CN210894372U

CN210894372U - Time-resolved fluorescence immunochromatography kit for rapidly detecting tacrolimus

Info

Publication number: CN210894372U
Application number: CN201920253457.1U
Authority: CN
Inventors: 刘斌虎; 吴云林; 傅琳
Original assignee: Shanghai Jianfeng Medical Science And Technology Co ltd
Current assignee: Shanghai Jianfeng Medical Science And Technology Co ltd
Priority date: 2019-02-28
Filing date: 2019-02-28
Publication date: 2020-06-30
Anticipated expiration: 2029-02-28

Abstract

The utility model provides a time resolution fluorescence immunochromatographic assay kit of short-term test tacrolimus, it is including detection card and the fluorescence immunoassay quantitative analysis appearance that can select, the detection card includes sample pad, combination pad, has detection pad, absorption pad and backing plate of detection line and quality control line, the combination pad spraying has nanometer microballon time resolution fluorescence-tacrolimus probe, nanometer microballon time resolution fluorescence-tacrolimus probe is prepared by nanometer fluorescence microballon and tacrolimus monoclonal antibody, the detection line is prepared by tacrolimus protein derivative spraying, the quality control line is prepared by goat anti mouse IgG antibody spraying. The kit is simple and easy to operate, can obtain an accurate and reliable determination result in only 5 minutes, and has good commercial application prospect.

Description

Time-resolved fluorescence immunochromatography kit for rapidly detecting tacrolimus

Technical Field

The utility model relates to a biological diagnosis technical field, concretely relates to time-resolved fluorescence immunochromatography kit of short-term test tacrolimus.

Background

Tacrolimus (Tacrolimus), also known as FK506, is a fermentation product isolated from streptomyces tsukubaensis, the chemical structure of which belongs to the 23-membered macrolide antibiotic. Is a strong novel immunosuppressant, mainly inhibits the release of interleukin-2 (L-2) to comprehensively inhibit the action of T lymphocytes, and is 100 times stronger than that of cyclosporine (CsA). In recent years, first line drugs for liver and kidney transplantation have been marketed in 14 countries such as japan and the usa. Clinical experiments show that the medicine has good curative effect when being applied to transplantation of heart, lung, intestine, bone marrow and the like. Meanwhile, FK506 also plays an active role in treating autoimmune diseases such as Atopic Dermatitis (AD), Systemic Lupus Erythematosus (SLE), autoimmune ocular diseases and the like.

Because different patients tend to show great individual differences in the absorption and metabolism of FK506, the plasma concentrations of FK506 often vary significantly from patient to patient even with the same drug dose; because of the liver and kidney toxicity of FK506 and the narrow therapeutic dose range thereof, the drug effect and drug toxicity of FK506 are closely related to the blood concentration, therefore, the accurate detection of the blood concentration of FK506 has important reference values for controlling the drug toxicity of FK506 and playing the anti-rejection role of FK 506.

Currently, methods for detecting the plasma concentration of FK506 mainly include high performance liquid chromatography-mass spectrometry (HPLC-MS), Microparticle Enzyme Immunoassay (MEIA), Chemiluminescent Microparticle Immunoassay (CMIA), enzyme-linked immunosorbent assay (ELISA), and the like. The HPLC-MS method is accurate and sensitive in blood concentration determination, but is complex in operation, long in detection time, high in detection cost and needs expensive equipment, and is mainly used in the field of scientific research or used as a reference method. The MEIA and CMIA are common detection methods at present, the detection automation degree is high, the detection result is accurate, but the reagent price is high, and a matched large instrument is required to be used. The ELISA method has the defects of long detection time, complex operation, poor repeatability and the like. The time-resolved fluoroimmunoassay has the characteristics of simple and rapid operation, no need of large-scale equipment and the like, and if a time-resolved fluoroimmunoassay reagent can be developed for detecting tacrolimus A in whole blood, the reagent can be immediately analyzed on a sampling site for a majority of transplanted patients using FK506 as an immunosuppressant, so that a complex processing procedure of a sample during laboratory test is omitted, a test result can be rapidly obtained, and a great amount of time for going to and from a hospital and waiting for the result is saved.

Disclosure of Invention

To the problem that above-mentioned prior art exists, the utility model aims at providing a time resolution fluorescence immunochromatography kit of short-term test tacrolimus.

In order to achieve the above purpose, the utility model adopts the technical proposal that: the time-resolved fluorescence immunochromatographic kit for rapidly detecting tacrolimus comprises a detection card and a selectable fluorescence immunoassay quantitative analyzer, wherein the detection card comprises a sample pad, a combination pad, a detection pad with a detection line and a quality control line, an absorption pad and a backing plate, the combination pad is sprayed with a nano microsphere time-resolved fluorescence-tacrolimus probe, the nano microsphere time-resolved fluorescence-tacrolimus probe is prepared from nano fluorescent microspheres and a tacrolimus monoclonal antibody, the detection line is prepared by spraying a tacrolimus protein derivative, and the quality control line is prepared by spraying a goat anti-mouse Ig G antibody.

Preferably, the tacrolimus antibody is a murine antibody.

Preferably, the nano fluorescent microsphere is prepared by utilizing carboxylated polystyrene nano microsphere, and the molar ratio of rare earth element ions, β -diketone chelate and fluorescence enhancement synergist in the nano fluorescent microsphere is 1.2: 3.

Preferably, the diameter of the carboxylated polystyrene nano-microsphere is between 80nm and 120nm, the rare earth element ions are europium ions or samarium ions, the β -diketone chelate is β -naphthoyl trifluoroacetone, and the fluorescence enhancement synergist is trioctylphosphine oxide.

Preferably, the width of the bonding pad is 8 mm.

Preferably, the detection line is located 20mm from the end of the detection pad near the sample pad, and the quality control line is located 30mm from the end of the detection pad near the absorbent pad.

Preferably, the detection pad is a nitrocellulose membrane and is a porous structure membrane with the pore diameter of 5-12 mu m; the material of sample pad, combination pad is glass cellulose membrane or non-woven fabrics, the material of absorption pad is water absorption filter paper.

Fluorescent microsphere time-resolved techniques and immunochromatographic detection techniques are known. The time-resolved fluorescent microsphere labeled antibody of the present invention can be prepared by methods well known in the art.

The utility model has the advantages that: the time-resolved fluorescence immunochromatographic kit for rapidly detecting tacrolimus provided by the utility model has high detection sensitivity, and the limit of quantification is at least 2 ng/mL; the detection range is wide, and the quantitative linear range can reach 2-30 ng/mL; the detection is stable and reliable, and the addition recovery rate is 85-115%. In addition, the kit is simple and easy to operate, can obtain an accurate and reliable determination result in only 5 minutes, and has good commercial application prospect.

The present invention will be further explained with reference to the drawings and examples.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

Fig. 1 is a schematic structural view of the present invention;

1. a sample pad; 2. a bonding pad; 3. detecting lines; 4. a quality control line; 5. a detection pad; 6. an absorbent pad; 7. a backing plate.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, more technical details of the present invention are described in detail with specific embodiments below.

Example 1

Referring to fig. 1, a time-resolved fluoroimmunoassay kit for rapidly detecting tacrolimus comprises a detection card and an optional fluoroimmunoassay quantitative analyzer, wherein the detection card comprises a sample pad 1, a binding pad 2, a detection pad 5 with a detection line 3 and a quality control line 4, an absorption pad 6 and a backing plate 7, the detection line 3 is 20mm away from the end of the detection pad 5 close to the sample pad, and the quality control line 4 is 30mm away from the end of the detection pad close to the absorption pad.

The nanometer fluorescent microsphere is prepared from carboxylated polystyrene nanometer microspheres, the diameter of the carboxylated polystyrene nanometer microspheres is about 80nm, the molar ratio of rare earth element ions, β -diketone chelate and a fluorescence enhancement synergist is 1.2: 3, the rare earth element ions are europium ions, the added raw material is europium trichloride, the β -diketone chelate is β -naphthoyl trifluoroacetone, and the fluorescence enhancement synergist is trioctylphosphine oxide.

The detection line 3 is prepared by spraying tacrolimus protein derivatives, and the quality control line 4 is prepared by spraying goat anti-mouse IgG antibodies.

The detection pad 5 is a nitrocellulose membrane and is a porous sample structure membrane with the aperture of 5 mu m; the material of sample pad 1, combination pad 2 is glass cellulose membrane or non-woven fabrics, the material of absorption pad 6 is water absorption filter paper.

The quantitative limit of the time-resolved fluorescence immunochromatographic kit for rapidly detecting tacrolimus provided by the embodiment is at least 2 ng/mL; the quantitative linear range can reach 2-30 ng/mL; the addition recovery rate is 85-115%.

Example 2

The nanometer fluorescent microsphere is prepared from carboxylated polystyrene nanometer microspheres, the diameter of the carboxylated polystyrene nanometer microspheres is about 120nm, the molar ratio of rare earth element ions, β -diketone chelate and a fluorescence enhancement synergist is 1.2: 3, the rare earth element ions are samarium ions, the added raw material is samarium trichloride, the β -diketone chelate is β -naphthoyl trifluoroacetone, and the fluorescence enhancement synergist is trioctylphosphine oxide.

The detection pad 5 is a nitrocellulose membrane and is a porous structure membrane with the aperture of 12 mu m; the material of sample pad 1, combination pad 2 is glass cellulose membrane or non-woven fabrics, the material of absorption pad 6 is water absorption filter paper.

Example 3

The nanometer fluorescent microsphere is prepared from carboxylated polystyrene nanometer microspheres, the diameter of the carboxylated polystyrene nanometer microspheres is about 100nm, the molar ratio of rare earth element ions, β -diketone chelate and a fluorescence enhancement synergist is 1.2: 3, the rare earth element ions are europium ions, the added raw material is europium trichloride, the β -diketone chelate is β -naphthoyl trifluoroacetone, and the fluorescence enhancement synergist is trioctylphosphine oxide.

The detection pad 5 is a nitrocellulose membrane and is a porous sample structure membrane with the aperture of 7 mu m; the material of sample pad 1, combination pad 2 is glass cellulose membrane or non-woven fabrics, the material of absorption pad 6 is water absorption filter paper.

The detection method of the kit of the utility model is as follows:

the sample to be detected is dripped into the sample pad area of the kit provided by the utility model, and is inserted into the fluorescence reading instrument after chromatography for 5min, and the result and the reading are calculated according to the standard curve to obtain the detection result.

The utility model discloses a detection mechanism is when having tacrolimus in the sample, tacrolimus in the sample will combine with the tacrolimus fluorescence microsphere that combines to the fluorescence microsphere that combines on the detection line 3 will reduce, and the fluorescence microsphere on the quality control line 4 will increase, thereby enlarges the difference of detection line 3 and quality control line 4, increases the sensitivity of whole test paper strip.

The utility model discloses the kit is applicable to whole blood detection, and the patient of being convenient for is gone out hospital and is carried out tacrolimus's detection in the blood at community health institution, alleviates to go the hospital to register, line up, chemical examination, gets loaded down with trivial details processes such as report, alleviates public medical institution's pressure, more is of value to patient's daily monitoring.

The utility model discloses structurally increased backing plate, can effectually get rid of the background interference to further improve diagnosis sensitivity and degree of accuracy, the boosting pad still helps accelerating the detection sample flow in addition, improves detection efficiency, shortens check-out time.

Compared with the prior art, the time-resolved fluorescence immunochromatographic kit for rapidly detecting tacrolimus provided by the utility model has high detection sensitivity, and the limit of quantification is as low as 2 ng/mL; the detection range is wide, and the quantitative linear range can reach 2-30 ng/mL; the detection is stable and reliable, and the addition recovery rate is 85-115%. In addition, the kit is simple and easy to operate, can obtain an accurate and reliable determination result in only 5 minutes, and has good commercial application prospect.

Although the present invention has been described with reference to particular embodiments, such description is not intended to be construed in a limiting sense. Other variations of the disclosed embodiments, as would be appreciated by those skilled in the art, are contemplated by reference to the description of the invention and are intended to be within the scope of the following claims.

Claims

1. A time-resolved fluoroimmunoassay kit for rapidly detecting tacrolimus, which comprises a detection card and an optional fluoroimmunoassay quantitative analyzer, the test card comprises a sample pad, a combination pad, a test pad with a test line and a quality control line, an absorption pad and a backing plate, it is characterized in that the binding pad is sprayed with a nano microsphere time-resolved fluorescence-tacrolimus probe, the nano-microsphere time-resolved fluorescence-tacrolimus probe is prepared from nano-fluorescent microspheres and a tacrolimus monoclonal antibody, the nano fluorescent microsphere is prepared by utilizing carboxylated polystyrene nano microsphere, the diameter of the carboxylated polystyrene nano microsphere is 80nm, the detection line is prepared by spraying tacrolimus protein derivatives, the quality control line is prepared by spraying goat anti-mouse IgG antibodies, and the detection pad is a nitrocellulose membrane and is a porous structure membrane with the pore diameter of 5-7 mu m.

2. The time-resolved fluoroimmunoassay kit for rapid detection of tacrolimus according to claim 1, wherein the tacrolimus antibody is murine antibody.

3. The time-resolved fluoroimmunoassay kit for rapid detection of tacrolimus according to claim 1, wherein the width of the binding pad is 8 mm.

4. The time-resolved fluoroimmunoassay kit for rapidly detecting tacrolimus according to claim 1, wherein the detection line is 20mm from the end of the detection pad close to the sample pad, and the quality control line is 30mm from the end of the detection pad close to the absorption pad.

5. The time-resolved fluoroimmunoassay kit for rapid detection of tacrolimus according to any one of claims 1 to 4, wherein the sample pad and the binding pad are made of glass cellulose membrane or non-woven fabric, and the absorption pad is made of absorbent filter paper.