method has the defects of long detection time, complex operation, poor repeatability and the like. The latex enhanced immunoturbidimetry has the advantages of simple and quick operation, high sensitivity, application to an automatic biochemical analyzer and the like, and can develop an immunoturbidimetry reagent which can be used on the full-automatic biochemical analyzer for detecting the tacrolimus in the whole blood, thereby providing powerful support for the clinical use of the tacrolimus as an immunosuppressant.

Disclosure of Invention

The invention aims to overcome the defects and the shortcomings of the prior art, and provides a tacrolimus detection kit with high sensitivity and wide linear range, and the tacrolimus detection kit has the advantages of accurate measurement value, simple and quick operation, wide instrument application range and the like, and can be used for emergency detection in an emergency department, wherein the kit comprises a reagent R1, a reagent R2 and a calibrator, wherein

a) Reagent R1 comprises tacrolimus protein complex, preservative, stabilizer and buffer;

b) Reagent R2 comprises latex particles combined with anti-tac Mo Sishan clone antibody, preservative, stabilizing agent, surfactant and buffer;

c) The calibrator comprises preservative, tac Mo Sichun and bovine whole blood matrix.

Preferably, the tacrolimus protein complex in the present invention is produced by coupling cyclic tacrolimus with BSA

Preferably, polystyrene latex microspheres crosslink the cloned antibody of tac Mo Sishan in the present invention.

According to the technical scheme, the tacrolimus antibody is crosslinked on the surface of the polystyrene latex microsphere and reacts with the tacrolimus protein complex to drive the polystyrene latex microsphere to aggregate to generate a certain turbidity, and the tacrolimus in the whole blood is combined with the monoclonal antibody on the surface of the polystyrene microsphere in a competitive manner, so that the turbidity is inversely proportional to the tacrolimus in the whole blood in a certain range, and the content of the tacrolimus can be detected by using a full-automatic biochemical analyzer under the wavelength of 400-800 nm.

The buffer solution 1 in the reagent R1 is one or more selected from MES buffer solution, tris-HCl buffer solution and PBS buffer solution, the pH value is 5.5-8.0, and the concentration is 25-200 mmol/L; the buffer solution 2 in the reagent R2 is one or more selected from PBS buffer solution, HEPES buffer solution and borax buffer solution, the pH value is 7.0-9.0, and the concentration is 25-200 mmol/L.

The stabilizer in the reagent R1 adopts ion stabilizer and suspension stabilizer to be matched for use: wherein the ion stabilizer is NaCl, KCl or MgCl ₂ One or more of the components with the content of 1 to 2 percent; the suspension stabilizer is PEG8000, PEG6000 or glucose with the content of 2-5%

The stabilizer 2 in the reagent R2 is ion stabilizer NaCl, KCl or K ₂ SO ₄ One or more of the components with the content of 0.5 to 5 percent

The preservative 1 in the reagent R1 is sodium azide, merthiolate or ProClin300; preservative 2 in the reagent R2 is sodium azide, thimerosal or ProClin300; preservative 3 in the calibrator is sodium azide, merthiolate or ProClin300.

The polystyrene latex microsphere with carboxyl as the surface functional group in the reagent R2 has the particle size of 200-400 nm.

The tacrolimus antibody in the reagent R2 is a mouse monoclonal antibody.

The surfactant in the reagent R2 is Tween-20, and the content is 0.05-2%.

The protective agent in the reagent R2 is bovine serum albumin with the content of 0.5 to 5 percent

The calibrator adopts cow whole blood frozen and thawed for 2-5 times as a matrix.

The invention relates to a preparation method of a kit for measuring an immunosuppressant tacrolimus in whole blood by a high-sensitivity latex enhanced immunoturbidimetry, which comprises the following steps:

(1) Preparation of reagent R1: tacrolimus 100-200 mg, succinic anhydride 25-50 mg, 4-Dimethylaminopyridine (DMAP) 5-10 mg, and Dichloromethane (DCM) 4-6 mL were put into a bottle and stirred at room temperature for reaction for 10 hours. Adding 2 times of 0.1N hydrochloric acid to wash for 2 times, and taking an organic phase (note: acid washing, the needed organic phase is on the lower layer); then, 2 times of the volume of saturated NaCl solution was added, and the mixture was washed 3 times to obtain an organic phase (note: wash with saturated NaCl, desired organic phase was the upper layer). And (3) pumping to dryness under negative pressure, then adding a small amount of dichloromethane, pumping again, and drying the product as much as possible. Weighing 5-10 mg of the product, adding 200 mu L N of N-Dimethylformamide (DMF), continuously adding 2-3 mg of EDCI and 10-20 mg of NHS, and stirring for reaction for 10min; adding 10mL of MES buffer solution containing 20mg of Bovine Serum Albumin (BSA) into another penicillin bottle, and uniformly mixing; under magnetic stirring, 200uL of activated tacrolimus DMF solution is slowly dripped into the BSA-containing solution, the reaction is carried out for 5 hours at room temperature, and the tacrolimus protein complex is obtained after purification. Adding the tacrolimus protein complex, the stabilizer 1 and the preservative 1 into the buffer solution 1, and uniformly stirring and mixing to obtain a reagent R1;

(2) Preparation of reagent R2: diluting polystyrene latex microspheres to 1% by mass concentration by using a marking buffer solution, adding EDC with 0.01% -0.1% by mass concentration, stirring at room temperature for reaction for 30min, centrifuging and washing at 12000rpm after the reaction is finished to remove unreacted EDC, adding a cloned antibody of Talcum Mo Sishan, stirring and reacting at 37 ℃ for 60min, adding 0.5% -2% BSA as a blocking agent for reacting for 60min, centrifuging and washing the obtained reaction solution at 12000rpm, and finally re-suspending the latex by using a buffer solution 2 comprising a stabilizing agent 2, a preservative 2 and a surfactant 2.

(3) Preparation of a calibrator: repeatedly freezing and thawing fresh anticoagulated whole blood for 2-5 times, filtering, adding 0.05-0.1 preservative, and adding tacks Mo Sichun according to the requirement. Obtaining tacrolimus calibrator with different concentrations

The invention adopts a 6-point calibration method, takes spline functions as calculation modes, and draws a calibration curve.

Compared with the prior art, the kit provided by the invention has the advantages of good specificity, high sensitivity, good accuracy, wide linear range and the like; in addition, the operation is simple and convenient when the detection is carried out, the method is suitable for a full-automatic biochemical analyzer, and the detection efficiency is greatly improved.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

Example 1:

a kit for detecting immunosuppressant tacrolimus in whole blood by a high-sensitivity latex-enhanced turbidimetric immunoassay comprises the following preparation steps:

(1) Tacrolimus 100mg, succinic anhydride 40mg, 4-Dimethylaminopyridine (DMAP) 10mg, and Dichloromethane (DCM) 6mL were placed in a flask and reacted at room temperature with stirring for 10 hours. Adding 2 times of 0.1N hydrochloric acid to wash for 2 times, and taking an organic phase (note: acid washing, the needed organic phase is on the lower layer); then, 2 times of the volume of saturated NaCl solution was added, and the mixture was washed 3 times to obtain an organic phase (note: wash with saturated NaCl, desired organic phase was the upper layer). And (3) pumping to dryness under negative pressure, then adding a small amount of dichloromethane, pumping again, and drying the product as much as possible. 10mg of the product is weighed, 200 mu LN and N-Dimethylformamide (DMF) are added, and 3mg EDCI,20mg NHS is added continuously to stir and react for 10min; adding 10mL of MES buffer solution containing 20mg of Bovine Serum Albumin (BSA) into another penicillin bottle, and uniformly mixing; under magnetic stirring, 200uL of activated tacrolimus DMF solution is slowly dripped into the BSA-containing solution, the reaction is carried out for 5 hours at room temperature, and the tacrolimus protein complex is obtained after purification. Reagent R1 was then prepared at the following concentrations:

(2) Preparation of reagent R2: diluting polystyrene latex microspheres to 1% by mass concentration by using a marking buffer solution, adding EDC with 0.05% by mass concentration, stirring at room temperature for reaction for 30min, centrifuging and washing at 12000rpm after the reaction is finished to remove unreacted EDC, adding a tac Mo Sishan clone antibody, stirring at 37 ℃ for reaction for 60min, adding 1% BSA as a blocking agent for reaction for 60min, centrifuging and washing the obtained reaction solution at 12000rpm, and finally re-suspending the latex by using buffer solution 2 comprising stabilizer 2, preservative 2 and surfactant 2, wherein the specific concentration is as follows:

(3) Preparation of a calibrator: fresh anticoagulated whole bovine blood is repeatedly frozen and thawed for 3 times, filtered, added with 0.05% preservative, and then added with Talcum Mo Sichun product according to the requirement. Obtaining tacrolimus calibrator with different concentrations

Example 2:

(1) Tacrolimus 200mg, succinic anhydride 50mg, 4-Dimethylaminopyridine (DMAP) 10mg, and Dichloromethane (DCM) 6mL were placed in a bottle and the reaction stirred at room temperature for 10 hours. Adding 2 times of 0.1N hydrochloric acid to wash for 2 times, and taking an organic phase (note: acid washing, the needed organic phase is on the lower layer); then, 2 times of the volume of saturated NaCl solution was added, and the mixture was washed 3 times to obtain an organic phase (note: wash with saturated NaCl, desired organic phase was the upper layer). And (3) pumping to dryness under negative pressure, then adding a small amount of dichloromethane, pumping again, and drying the product as much as possible. 10mg of the product is weighed, 200 mu LN and N-Dimethylformamide (DMF) are added, and 1.5mg EDCI,10mg NHS is added continuously to stir and react for 10min; adding 10mL of MES buffer solution containing 20mg of Bovine Serum Albumin (BSA) into another penicillin bottle, and uniformly mixing; under magnetic stirring, 200uL of activated tacrolimus DMF solution is slowly dripped into the BSA-containing solution, the reaction is carried out for 5 hours at room temperature, and the tacrolimus protein complex is obtained after purification. Reagent R1 was then prepared at the following concentrations:

Example 3:

(1) Tacrolimus 100mg, succinic anhydride 35mg, 4-Dimethylaminopyridine (DMAP) 10mg, and Dichloromethane (DCM) 4mL were placed in a bottle and the reaction stirred at room temperature for 10 hours. Adding 2 times of 0.1N hydrochloric acid to wash for 2 times, and taking an organic phase (note: acid washing, the needed organic phase is on the lower layer); then, 2 times of the volume of saturated NaCl solution was added, and the mixture was washed 3 times to obtain an organic phase (note: wash with saturated NaCl, desired organic phase was the upper layer). And (3) pumping to dryness under negative pressure, then adding a small amount of dichloromethane, pumping again, and drying the product as much as possible. 10mg of the product is weighed, 200 mu LN and N-Dimethylformamide (DMF) are added, and 1.5mg EDCI,10mg NHS is added continuously to stir and react for 10min; adding 10mL of MES buffer solution containing 20mg of Bovine Serum Albumin (BSA) into another penicillin bottle, and uniformly mixing; under magnetic stirring, 200uL of activated tacrolimus DMF solution is slowly dripped into the BSA-containing solution, the reaction is carried out for 5 hours at room temperature, and the tacrolimus protein complex is obtained after purification. Reagent R1 was then prepared at the following concentrations:

(3) Preparation of a calibrator: repeatedly freezing and thawing fresh anticoagulated whole blood for 2-5 times, filtering, adding 0.05% preservative, and adding Mo Sichun tacks as required. Obtaining tacrolimus calibrator with different concentrations

Example 4:

(1) Tacrolimus 100mg, succinic anhydride 35mg, 4-Dimethylaminopyridine (DMAP) 5mg, and Dichloromethane (DCM) 4mL were placed in a bottle and the reaction stirred at room temperature for 10 hours. Adding 2 times of 0.1N hydrochloric acid to wash for 2 times, and taking an organic phase (note: acid washing, the needed organic phase is on the lower layer); then, 2 times of the volume of saturated NaCl solution was added, and the mixture was washed 3 times to obtain an organic phase (note: wash with saturated NaCl, desired organic phase was the upper layer). And (3) pumping to dryness under negative pressure, then adding a small amount of dichloromethane, pumping again, and drying the product as much as possible. 10mg of the product is weighed, 200 mu LN and N-Dimethylformamide (DMF) are added, and 1.5mg EDCI,10mg NHS is added continuously to stir and react for 10min; adding 10mL of MES buffer solution containing 20mg of Bovine Serum Albumin (BSA) into another penicillin bottle, and uniformly mixing; under magnetic stirring, 200uL of activated tacrolimus DMF solution is slowly dripped into the BSA-containing solution, the reaction is carried out for 5 hours at room temperature, and the tacrolimus protein complex is obtained after purification. Reagent R1 was then prepared at the following concentrations:

Test results:

the reagents prepared in the above 4 examples and comparative examples are tested by a Beckmann AU480 full-automatic biochemical analyzer, the test wavelength is 600nm, a sample or a calibrator 4uL is taken, 160uL of reagent R1 is added, the temperature is kept constant at 37 ℃ for 5min, 40uL of reagent R2 is then added, absorbance A1 is read after 20s, absorbance A2 is read after incubation for 4 minutes and 40 seconds at 37 ℃, and the reaction absorbance DeltaA=A2-A1; firstly, performing multi-point calibration by using a standard substance, and calculating by using a spline function to obtain a calibration curve. And comparing the absorbance change of the sample with a standard curve to obtain the PCT concentration in the sample. The 4 examples and the comparative examples were subjected to verification of analysis sensitivity, accuracy, precision, stability and the like, and the results were verified

As shown in table 1:

according to the test results, the detection reagent prepared in the parameter range of the invention has better analysis sensitivity, accuracy, precision and stability, wherein the implementation 2 is the optimal embodiment, and the reagent test effect can be best improved.

In summary, the present invention effectively overcomes the disadvantages of the prior art and has high industrial utility value.

Claims

1. A kit for measuring an immunosuppressant tacrolimus in whole blood by a high-sensitivity latex-enhanced turbidimetric immunoassay comprises the following preparation steps:

(1) Tacrolimus 200mg, succinic anhydride 50mg, 4-dimethylaminopyridine 10mg and dichloromethane 6mL are put into a bottle and stirred at room temperature for reaction for 10 hours; adding 2 times of 0.1N hydrochloric acid to wash for 2 times, and taking an organic phase; adding 2 times of saturated NaCl solution, washing for 3 times, and taking an organic phase; negative pressure pumping, adding a small amount of dichloromethane, pumping again,drying the product as much as possible; weighing 10mg of the product, adding 200 mu L N of N-dimethylformamide, continuously adding 1.5mg EDCI,10mg NHS of the mixture, and stirring and reacting for 10min; adding 10mL MES buffer solution containing 20mg of bovine serum albumin into another penicillin bottle, and uniformly mixing; under magnetic stirring, taking 200 mu L of activated tacrolimus DMF solution, slowly dripping the solution into the solution containing BSA, reacting for 5 hours at room temperature, and purifying to obtain tacrolimus protein complex; reagent R1 was then prepared at the following concentrations: 1mL/L tacrolimus protein complex, 32g/L PEG-6000,5.33g/L MES monohydrate, 10g/L KCl,10g/L BSA,4.5g/L MgCl ₂ ，5mL/L 10%NaN ₃ A solution;

(2) Preparation of reagent R2: diluting polystyrene latex microspheres to 1% by mass concentration with a labeling buffer solution, adding EDC with 0.05% by mass concentration, stirring at room temperature for reaction for 30min, centrifuging and washing at 12000rpm after the reaction is finished to remove unreacted EDC, adding a tac Mo Sishan clone antibody, stirring at 37 ℃ for reaction for 60min, adding 1% BSA as a blocking agent for reaction for 60min, centrifuging and washing the obtained reaction solution at 12000rpm, and resuspending the latex with a buffer solution 2 comprising a stabilizer 2, a preservative 2 and a surfactant 2 according to the following concentrations: 1mL/L antibody-labeled microspheres, 11.915g/L HEPES,10g/L KCl,10g/L BSA,1g/L Tween-20,5 mL/L10% NaN ₃ A solution;

(3) Preparation of a calibrator: repeatedly freezing and thawing fresh anticoagulated whole blood for 3 times, filtering, adding 0.05% preservative, and adding Talcum Mo Sichun product as required; different concentrations of tacrolimus calibrator were obtained.

2. A preparation method of a kit for measuring an immunosuppressant tacrolimus in whole blood by a high-sensitivity latex-enhanced turbidimetric immunoassay, which is characterized by comprising the following preparation steps:

(1) Tacrolimus 200mg, succinic anhydride 50mg, 4-dimethylaminopyridine 10mg and dichloromethane 6mL are put into a bottle and stirred at room temperature for reaction for 10 hours; adding 2 times of 0.1N hydrochloric acid to wash for 2 times, and taking an organic phase; adding 2 times of saturated NaCl solution, washing for 3 times, and taking an organic phase; negative pressure pumping, and adding a small amount of twoMethyl chloride is pumped down again, and the product is dried as much as possible; weighing 10mg of the product, adding 200 mu L N of N-dimethylformamide, continuously adding 1.5mg EDCI,10mg NHS of the mixture, and stirring and reacting for 10min; adding 10mL MES buffer solution containing 20mg of bovine serum albumin into another penicillin bottle, and uniformly mixing; under magnetic stirring, taking 200 mu L of activated tacrolimus DMF solution, slowly dripping the solution into the solution containing BSA, reacting for 5 hours at room temperature, and purifying to obtain tacrolimus protein complex; reagent R1 was then prepared at the following concentrations: 1mL/L tacrolimus protein complex, 32g/L PEG-6000,5.33g/L MES monohydrate, 10g/L KCl,10g/L BSA,4.5g/L MgCl ₂ ，5mL/L 10%NaN ₃ A solution;