Lipoprotein (a) detection kit and application

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a lipoprotein (a) detection kit. The lipoprotein (a) detection kit provided by the invention comprises a reagent R1, a reagent R2 and a standard substance, the reagent R1 comprises trihydroxymethyl aminomethane (Tris Ultra Pure), hydrochloric acid, 1-oxo-2-hydroxypyridine (HPO), sodium chloride, polyglycolate (Thick), bovine serum albumin (BSA firv) and horse serum (carbon adsorption); and the reagent R2 is prepared from tris (hydroxymethyl) aminomethane (Tris Ultra Pure), hydrochloric acid, 1-oxo-2-hydroxypyridine (HPO), sodium chloride, Thick, bovine serum albumin (BSA firv), horse serum (carbon adsorption) and an LPa latex antibody. The standard substance is human serum added with human recombinant apolipoprotein (a) or liquid similar to a serum matrix. The method comprises the following steps: mixing a sample and a reagent according to a certain volume ratio, carrying out a series of reactions, placing reactants under a microplate reader, and detecting the absorbance change speed at a dominant wavelength of 700nm so as to measure the concentration of lipoprotein (a). The method has the advantages of good precision, good linearity in a detection range, good stability, low production cost, low blank absorbance and anti-interference performance.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a lipoprotein (a) detection kit and application thereof. Background Art

[0002] In my country, the patient base in the cardiovascular field is large, and the drug market has great potential. According to the "China Cardiovascular Health and Disease Report 2023", there are 330 million people suffering from cerebrovascular disease (CVD) in my country, including 13 million strokes and 11.39 million coronary heart disease. Lipid-lowering drugs are mainly used to prevent and treat cardiovascular diseases by lowering cholesterol and triglyceride levels in the blood. According to relevant data, looking at the entire lipid-lowering drug track, the ceiling of my country's lipid-lowering drug market continues to rise over time. From 2017 to 2022, the scale of my country's lipid-lowering drug market increased from 27.44 billion yuan to 47 billion yuan, with an annual compound growth rate of 11.36%. In 2023, the market size has exceeded 50 billion yuan. This also shows the rapid growth of innovative drugs in the lipid-lowering drug market and the increasing market demand for new lipid-lowering drugs.

[0003] Lipoprotein (a) [Lp(a)] has been found to be a risk factor for cardiovascular disease (CVD). Lp(a) has been associated with an increased risk of coronary heart disease, ischemic stroke, aortic stenosis, heart failure, atrial fibrillation, and peripheral arterial disease. There are many pathophysiological pathways involved in the increased risk of CVD when plasma Lp(a) levels are high. Lp(a) was discovered and named in 1963 by the Norwegian geneticist Kare Berg in an immunochemical study. Lp(a) is a subtype / variant of LDL-C that is distinguished by the fact that apolipoprotein (a) poB100 is covalently bound to a unique glycoprotein called apo(a) via its disulfide bonds. Lp(a) exerts proatherogenic effects through its low-density lipoprotein cholesterol (LDL-C) portion and proinflammatory effects through its oxidized phospholipid content, as well as antifibrinolytic effects (competitively blocking the binding of plasminogen to fibrin) through the apolipoprotein [apo(a)] component. Approximately 20% of the world's population has elevated Lp(a) levels, which are primarily determined by genetic factors. The mechanisms of action of Lp(a) in mediating atherosclerotic cardiovascular disease (ASCVD) include contributions to atherosclerosis through the lipoprotein portion and potential contributions to thrombosis through the apo(a) portion, which has structural similarities to plasminogen.

[0004] However, further research on Lp(a) is needed to determine the potential clinical benefits for cardiovascular events and its effects in long-term treatment. Currently, the known methods for measuring lipoprotein (a) include radioimmunoassay, fluorescence immunoassay, time-resolved fluorescence immunoassay, and enzyme-linked immunosorbent assay. These conventional methods are cumbersome to operate, time-consuming, have large detection deviations, and are easily interfered with and cause detection errors, affecting the user's detection efficiency, detection accuracy, and detection stability of lipoprotein (a). They are not suitable for routine testing. Summary of the invention

[0005] The present invention provides a lipoprotein (a) detection kit and application; Another object of the present invention is to provide a method for using a lipoprotein (a) detection kit; Another object of the present invention is to use the detection of lipoprotein (a) in detecting the content of lipoprotein (a) in human plasma.

[0006] The above-mentioned purpose of the present invention is achieved by the following technical means: Provided is a lipoprotein (a) detection kit, which comprises a reagent R1, a reagent R2 and a standard substance.

[0007] The reagent R1 is purchased from a commercial source; The reagent R2 is purchased from a commercial source; The standard products were purchased from commercial sources.

[0008] The invention also provides a method for measuring the content of lipoprotein (a) in human serum, plasma, or in vitro assembled cell supernatant by using lipoprotein (a) detection, comprising the following steps: In a light-proof environment, dilute the standard of corresponding concentration with deionized water, add reagent R1 (100μl) and standard / sample (3μl) to the 96-well plate of enzyme labeling, and keep warm at 37℃ for 4-5 min. After taking out, add reagent R2 (25μl) to the mixed solution and keep warm at 37℃ for 5-10min. Measure the OD value of each well at a wavelength of 700nm. Draw a standard curve of lipoprotein (a), and calculate the concentration of lipoprotein (a) in the sample to be tested by the standard curve.

[0009] The lipoprotein (a) detection kit provided by the present invention has a linear range of 22.89 mg / mL-586.67 mg / L for determining lipoprotein (a) in human serum or plasma.

[0010] The quantitative lipoprotein (a) detection kit provided by the present invention provides an effective tool for rapid, stable and reliable lipoprotein (a) detection in the preclinical stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The standard curve is for the detection of lipoprotein (a) standards. DETAILED DESCRIPTION

[0012] The present invention is further described below in conjunction with experimental examples and specific implementation methods. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0013] (1) Dilute the standard sample of corresponding concentration with deionized water in a light-proof environment; (2) Add reagent R1 (100 μl) and standard / sample (3 μl) to the ELISA 96-well plate and incubate at 37°C for 4-5 min. (3) After removal, add reagent R2 (25 μl) and incubate at 37°C for 5-10 min; (4) Measure the OD value of each well at a wavelength of 700 nm and draw a standard curve for the detection of lipoprotein (a) standards, such as Figure 1 shown.

[0014] Blood samples were collected using blood collection tubes, and plasma was collected and frozen at -80°C for plasma lipoprotein (a) detection.

[0015] All the above plasma samples were tested collectively according to the operation steps shown in Example 1. Reagent R1 was added to a 96-well plate, 100 μl per well, and human plasma samples were added to a 96-well plate, 3 μl per well. Positive and negative controls were set at the same time, and the samples were kept at 37°C for 4-5 min. After removal, reagent R1 was added to a 96-well plate, 100 μl per well, and kept at 37°C for 5-10 min. The OD value at 700 nm was detected by an enzyme marker, and the content of lipoprotein (a) in human plasma was calculated to be 107.53 mg / L according to the standard curve.

Claims

1. A lipoprotein (a) detection kit, comprising: reagent R1, reagent R2, and a standard substance, wherein: a. Reagent R1 includes: 6.05 g / L tris(hydroxymethyl)aminomethane, 4.3 ml / L hydrochloric acid, 1.3 g / L 1-oxo-2-hydroxypyridine, 12.58 g / L sodium chloride, 1 ml / L poly(ethylene glycol), 10 g / L bovine serum albumin, and 50 ml / L horse serum; b. Reagent R2: 6.05 g / L tris(hydroxymethyl)aminomethane, 4.3 ml / L hydrochloric acid, 1.3 g / L 1-oxo-2-hydroxypyridine, 12.58 g / L sodium chloride, 1 ml / L poly(ethylene glycol), 10 g / L bovine serum albumin, 50 ml / L horse serum, 550 ml / L LPa latex antibody.

2. The lipoprotein (a) detection kit according to claim 1, for use in in vitro detection of lipoprotein (a), characterized in that: The following steps are involved: (a) Mix reagent R1 and the sample to be tested in a certain proportion and allow them to react completely at a certain temperature; (b) After adding reagent R2, it is allowed to react completely at a certain temperature; (c) Determine the absorbance change using an enzyme-labeled instrument; (d) Calculate the concentration of lipoprotein (a) in the sample based on the absorbance change.

3. The use according to claim 2, characterized in that The ratio of the sample to be tested and reagent R1 should be controlled at (1.5~3):

100.

4. The use according to claim 2, characterized in that The mixing ratio of reagent R1 and the sample to be tested should be controlled at 1:

4.

5. The method of claim 2, characterized in that The temperature is 37 (±1)°C.

6. The method of claim 2, characterized in that The reaction time of the reaction is 5-10 minutes.

7. The method of claim 2, characterized in that The dominant wavelength for detection with an ELISA reader is 500-800 nm.

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