CN114966030B

CN114966030B - Drug-protein dissociation composition, tacrolimus detection kit containing same and application of tacrolimus detection kit

Info

Publication number: CN114966030B
Application number: CN202111565284.5A
Authority: CN
Inventors: 陈国锋; 林巍靖; 王西
Original assignee: Shanghai Jianfeng Medical Science And Technology Co ltd; Shanghai Inzex Biotechnology Co ltd
Current assignee: Shanghai Jianfeng Medical Science And Technology Co ltd; Shanghai Inzex Biotechnology Co ltd
Priority date: 2021-12-20
Filing date: 2021-12-20
Publication date: 2024-10-25
Anticipated expiration: 2041-12-20
Also published as: CN114966030A

Abstract

The invention discloses a medicine-protein dissociation composition, which comprises 25-150mM of buffer solution with pH of 6.5-9.0, 0.8-1.4% of metal chelating agent, 0.4-1.2% of ammonium chloride, 0.8-1.2% of nonionic surfactant, 0.05-0.3% of fatty acid, 0.2-1.5% of sodium cholate, 0.05-0.15% of salicylic acid and 0.05-0.5% of glutathione. The invention also discloses an extraction-free blood concentration tacrolimus detection kit, which is simple and convenient to use, does not need a complex sample pretreatment process, reduces errors caused by manual operation in pretreatment and the influence of the errors on the accuracy of a measured value, does not contain volatile organic reagents, reduces the pollution of uncapping volatilization to the environment, and further ensures the accuracy of the measured value.

Description

Drug-protein dissociation composition, tacrolimus detection kit containing same and application of tacrolimus detection kit

Technical Field

The invention belongs to the field of medical inspection, relates to medicine concentration detection, and in particular relates to a medicine-protein dissociation composition, a tacrolimus detection kit containing the same and application thereof.

Background

The Therapeutic Drug Monitoring (TDM) is to measure the drug concentration in blood or other body fluids of drugs with narrow therapeutic index, strong toxic effect and large individual difference, so as to evaluate the curative effect, and to formulate individual dosing schemes according to the principle of pharmacokinetics, so as to improve the therapeutic level of the drugs and achieve the purposes of clinical safety, effectiveness and reasonable dosing. Clinical practice of blood concentration monitoring has fully established its role in guiding and evaluating drug treatment, as well as improving rational drug use level. For example, the rate of seizure control is increased from 47% to 74% by TDM and personalized dosing regimen. TDM has become a daily medical task in countries such as america, english, canada, etc. The common methods for monitoring the drug concentration in the laboratory are liquid phase mass spectrum, gas phase mass spectrum and the like, but the methods have the defects of complex operation, expensive instruments, high requirements on professional knowledge of technicians and difficulty in popularization in hospitals. Currently, various diagnostic reagent manufacturers have successively introduced a partial therapeutic drug monitoring kit comprising: tacrolimus, cyclosporin A, voriconazole, itraconazole, digoxin, oxcarbazepine, valproic acid, carbamazepine and the like, and the diagnostic reagent products are simple and convenient to use, short in reaction time and accurate in result, greatly facilitate the development of monitoring work, and are safer and more effective for future patient administration as more and more drug monitoring kits are developed.

After the medicine enters the human body, most of the medicine is combined with plasma proteins in blood, part of the medicine can be combined with blood cells or enter the blood cells, the medicine-protein combination body and the free medicine achieve dynamic balance, the slow release and transportation functions are achieved, and pharmacological action is achieved after the medicine reaches an action part. The drug concentration detection kit generally combines an antibody of a specific drug with the drug in a blood sample of a patient to obtain a concentration value thereof. The binding rate of different drugs to proteins in blood plasma is different, the higher the binding rate is, the more firmly the drug is bound, the more difficult the antibody recognition process is, and the concentration of the drug is not easy to detect. For example, the first-line anti-rejection medicine in organ transplantation has extremely high binding rate of tacrolimus and carrier protein, especially tacrolimus, and in the systemic circulation, the tacrolimus is highly bound with erythrocytes, the whole blood/plasma concentration distribution ratio is about 20:1, and in plasma, the binding rate of tacrolimus and plasma protein is as high as 98.8% or more, and is mainly bound with serum albumin and alpha-1-acid glycoprotein.

The high protein binding rate of tacrolimus makes the concentration detection difficult, and although commercial kits exist at present, certain disadvantages exist, such as: the complex pretreatment process is needed, a certain amount of special extractant is needed to be added into the sample firstly, after shaking and mixing for tens of seconds, the sample is centrifuged for a few minutes, then the sample is sampled and detected on the machine, the processing operation of the sample is more complicated and time-consuming, the more experimental steps are, the more the error of manual operation is, the more obvious the accumulation effect is, the accuracy of the measured value is affected, in addition, the extractant contains volatile organic components, the environment is polluted, and the effective components are also changed along with the reduction of the effective components along with the continuous cover opening in the use process.

Disclosure of Invention

In order to solve the problems, the invention provides a medicine-protein dissociation composition and a method for applying the composition to a latex-enhanced turbidimetry tacrolimus detection kit, and the medicine-protein dissociation composition can remarkably simplify the detection operation of a medicine with high protein binding rate, improves the detection efficiency, has wide applicability and has a great application prospect.

To solve the above problems, the first aspect of the present invention provides a drug-protein dissociation composition comprising 25 to 150mM buffer with pH of 6.5 to 9.0, 0.8 to 1.4% metal chelator, 0.4 to 1.2% ammonium chloride, 0.8 to 1.2% nonionic surfactant, 0.05 to 0.3% fatty acid, 0.2 to 1.5% sodium cholate, 0.05 to 0.15% salicylic acid and 0.05 to 0.5% glutathione; the percentages are mass percentages of each component in the medicine-protein dissociation composition.

In a specific embodiment, the buffer is phosphate buffer, HEPES buffer, MES buffer, or MOPS buffer.

In one embodiment, the concentration of the buffer is 50-100mM and the pH value is 6.8-7.4;

preferably, the buffer is HEPES buffer, the concentration of the buffer is 50mM, and the pH value of the buffer is 7.4.

In a specific embodiment, the metal chelator is EDTA, EGTA or a salt thereof; preferably, the metal chelator is an EDTA salt such as EDTA-2Na.

In a specific embodiment, the metal chelator is 0.8% by mass.

In a specific embodiment, the mass fraction of the ammonium chloride is 0.8% -1%. Preferably, the mass fraction of the ammonium chloride is 1%.

In a specific embodiment, the nonionic surfactant is polyoxyethylene polyoxypropylene block polyether; preferably, the nonionic surfactant is one or more of F68, F108, L61 or L64.

In a specific embodiment, the mass fraction of the nonionic surfactant is 0.8%.

In a specific embodiment, the mass fraction of the sodium cholate is 0.6% -1%. Preferably, the mass fraction of the sodium cholate is 0.6%.

In a particular embodiment, the fatty acid comprises one or more of valeric acid, caproic acid, and heptanoic acid; preferably, the fatty acid is valeric acid.

In a specific embodiment, the mass fraction of the fatty acid is 0.1% -0.2%. Preferably, the mass fraction of the fatty acid is 0.2%.

In a specific embodiment, the mass fraction of the salicylic acid is 0.1% -0.2%. Preferably, the mass fraction of the salicylic acid is 0.1%.

In a specific embodiment, the mass fraction of the glutathione is 0.15% -0.4%. Preferably, the mass fraction of the glutathione is 0.15%.

In a second aspect, the invention provides an extraction-free blood concentration detection kit comprising a drug-protein dissociation composition according to the first aspect of the invention.

Preferably, it comprises: reagent R1 and reagent R2; wherein:

(1) Reagent R1 comprises: tacrolimus protein complex, drug-protein dissociating composition according to the first aspect of the present invention, preservative;

(2) Reagent R2 comprises: latex microspheres sensitized by anti-tacks Mo Sishan clone antibody, buffer solution, stabilizer and preservative.

More preferably, the extraction-free blood concentration detection kit further comprises a calibrator;

The calibrator is preferably whole blood containing tacrolimus.

In the medicine-protein dissociation composition, metal ion chelating agent such as ethylenediamine tetraacetic acid (EDTA) salt creates hypotonic environment, promotes cell rupture in blood, and ammonium chloride mainly lyses red blood cells, and polyoxyethylene polyoxypropylene block polyether surfactant can accelerate cell lysis and improve the anti-interference capability of the reagent. Fatty acid, sodium cholate, salicylic acid and plasma protein have strong binding force, and induced conformational change of the plasma protein promotes rapid release of the drug combined with the protein. Glutathione can form disulfide bonds with the drug binding active site of plasma albumin, and can prevent the recombination of free drugs to a certain extent.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

1. the kit prepared by the invention is simple and convenient to use, does not need a complex sample pretreatment process (about 10 minutes), and reduces errors caused by manual operation in pretreatment and the influence of the errors on the accuracy of measured values

2. As the extractant of the commercial kit contains volatile organic reagent, the environment is polluted in the using process, and the effective components of the extractant are reduced along with the continuous cover opening in the using process, the extraction effect of the extractant on the medicine is changed, and the accuracy of the measured value is further affected.

Drawings

FIG. 1 is a comparison of the example 1 kit and a commercial kit sample.

Fig. 2 is a comparison of test group 3 kit and commercial kit samples.

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.

Tacrolimus assay kit (latex enhanced immunosuppression method)

The tacrolimus determination kit (latex enhanced immunosuppression method) consists of a reagent R1, a reagent R2 and a calibrator, wherein the volume ratio of the reagent R1 to the reagent R2 is 1:1.

Reagent R1 includes: tacrolimus protein complex, drug-protein dissociating composition, preservative.

Reagent R2 includes: latex microspheres sensitized by anti-tacks Mo Sishan clone antibody, buffer solution, stabilizer and preservative.

The calibrator is whole blood containing tacrolimus.

The preparation method of the reagent R2 comprises the following steps:

(1) Activation of polystyrene latex microspheres: 35g of 310nm latex microspheres (concentration 10%) are taken, 10mL of 500mM HEPES solution with pH value of 7.8 is added, 6mL of freshly prepared EDC solution with concentration of 20mg/mL, purified water is added to a final volume of 100mL, fully and uniformly mixed, and mixed for 30min at room temperature (20-25 ℃);

(2) Coupling: the preparation method comprises the following steps of: adding tacrolimus antibody into the reaction solution of the previous step according to the mass ratio of the antibody=15:1, and mixing and coupling for 2 hours at room temperature;

(3) Closing: ultrasonic processing the reacted solution for 5min, adding 6mL of 20% bovine serum albumin solution, and mixing for 1h at room temperature;

(4) Washing: centrifuging the solution obtained in the previous step, removing the supernatant, adding 100mM tris8.0 in equal volume, performing ultrasonic treatment until the solution is uniformly dispersed, centrifuging to remove the supernatant, finishing one-time washing, and repeating the washing for 2 times;

(5) Mixing and preserving: after washing for 2 times, centrifuging again to remove the supernatant, adding 30mL of R2 diluent (containing 100mM glycine buffer solution, 4% bovine serum albumin, 0.5% trehalose, 0.05% sodium azide and pH value of 8.0), performing ultrasonic treatment for 15min, and fixing the volume with 500mL of the R2 diluent to obtain the reagent R2.

The preparation method of the calibrator comprises the following steps:

taking 40mL of whole blood, adding disodium ethylenediamine tetraacetate with the mass fraction of 0.4%, filtering with a 0.22 μm filter membrane, adding tacrolimus Mo Sichun, uniformly mixing, and carrying out assignment according to an assignment program to finally obtain the tacrolimus calibrator.

The preparation method of the reagent R1 comprises the following steps:

Example 1

450ML of purified water was taken and each material was weighed according to the following table

After the dissolution of each substance was completed, the pH was adjusted to 7.4, the volume was set to 500mL, and 14. Mu.L of tacrolimus protein complex (mother liquor concentration 5 mg/mL) was added

Example 2

Substance name	Weigh g	Final concentration
			HEPES	5.95	50mM
EDTA-2Na	4.00	0.8％
			Ammonium chloride	2.00	0.4％
F68	1.50	0.3％
			Sodium cholate	3.00	0.6％
Valeric acid	1.00	0.2％
			Salicylic acid	0.25	0.05％
Glutathione	0.50	0.1％
			Sodium azide	0.50	0.1％

After the dissolution of each substance was completed, the pH was adjusted to 7.4, the volume was set to 500mL, and 12.5. Mu.L of tacrolimus protein complex (mother liquor concentration 5 mg/mL) was added

Example 3

Substance name	Weigh g	Final concentration
			HEPES	5.95	50mM
EDTA-2Na	5.00	1％
			Ammonium chloride	6.00	1.2％
L61	4.00	0.3％
			Sodium cholate	3.00	1％
Valeric acid	0.50	0.1％
			Salicylic acid	0.25	0.05％
Glutathione	2.50	0.5％
			Sodium azide	0.50	0.1％

R1 and R2 are split-packed according to a volume ratio of 1:1, and are assembled together with a calibrator to form the tacrolimus assay kit (latex-enhanced immunosuppression method).

Comparative example 1 performance analysis was performed on the kit prepared in example 1:

table 1 kit linear evaluation

11 Gradient dilutions were performed and tested on each of the high and low plasma:

commercial kits linear range 1-30ng/mL, and the example 1 kit prepared by the invention has good recovery rate in this range.

Table 2 sample alignment with commercial kits

To evaluate the effect of the drug-protein dissociation composition components on the test, 4 sets of reagents were co-formulated with commercial reagents to test samples for comparison testing

The reagent R1 of the experiment group 1 is not added with a medicine-protein dissociation composition, namely, the reagent R1 contains tacrolimus protein complex, preservative and buffer solution, and the reagent R2 and calibrator share the same batch with the experiment example 1.

The reagent R1 of the experimental group 2 contains partial medicine-protein dissociation composition, namely, the R1 contains tacrolimus protein complex, preservative, buffer solution, EDTA-2Na and ammonium chloride, the content of each component is the same as that of the example 1, but the following substances are not included: f108, sodium cholate, valeric acid, salicylic acid, glutathione, reagent R2, calibrator share the same batch as example 1.

In the reagent R1 of the experiment group 3, the nonionic surfactant F108 in the medicine-protein dissociation composition is replaced by the relatively common nonionic surfactant Tween 20 in the industry, namely R1 contains tacrolimus protein complex, preservative and buffer solution, EDTA-2Na, ammonium chloride, sodium cholate, valeric acid, salicylic acid, glutathione and Tween 20, the contents of the components are the same as those of the example 1, and the reagent R2 and the calibrator share the same batch with the example 1.

Example 1 kit reagent R1 contains a drug-protein dissociation composition, the reagent R2 and calibrator of which share the same batch as the experimental group 1, the experimental group 2 and the experimental group 3.

Sample processing

Test group 1, test group 2, test group 3 and test group 1 kit samples were tested on a Biochemical instrument without pretreatment

Commercial kits extract samples according to instructions using pretreatment fluids in their packaging: 200uL of sample is taken, equal amount of extract is added, evenly mixed for 30 seconds, centrifuged for 10 minutes, supernatant is taken for detection, and the test result is as follows.

Table 2 comparison of test results for experimental set, example 1 kit and commercial kit

The test results show that the test results of the test group 1 and the test group 2 show that the test values are far lower, the correlation with the commercialized kit is very poor, the test group 2 is combined with the carrier protein, the red blood cell lysis component is added, but the medicine still combined with the carrier protein can not be detected, the test group 3 is added with fatty acid, sodium cholate and salicylic acid on the basis of the test group 2, and the glutathione releases the medicine combined with the protein, so that a certain content of tacrolimus can be detected, but compared with the commercialized reagent, the correlation and the test values are lower.

From tables 1 and 2 and fig. 1 and 2, the detection effect of the kit of example 1 achieved recovery and detection effect comparable to commercial kits. The kit of example 1 and the kits of experimental groups 1-3 do not require complex pretreatment when in use, and the only difference is the drug-protein dissociation composition, the effect of the kit of example 1 is comparable to that of the commercial kit, and the effect of the kits of experimental groups 1-3 is far lower than that of the kit of example 1.

Table 3 example 1 stability tracking of kit

As is clear from Table 3, the tacrolimus assay kit prepared in example 1 has good stability and can be stored at 4℃for 12 months.

Claims

1. A composition for drug-protein dissociation, characterized in that it comprises 25-150mM buffer with pH 6.5-9.0, 0.8% -1.4% metal chelator, 0.4% -1.2% ammonium chloride, 0.8% -1.2% nonionic surfactant, 0.05% -0.3% fatty acid, 0.2% -1.5% sodium cholate, 0.05% -0.15% salicylic acid and 0.05% -0.5% glutathione; the percentages are mass volume percentages of the components in the medicine-protein dissociation composition; the drug-protein is tacrolimus-protein; the nonionic surfactant is polyoxyethylene polyoxypropylene block polyether.

2. The composition for drug-protein dissociation of claim 1, wherein the buffer is phosphate buffer, HEPES buffer, MES buffer, or MOPS buffer;

And/or the concentration of the buffer solution is 50-100mM, and the pH value is 6.8-7.4;

and/or the nonionic surfactant is one or more of F68, F108, L61 or L64.

3. The composition for drug-protein dissociation of claim 2, wherein the buffer is HEPES buffer.

4. The composition for drug-protein dissociation of claim 1, wherein said metal chelator is EDTA, EGTA or a salt thereof.

5. The composition for drug-protein dissociation of claim 4, wherein said metal chelator is EDTA salt.

6. The composition for drug-protein dissociation of claim 5, wherein said EDTA salt is EDTA-2Na.

7. The composition for drug-protein dissociation of claim 1, wherein the mass fraction of ammonium chloride is between 0.8% and 1%.

8. The composition for drug-protein dissociation of claim 1, wherein the mass fraction of sodium cholate is between 0.6% and 1%.

9. The composition for drug-protein dissociation of claim 1, wherein said fatty acids comprise one or more of valeric acid, caproic acid and heptanoic acid.

10. The composition for drug-protein dissociation of claim 9, wherein said fatty acid is valeric acid;

And/or the mass fraction of the fatty acid is 0.1% -0.2%.

11. The composition for drug-protein dissociation of claim 1, wherein said salicylic acid is present in an amount of 0.1% to 0.2% by mass.

12. The composition for drug-protein dissociation of claim 1, in which the glutathione is present in a mass fraction of 0.15% to 0.4%.

13. An extraction-free blood concentration detection kit comprising a composition for drug-protein dissociation according to any one of claims 1 to 12.

14. The extraction-free blood concentration detection kit of claim 13, wherein the extraction-free blood concentration detection kit comprises: reagent R1 and reagent R2;

(1) Reagent R1 comprises: tacrolimus protein complex, composition for drug-protein dissociation according to claim 1 and preservative;

(2) Reagent R2 comprises: latex microspheres sensitized with anti-tac Mo Sishan clone antibody, buffer, stabilizer and preservative.

15. The extraction-free blood concentration detection kit of claim 14, the extraction-free blood concentration detection kit also comprises a calibrator.

16. The extraction-free blood concentration detection kit of claim 15 wherein the calibrator is whole blood containing tacrolimus.