CN107271416B

CN107271416B - Reagent system for detecting myoglobin

Info

Publication number: CN107271416B
Application number: CN201710550887.5A
Authority: CN
Inventors: 陈静; 谭有将; 戴尽波
Original assignee: Guangdong Shunde Industrial Design Institute
Current assignee: Foshan Mosai Biotechnology Co.,Ltd.
Priority date: 2017-07-07
Filing date: 2017-07-07
Publication date: 2020-02-11
Anticipated expiration: 2037-07-07
Also published as: CN107271416A

Abstract

The invention discloses a myoglobin detection reagent system based on time-resolved microspheres, which comprises the following components: adding 0.05-0.5 wt% of time resolution microsphere marked with myoglobin antibody, 0.1-0.5 wt% of bovine serum albumin, 0.1-5 wt% of polyvinylpyrrolidone, 0.1-1 wt% of ribosomal protein S7 and 0.01-0.5 wt% of tween-20 into the buffer solution. The reagent system can react after being mixed with a sample, and the operation is simple; under the action of the reagent system, the time-resolved microspheres labeled with the antibody do not remain on the sample pad, nor remain at the joint of the sample pad and the Nitrocellulose (NC) membrane. The antibody in the reagent system has high specificity and high sensitivity in reaction with myoglobin, and the detection system has high stability, high accuracy, high precision and wide detection range.

Description

Reagent system for detecting myoglobin

Technical Field

The invention relates to the technical field of biology, in particular to a myoglobin detection reagent system based on time-resolved microspheres and a preparation method and application thereof.

Background

The time-resolved fluorescence biochemical analysis technology is established based on the special fluorescence property of the rare earth fluorescent complex, the time-resolved fluorescence determination technology utilizes the characteristic that the rare earth complex has long-life fluorescence, certain delay time (delay time) is introduced according to the difference of the fluorescence life of fluorescent substances contained in a sample after the sample is excited by pulsed light and before fluorescent signals are collected, and after the short-life background fluorescence is completely quenched, the long-life specific fluorescent signals are determined.

Myoglobin (MYO) is a heme protein present in muscle tissue, and is rapidly released into the blood after muscle cell injury due to its small molecular weight. In acute myocardial injury, myoglobin is released into blood firstly, and MYO in blood can exceed the upper limit of normal after symptoms appear for 2-3 hours, reaches the peak value in 9-12 hours, and recovers after 24-36 hours. MYO is the earliest abnormal cardiovascular marker after myocardial damage at present, while MYO positive cannot diagnose Acute Myocardial Infarction (AMI), but can be used for early eliminating important indexes for AMI diagnosis, and if MYO negative, myocardial infarction is basically eliminated. Various myopathies, such as acute muscle injury, muscular dystrophy, muscular atrophy, and polymyositis, result in elevated levels of myoglobin in the blood. Acute and chronic renal failure, severe congestive heart failure and long-term shock also lead to elevated myoglobin. Monitoring myoglobin levels is of great importance for the diagnosis of a variety of diseases.

Myoglobin is usually measured by chemiluminescence, latex turbidimetry, colloidal gold immunochromatography, or fluorescence immunochromatography, but these methods are complicated to operate, have low stability, have low sensitivity, and are prone to false negatives and false positives. The antigen-antibody combination specificity is not strong, the accuracy of the detection result is not high, the precision is low, and the detection range is small.

Disclosure of Invention

Therefore, it is necessary to provide a time-resolved microsphere-based reagent system for myoglobin detection, aiming at the problems of low sensitivity, low precision, low stability, complex operation, too high detection limit and the like in myoglobin detection, so as to promote the specificity of antigen-antibody combination, realize simple and rapid detection of myoglobin content, and simultaneously have high sensitivity, high stability, good precision, small detection limit, large detection range and capability of detecting samples with different concentrations.

The specific technical scheme is as follows:

a myoglobin detection reagent system based on time-resolved microspheres comprises the following components: adding 0.05-0.5 wt% of time resolution microsphere marked with myoglobin antibody, 0.1-0.5 wt% of bovine serum albumin, 0.1-5 wt% of polyvinylpyrrolidone, 0.1-1 wt% of ribosomal protein S7 and 0.01-0.5 wt% of tween-20 into the buffer solution.

In some of these embodiments, the time-resolved microsphere-based reagent system for myoglobin detection comprises the following components: adding 0.15-0.25 wt% of time resolution microsphere marked with myoglobin antibody, 0.2-0.3 wt% of bovine serum albumin, 0.5-1.5 wt% of polyvinylpyrrolidone, 0.4-0.6 wt% of ribosomal protein S7 and 0.05-0.15 wt% of tween-20 into the buffer solution.

In some of these embodiments, the time-resolved microsphere-based reagent system for myoglobin detection comprises the following components: the buffer solution was added with 0.2 wt% time-resolved microspheres labeled with myoglobin monoclonal antibody, 0.25 wt% bovine serum albumin, 1 wt% polyvinylpyrrolidone, 0.5 wt% ribosomal protein S7, 0.1 wt% Tween-20.

In some of these embodiments, the mass ratio of bovine serum albumin to ribosomal protein S7 is 1: 2.

In some of these embodiments, the myoglobin antibody is selected from one or more of myoglobin monoclonal antibody, myoglobin polyclonal antibody.

In some of these embodiments, the reagent system has a pH of 7-8 (preferably 7.4).

In some of these embodiments, the buffer solution is a phosphate buffer or a Tris buffer.

The reagent system for detecting myoglobin has the following beneficial effects:

the reagent system can react with the sample after being mixed, and the operation is simple; under the action of the reagent system, the time-resolved microspheres labeled with the antibody do not remain on the sample pad, nor remain at the joint of the sample pad and the Nitrocellulose (NC) membrane. The antibody in the reagent system has high specificity and high sensitivity in reaction with myoglobin, and the detection system has high stability, high accuracy, high precision and wide detection range.

Drawings

Fig. 1 is a standard graph.

Wherein the abscissa is the concentration of myoglobin standard (ng/mL) and the ordinate is the detected fluorescence signal (T/C value).

Detailed Description

The time-resolved microsphere-based reagent system for myoglobin assay according to the present invention will be described in detail with reference to the following specific examples.

The reagent specification, instrument type and manufacturer used in the invention are as follows:

phosphate Buffered Saline (PBS), specific composition: NaCl 8.5g, NaHPO ₄2.2g，NaH ₂PO ₄0.2g，H ₂O1000mL；

Bovine Serum Albumin (BSA) available from west bao biotechnology limited;

polyvinylpyrrolidone (PVP), available from alatin;

ribosomal protein S7, available from shanghai jeldahl biotechnology limited;

time-resolved microspheres labeled with myoglobin antibodies, purchased from Bangslab;

myoglobin standard, purchased from Shanghai Guduo Biotech, Inc.;

the sample is sourced from north Kau Hospital of Shuidedistrict, Guangdong province;

fluorescence detector Microdetection Nanjing micro-assay Biotechnology Ltd.

Example 1

In this example, the stability of three groups of solutions containing time-resolved microspheres labeled with myoglobin monoclonal antibodies was examined, specifically, the stability of the solutions was determined by detecting the change of fluorescence signals of the solutions after standing for different times. The compositions of the solutions of each group are shown in table 1:

TABLE 1

Three groups of solutions were prepared according to the compositions in table 1, and fluorescence signals were detected after the solution preparation was completed and left for 1 day, 3 days, 5 days, and 7 days, with the detection results shown in table 2:

TABLE 2

As can be seen from the test results in Table 2, the T/C value change rate of the solution ① in 7 days of standing was more than 10%, the T/C value change rate of the solution ② in 3 days and 5 days of standing was small, the T/C value change rate after 5 days of standing was as low as 0.52%, the T/C value change rate of the solution ③ in 3 days of standing was as low as 1.52%, and the T/C value change rate after standing was more than 22%, indicating that polyvinylpyrrolidone PVP contributes to the stability of the solution.

Example 2

The above test was carried out, and the conditions were as follows: BSA: 0.25%, PVP: 1%, S7: the result of 0.5% has better stability and smaller decline amplitude, and can be repeated after a plurality of subsequent tests.

The composition of a reagent system for detecting myoglobin is determined as follows: 10mM PBS containing 0.25 wt% BSA, 1 wt% PVP, 0.5 wt% S7, 0.1 wt% Tween-20 and 0.2 wt% myoglobin time resolved microspheres.

Example 3

Drawing of standard curve and sample testing

The standard substance is diluted to the following concentration, 2000ng/ml, 1000ng/ml, 750ng/ml, 500ng/ml, 250ng/ml, 100ng/ml, 75ng/ml, 50ng/ml, 25ng/ml, 12.5ng/ml and 6.25ng/ml for fluorescence detection respectively, and corresponding fluorescence values are obtained. The standard curve is plotted as shown in fig. 1.

Concentration of standard substance	Testing T/C value
		2000	3.50756
1000	2.91702
		750	2.78745
500	2.55058
		250	2.05047
100	1.399803
		75	1.31654
50	1.06481
		25	0.63259
12.5	0.339408
		6.25	0.216751

Example 4

Limit of detection experiment

The lowest detection value of the sampling sample is 21, and the method can detect a lower value.

Example 5

Recovery rate experiment (accuracy)

Different samples of multiple values were selected for repeated testing with the following results:

example 6

Precision experiment

Taking three samples with different concentrations, testing the same sample five times, and observing five changes

Comparative example 1

The composition of the reagent system for detecting myoglobin is as follows: 10mM PBS contained 1% PVP, 0.5% S7, 0.1% Tween-20, and 0.2% time resolved microspheres labeled myoglobin.

Comparative example 2

The composition of the reagent system for detecting myoglobin is as follows: 10mM PBS containing 0.25% BSA, 1% PVP, 0.1% Tween-20 and 0.2% myoglobin time resolved microspheres.

Each average is the average of three measurements.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A myoglobin detection reagent system is characterized by comprising the following components: adding 0.15-0.25 wt% of time resolution microsphere marked with myoglobin antibody, 0.2-0.3 wt% of bovine serum albumin, 0.5-1.5 wt% of polyvinylpyrrolidone, 0.4-0.6 wt% of ribosomal protein S7 and 0.05-0.15 wt% of tween-20 into the buffer solution.

2. The myoglobin detection reagent system of claim 1, comprising the following components: the buffer solution was added with 0.2 wt% time-resolved microspheres labeled with myoglobin monoclonal antibody, 0.25 wt% bovine serum albumin, 1 wt% polyvinylpyrrolidone, 0.5 wt% ribosomal protein S7, 0.1 wt% Tween-20.

3. The myoglobin assay reagent system of claim 1, wherein the mass ratio of bovine serum albumin to ribosomal protein S7 is 1: 2.

4. The myoglobin detection reagent system of any one of claims 1-2, wherein said myoglobin antibody is selected from the group consisting of myoglobin monoclonal antibody, myoglobin polyclonal antibody, and combinations thereof.

5. The myoglobin assay reagent system of any one of claims 1-2, wherein the reagent system has a pH of 7-8.

6. The myoglobin detection reagent system of claim 5, wherein said reagent system has a pH of 7.4.

7. The myoglobin detection reagent system of any one of claims 1-2, wherein said buffer solution is phosphate buffer or Tris buffer.