CN217033965U

CN217033965U - Kit for combined detection of catastatin, BNP and NT-proBNP in sample

Info

Publication number: CN217033965U
Application number: CN202123088876.0U
Authority: CN
Inventors: 朱丹
Original assignee: Peking University Third Hospital Peking University Third Clinical Medical College
Current assignee: Peking University Third Hospital Peking University Third Clinical Medical College
Priority date: 2021-12-09
Filing date: 2021-12-09
Publication date: 2022-07-22
Anticipated expiration: 2031-12-09

Abstract

The utility model relates to a kit for combined detection of catastin, BNP and NT-proBNP in a sample. The kit comprises an outer box, a test board, an analysis membrane, a binding membrane and an absorption membrane. The fluorescence immunochromatography technology is adopted for simultaneously detecting the levels of catastin, BNP and NT-proBNP clinically, and provides a basis for diagnosis and prognosis judgment of clinical diseases.

Description

Kit for combined detection of catastin, BNP and NT-proBNP in sample

Technical Field

The utility model relates to a kit for combined detection of catastin, BNP and NT-proBNP in a sample.

Background

Heart Failure (HF) is a major cause of death in humans. Heart failure patients in the united states can take over 500 million new occurrences in 55 million new years, increasing at a rate of 1506 cases per day. Effectively reducing the morbidity and mortality of heart failure has been a dream to be realized in clinical science. The key to achieving this dream lies in early diagnosis, early prevention and early intervention. At present, the clinical diagnosis of heart failure is mainly based on clinical characteristics, echocardiograms and biomarkers, and no method with high specificity and easy detection is used for early diagnosis of heart failure. Therefore, the joint and parallel detection of various different heart failure markers has become a necessity, and the existence of a joint kit is crucial for the diagnosis of diseases.

Catecholamine (CST) is a polypeptide with cardiovascular activity, derived from chromophorin a, consisting of 21 amino acids. Both in animal experiments and in vitro studies confirm that: catestatin acts on neuronal nicotinic acetylcholine receptors (nAChR) in an autocrine manner, inhibits catecholamine release, and acts on G protein in a paracrine manner to promote histamine release in mast cells. Catecholamine statins inhibit the release of catecholamines and are able to reflect the activity of the sympathetic nervous system.

Pro-brain natriuretic peptide (ProBNP) secretes more during ventricular dilatation and volume overload, and degrades in vivo to yield C-terminal BNP of 32 amino acids and N-terminal fragment of 76 amino acids pro-brain natriuretic peptide (NT-ProBNP). BNP has the biological activities of natriuresis and vasodilatation, and the half-life period of NT-proBNP is about 15 times longer and the clearance time is long.

Catestatin, BNP and NT-proBNP all have certain predictive value for the occurrence and the development of cardiovascular diseases, but have respective emphasis, and researches prove that the catastin can change the level at the early stage of cardiac insufficiency, and the change is earlier than that of the BNP and NT-proBNP, and the BNP and NT-proBNP are considered as the most sensitive indexes capable of indicating clinical heart failure, namely the later stage of the cardiac insufficiency. The combination of catastatin, BNP and NT-proBNP is therefore of particular importance for the diagnosis, especially early diagnosis, of heart failure. At present, no kit capable of simultaneously detecting catastin, BNP and NT-proBNP exists, three markers need to be separately detected, three blood samples are needed, and the diagnosis and treatment time of a patient is delayed, so that a combined kit capable of simply, conveniently and quickly obtaining a detection result is urgently needed. A kit for jointly detecting catastin, BNP and NT-proBNP in a sample is to be established, so that the clinical needs are better solved.

Disclosure of Invention

In order to better realize the aim of jointly detecting the levels of catetin, BNP and NT-proBNP in a sample in clinic, the utility model provides a combined kit which is clinically used for simultaneously detecting the levels of catetin, BNP and NT-proBNP and provides a basis for the diagnosis and prognosis judgment of clinical diseases.

1. Principle of combined kit detection

All samples are detected by adopting a fluorescence immunochromatography technology, the technology is based on antigen-antibody specific immunoreaction, strip-shaped fiber chromatography materials fixed with a detection line (coated antibody or coated antigen) and a quality control line (anti-antibody) are used as stationary phases, and a test solution is used as a mobile phase. For macromolecular antigens with a plurality of antigenic determinants, a sandwich-type double-antibody sandwich immunochromatography method is generally adopted, namely, an object to be detected is firstly combined with a fluorescence labeling antibody under the action of a mobile phase, and then is combined with a coating antibody to form a sandwich-type double-antibody sandwich when reaching a detection line.

2. Features of the kit

1) A kit for combined detection of catastin, BNP and NT-proBNP in a sample, which is characterized in that: the kit comprises an outer box, wherein the outer box comprises: the test board and the test board are sequentially provided with an analysis film, a combination film and an absorption film.

2) The analysis membrane is provided with three detection lines and a quality control line, wherein the three detection lines are a catastin detection line, a BNP detection line and an NT-proBNP detection line. The catestin detection line is coated with a catestin detection antibody, the BNP detection line is coated with a BNP detection antibody, and the NT-proBNP detection line is coated with an NT-proBNP detection antibody.

3) The binding membrane is provided with a catastin antibody marker, a BNP antibody marker and an NT-proBNP antibody marker.

4) The catestin detection antibody and the catestin antibody of the catestin antibody marker have different antigen binding sites, the BNP detection antibody and the BNP antibody of the BNP antibody marker have different antigen binding sites, and the NT-proBNP detection antibody and the NT-proBNP antibody of the NT-proBNP antibody marker have different antigen binding sites.

5) Wherein the sample to be tested is a human blood sample.

3. Kit composition

The kit includes an outer box (fig. 1). The outer box comprises: the test board and the test board are sequentially provided with an analysis film, a combination film and an absorption film.

The utility model patent is preferably prepared by the following steps:

the first step is as follows: antibody labeling:

respectively labeling the catastin, BNP and NT-proBNP antibodies and the markers by using an antibody labeling technology to form an antibody marker compound solution or suspension, wherein the antibody concentration is 0.05 mg/mL-5 mg/L;

the antibody labeling technology adopts an antibody labeling fluorescent microsphere: activation of microspheres: adding 0.1-10 mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) and 0.1-10 mg of NHS (N-hydroxysuccinimide) dissolved by 10-100 mM of MES (2- (N-morpholine) ethanesulfonic acid monohydrate) at pH of 5.0-9.0 into 0.1-10 mg/ml of fluorescent microspheres, activating for 10-600 min, centrifuging at 7000-15000 rpm for 5-30 min, discarding supernatant, and re-suspending precipitate by 10-100 mM of PBS at pH of 5.0-9.0 to obtain 0.1-10 mg/ml activated fluorescent microsphere solution; antibody coupling: respectively adding 0.1-10.0 mg of catastin, BNP and NT-proBNP labeled antibody into 0.1-10 mg/ml of activated fluorescent microsphere solution, uniformly mixing for 0.5-24 h at room temperature, centrifuging at 7000-15000 rpm for 5-30 min, discarding supernatant, sealing the precipitate with 10-100 mM PBS (phosphate buffer) solution containing 0.5-50 mg of BSA (bovine serum albumin) for 0.5-24 h, stirring, uniformly mixing, centrifuging at 7000-15000 rpm for 5-30 min, and storing the collected precipitate in 0.0-8.0 and 0-100 mM Tris buffer solution to obtain the catastin antibody labeled fluorescent microsphere solution, the BNP antibody labeled fluorescent microsphere solution and the NT-proBNP labeled fluorescent microsphere solution.

The second step: preparation of analytical membranes:

respectively diluting the catastin detection antibody, the BNP detection antibody, the NT-proBNP detection antibody and the quality control line to 0.1-5 mg/ml, spraying the catastin detection antibody, the BNP detection antibody, the NT-proBNP detection antibody and the quality control line on an analysis membrane material on a membrane spraying instrument in a spraying amount of 0.25-4.0 muL/cm to form a catastin detection line, a BNP detection line, an NT-proBNP detection line and the quality control line, namely the analysis membrane, and drying for later use.

The third step: preparing a bonding film:

mixing the catestin antibody marker, the BNP antibody marker and the NT-proBNP antibody marker obtained in the first step according to the volume ratio to form an antibody marker mixed solution, wherein the volume of the catestin antibody marker solution is 0.5-90%, the volume of the BNP antibody marker solution is 0.5-90%, and the volume of the NT-proBNP antibody marker solution is 0.5-90%; spraying the mixture on the binding membrane material according to 0.25-15 mu L/cm to obtain the binding membrane, and drying for later use.

The main innovation of the utility model is that:

1. the kit can jointly detect the catastatin, the BNP and the NT-proBNP, and simultaneously obtain the detection results of the three items of the catastatin, the BNP and the NT-proBNP, thereby simplifying the operation steps, reducing the sample dosage and saving the time and the cost.

2. The test board in the kit can stably analyze the membrane, combine the membrane and absorb the membrane, reduce the loss in the transportation process and improve the test accuracy.

Drawings

FIG. 1: reagent box shape

FIG. 2 is a drawing: patient catastatin and NT-proBNP levels

Detailed Description

The kit comprises an outer box, wherein the outer box comprises: the test board and the test board are sequentially provided with an analysis film, a combination film and an absorption film.

The analysis membrane is provided with three detection lines and a quality control line, wherein the three detection lines are a catastin detection line, a BNP detection line and an NT-proBNP detection line respectively. The catestin detection line is coated with a catestin detection antibody, the BNP detection line is coated with a BNP detection antibody, and the NT-proBNP detection line is coated with an NT-proBNP detection antibody. The binding membrane is provided with a catastin antibody marker, a BNP antibody marker and an NT-proBNP antibody marker. The catastin detection antibody and the catastin antibody of the catastin antibody marker have different antigen binding sites; the BNP detection antibody and the BNP antibody of the BNP antibody marker have different antigen binding sites; the NT-proBNP antibody detecting antibody and the NT-proBNP antibody labeled with the NT-proBNP antibody have different antigen binding sites. The reagent strip can be used for quantitatively detecting catastin/BNP/NT-proBNP in samples of serum, plasma, whole blood and peripheral blood, does not need to pretreat the samples, and is simple and convenient to operate, convenient and fast. Adding the sample into a sample port, adding a certain volume of 0-200 mu L of sample diluent (such as 0.09% NaCl), detecting by a matched instrument after 30min, and automatically calculating to obtain the concentration of the catastin/BNP/NT-proBNP in the sample. And one sample is loaded at one time, and is detected at one time, so that detection results of three items, namely catastatin, BNP and NT-proBNP are obtained, the operation steps are simplified, the sample dosage is reduced, the time is saved, and the cost is saved.

The kit is used for testing samples, ROC curves of catestin, BNP and NT-proBNP are drawn, and the ROC curves are shown in table 1. As can be seen from Table 1, the area under the ROC curve of the combined detection is larger, and the combined detection has higher accuracy in the diagnosis of heart failure. Table 1 is as follows:

variables of	AUC	SE	95％CI
				Joint detection	0.916	0.0307	0.832-0.958
catestatin	0.842	0.0354	0.801-0.927
				BNP	0.816	0.0276	0.764-0.886
NT-proBNP	0.896	0.0348	0.802-0.943

The study enrolled patients who were admitted to the hospital in cardiology department of the third hospital of Beijing university between 2017 and 2018 and signed informed consent in accordance with the enrollment criteria, between 9 months. Collecting three groups of patients by ACC/AHA cardiac function stage, wherein the sex constitution, body mass index, blood pressure, heart rate and disease course of each group of patients have no statistical difference, and cardiovascular disease risk factors have no statistical difference (P is more than 0.05); however, the catastin level is reduced in the B period of cardiac function, and the NT-proBNP level is reduced in the C period of cardiac function, which indicates that the combined detection of catastin and NT-proBNP can reflect the change of cardiac function.

Claims

1. A kit for jointly detecting catastatin, BNP and NT-proBNP in a sample is characterized in that: the kit comprises an outer box (1), wherein the outer box comprises: the test board (2) and the test board are sequentially provided with an analysis film, a binding film and an absorption film, the analysis film is provided with three detection lines and a quality control line, the three detection lines are a catastin detection line, a BNP detection line and an NT-proBNP detection line, the catastin detection line is coated with a catastin detection antibody, the BNP detection line is coated with a BNP detection antibody, and the NT-proBNP detection line is coated with an NT-proBNP detection antibody.

2. The kit of claim 1, wherein: the binding membrane is provided with a catastin antibody marker, a BNP antibody marker and an NT-proBNP antibody marker.

3. The kit of claim 1, wherein: the catestin detection antibody and the catestin antibody of the catestin antibody marker have different antigen binding sites, the BNP detection antibody and the BNP antibody of the BNP antibody marker have different antigen binding sites, and the NT-proBNP detection antibody and the NT-proBNP antibody of the NT-proBNP antibody marker have different antigen binding sites.

4. The kit of claim 1, wherein the sample to be tested is a human blood sample.