CN101183108A - A colloidal gold test strip for the detection of trace amounts of oxidized low-density lipoprotein by an indirect competitive method - Google Patents

Abstract

The invention provides a colloidal gold test strip for detecting micro-oxidized low-density lipoprotein by an indirect competitive method. The test strip adopts the competition method, and a certain amount of gold-labeled antibody is coated on the gold-labeled antibody pad, and oxidized low-density lipoprotein is coated on the detection area. By observing whether the detection area develops color, it can detect whether the oxidized low-density lipoprotein in human serum or plasma is higher than the normal level. The invention can detect the oxidized low-density lipoprotein conveniently, quickly and intuitively, thereby providing a reliable basis for early diagnosis of atherosclerotic diseases.

Description

A colloidal gold test strip for the detection of trace amounts of oxidized low-density lipoprotein by an indirect competitive method

1. Technical field:

The invention relates to the detection of oxidized low-density lipoprotein, in particular to a colloidal gold test paper for detecting oxidized low-density lipoprotein.

2. Background technology:

According to the latest statistics of the American Heart Association in 2001, cardiovascular disease has become the number one killer of human beings. Atherosclerosis is the pre-pathological basis of most cardiovascular and cerebrovascular diseases, so the research on the pathogenesis, diagnosis and prevention methods of atherosclerosis has become a hot spot in the medical field.

Lipoproteins are the major carriers of plasma cholesterol and triglycerides. Low-density lipoprotein (LDL) is composed of phospholipids, apolipoprotein (Apo) B polypeptide chain, cholesterol, triglycerides, carbohydrates, etc. Oxidized low-density lipoprotein (oxLDL) is formed by oxidative modification of LDL. In recent years, it has been found that oxLDL is closely related to atherosclerosis. oxLDL promotes the formation of foam cells, and the death of foam cells will lead to the deposition of lipids on the arterial wall, which is one of the main causes of atherosclerosis. Recent studies have shown that oxLDL deposition is found in atherosclerotic lesions, but not in normal arterial walls, which is a unique component of atherosclerotic lesions. Therefore, the amount of oxLDL in the patient's body is much higher than that of normal people, and its concentration is proportional to the extent of the lesion. It is clinically believed that the content of oxLDL in healthy people is 200-500 μg/L, and when the content of oxLDL is greater than 600 μg/L in clinical detection, it is abnormal. Therefore, the detection of oxLDL is of great significance for the early warning and early auxiliary diagnosis of atherosclerotic cardiovascular disease.

There are many methods for detecting oxLDL at present, and the most commonly used method is to detect the content of serum oxLDL by enzyme-linked immunosorbent assay (ELISA) using oxLDL monoclonal antibody or polyclonal antibody. The disadvantages of this method are: the operation process is relatively complicated, and special instruments such as microplate readers are required to operate, and professional inspectors are required to operate; the operation time is long, and the whole process takes more than one hour; the detection cost is relatively high, and cannot Realize single-person detection, etc.

As a rapid clinical diagnostic technology, colloidal gold immunochromatography is characterized by single-component detection, which is convenient and fast. It does not require any equipment except commercial reagents. The results can be observed with naked eyes in a few minutes and have a long shelf life.

Chinese patent 200510200394.6 discloses an oxidized low-density lipoprotein colloidal gold test strip, which establishes the relationship between color and concentration according to the amount of antigen captured by oxidized low-density lipoprotein antibodies. However, there are certain limitations in the naked eye's discrimination of color shades, so it forms a limit to the sensitivity of the semi-quantitative function of this detection method, and individual differences may occur depending on the individual's different discrimination of color shades.

The purpose of the present invention is to provide an effective, rapid and accurate method for measuring low-density oxidized lipoprotein without special equipment, and can simply and directly determine whether the oxidized low-density lipoprotein in the human body is higher than the normal level through this method. At the same time, the detection cost is effectively reduced, the operation process is simplified, and the detection time is shortened.

3. Contents of the invention:

The invention establishes a method for measuring the content of oxidized low-density lipoprotein in a human body through a competition method. Through the presence or absence of the color of the detection area, it can be judged whether the oxidized low-density lipoprotein in the human body is higher than the normal level. Thus, early warning and early auxiliary diagnosis of atherosclerotic cardiovascular diseases are realized.

The invention comprises a basic supporting sheet, and sample absorbing pads, gold-labeled antibody pads, chromatography color-developing membranes and water-absorbing pads arranged in sequence on the basic supporting sheet.

The basic support sheet is a non-absorbent flexible material coated with self-adhesive on one side, such as a PVC board, which plays the role of fixing and supporting other components of the test paper.

The sample absorbent pad is non-woven fabric or glass cellulose membrane.

The gold-labeled antibody pad is provided with a glass cellulose membrane coated with the gold-labeled antibody. The above gold-labeled antibody is a certain amount of mouse monoclonal or polyclonal antibody against oxidized low-density lipoprotein.

Chromogenic membrane is nitrocellulose membrane or cellulose acetate membrane. A detection zone and a quality control zone are sequentially set up on the color developing area. The detection zone is coated with oxidized low-density lipoprotein as an antigen. The control strip is coated with a second animal anti-mouse IgG.

The absorbent pad is filter paper or glass cellulose membrane.

The principle of the invention is that a certain amount of gold-labeled antibody is coated on the glass cellulose membrane, and the oxidized low-density lipoprotein coated on the detection belt forms a competitive relationship with the oxidized low-density lipoprotein in the sample. The oxidized low-density lipoprotein in the sample to be tested combines with the gold-labeled antibody to form a gold-labeled antigen-antibody conjugate. The gold-labeled antigen-antibody and the gold-labeled antibody unbound to the antigen are chromatographed along the chromatographic membrane to the detection zone due to capillary action. If the detection band does not develop color, it proves that the oxidized low-density lipoprotein in the sample exceeds the limit value, that is, all the gold-labeled antibodies bind to the oxidized low-density lipoprotein in the sample and cannot bind to the oxidized low-density lipoprotein on the detection band. If the detection band develops color, it proves that the concentration of oxidized low-density lipoprotein in the sample has not reached the limit value, and the gold-labeled antibody to oxidized low-density lipoprotein in the unbound sample binds to the oxidized low-density lipoprotein on the detection band to develop color.

The present invention has the following advantages:

1. High detection accuracy and strong specificity.

2. Fast detection: It only takes 5 minutes to observe the result.

3. Easy to carry and easy to operate: the detection does not require any special instruments or equipment, and the operation steps are simple, which is convenient for medical staff to carry, and can also be used for self-detection and monitoring of perennial patients, so as to achieve early warning.

4. The production process of the oxidized low-density lipoprotein test paper is simple and the cost is low.

5. Colloidal gold test paper can be stored at room temperature without special instruments and equipment. The storage period is up to one year, and the repeatability is good.

4. Description of drawings:

Figure 1 is a front schematic view and a side schematic view of the structure of the present invention. In Figure 1, 1 is the absorbent pad, 2 is the quality control belt, 3 is the detection belt, 4 is the chromatography film, 5 is the gold label antibody pad, 6 is the sample absorbent pad, and 7 is the basic support sheet, such as PVC board, etc. .

Figure 2 is an explanatory diagram of the results of use of the present invention. In Figure 2, the quality control band is colored but the detection band is not, which is a positive result, indicating that the oxidized low-density lipoprotein content in the sample is higher than or equal to 600 μg/L, and the patient is sick; b. Both the quality control band and the detection band are colored , which is a negative result, indicating that the content of oxidized low-density lipoprotein in the sample is lower than the normal value μg/L, which is normal; neither the quality control band nor the detection band develops color, indicating that the test strip is invalid.

Five. Specific implementation plan:

Embodiment one

Preparation of Monoclonal Antibody to Oxidized Low Density Lipoprotein

Mice were immunized with oxLDL (purchased from Beijing Xiehe Sanyou Technology Development Company). Splenocytes from immunized mice were hybridized with mouse myeloma cells to prepare hybridoma cells. The hybridoma cells with high positivity were selected for cloning to prepare monoclonal hybridoma cells. Continue to culture for 20 generations, screen and finally establish a hybridoma cell line that can stably secrete monoclonal antibody.

Ascites is prepared by inoculating hybridoma cells into the peritoneal cavity of mice, and then monoclonal antibodies are prepared by extracting antibodies from the ascites.

Embodiment two

Colloidal gold preparation and method for labeling monoclonal antibody

Colloidal gold particles were prepared by trisodium citrate reduction method. Take 100ml of 0.01% HAuCl ₄ aqueous solution, heat to boil. Quickly add 2ml of 1% trisodium citrate in water as needed, continue to boil for about 5min, wine red appears. Leave it to cool, and thus make colloidal gold particles with a diameter of about 20 nm.

The colloidal gold optimal binding protein amount was determined by coagulation colorimetry. Adjust the pH of the colloidal gold to 9.0 with 0.1Mol/L K ₂ CO ₃ , and divide it into ten tubes, 1ml in each tube. After gradiently diluting the protein concentration, add it to the above ten tubes respectively, and add 1ml to each tube. After 5 minutes, add 1ml of 10% NaCl solution to each of the above tubes, mix well and let stand for 2 hours to measure the OD value. According to the OD value, the amount of antibody needed to stabilize 1ml of colloidal gold can be observed.

Antibodies are labeled with colloidal gold particles. Colloidal gold and a certain concentration of antibody solutions were adjusted to pH 9.0 with 0.1Mol/L K ₂ CO ₃ . The antibody solution was stirred electromagnetically, and the colloidal gold solution was added dropwise. Continue to stir for 10 min, add 5% stabilizer bovine serum albumin (BSA) to prevent precipitation of antibody protein and colloidal gold polymerization, and stir for another 10 min.

Colloidal gold-labeled antibodies were purified by ultracentrifugation. Centrifuge at 1200r/min for 20min to remove large polymers (this step can be omitted according to the situation). Centrifuge at 13000 g at 4°C for 40 min, carefully suck out the supernatant, resuspend the precipitate in PBS solution containing 1% BSA (containing 0.02% NaN ₃ ), and centrifuge repeatedly 3 times. Resuspend the pellet to 1/10 of its original volume and store at 4°C. If 50% glycerol is added to the conjugate, it can be stored at 20°C for more than one year.

Embodiment three

Preparation and assembly of colloidal gold test strips for detecting oxidized low-density lipoprotein

Sample absorbent pad: Cut the glass fiber into a size of 1.9cm×30cm for use.

Gold pad: Soak glass fibers cut into 0.5mm×30cm in a certain concentration of gold-labeled antibody solution for 2 hours, dry at 37°C for 1 hour, seal, and store at 4°C for later use.

Chromatographic membrane: use nitrocellulose membrane or cellulose acetate membrane, cut into 2.5cm×30cm size. A certain concentration of oxidized low-density lipoprotein is coated at 1 cm from the lower end of the membrane as a detection band with a bandwidth of 2 mm, and 1 mg/ml animal anti-mouse (mostly goat anti-mouse) IgG is coated at an upward interval of 0.5 cm as a quality control band. After natural drying in the shade, it was blocked with PBS containing 1% BSA, 0.01mol/L, pH 7.4 for 2 hours, dried in the shade at room temperature, and sealed for later use.

Absorbent pad: Cut the glass fiber into 4cm×30cm size for later use.

The absorbent pad, chromatographic membrane, gold pad and sample absorbent pad are stacked on the PVC board from top to bottom in order, and the overlap is about 2mm wide. And starting from the 2mm lower end of the chromatographic membrane, including the gold pad and some absorbent pads, use adhesive tape to fix the connection between each other, and paste the MAX line label. Then stick a color label on the surface of the absorbent pad. Finally, seal the test strips cut into 4mm and store them in a desiccant.

Embodiment four

Detailed rules for the use of colloidal gold test strips

Sampling: Draw blood from the forearm on an empty stomach between 7 and 10 in the morning, and immediately add antioxidants to prevent low-density lipoproteins from being oxidized in vitro. Plasma was collected after centrifugation.

Detection: Before testing, take out the test paper and package, leave it at room temperature for about 5 minutes, and take out the test paper. Immerse the sample absorbent pad in the sample solution, and the liquid level should not exceed the MAX line label, or place the test strip flat on a clean workbench, and drop the sample solution onto the sample absorbent pad. The results were observed visually after 10 minutes.

Observation: If the detection band does not develop color and the quality control band develops color, it proves that the oxidized low-density lipoprotein in the sample exceeds the limit value, which is positive. If the detection band develops color and the quality control band develops color, it proves that the concentration of oxidized low-density lipoprotein in the sample does not reach the limit value, which is negative. If neither the test strip nor the quality control strip develops color, it proves that the test strip is effective.

Claims (4)

1. A semi-quantitative detection trace oxidized low-density lipoprotein colloidal gold test strip, the test strip comprises a basic support sheet, and a water-absorbent pad arranged in sequence on the basic support sheet, a chromatographic membrane, a gold pad and a sample absorption pad. It is characterized in that the gold-labeled antibody that can only bind to a concentration of about 600 μg/L is fixed on the gold pad, and the oxidized low-density lipoprotein immobilized on the chromatographic membrane competes with the oxidized low-density lipoprotein in the sample to be tested by using the indirect competition method color. 2. a kind of semi-quantitative detection trace oxidized low-density lipoprotein colloidal gold test strip according to claim 1 is characterized in that a detection zone and a quality control zone are set up successively on the color developing area. The detection zone is coated with oxidized low-density lipoprotein as an antigen. The control strip is coated with a second animal anti-mouse IgG. The gold-labeled antibody is a certain amount of mouse monoclonal or polyclonal antibody against oxidized low-density lipoprotein. 3. a kind of semi-quantitative detection trace oxidized low-density lipoprotein colloidal gold test strip according to claim 1, it is characterized in that basic support sheet is the non-absorbent tenacity material that one side is coated with self-adhesive, as PVC plate. The absorbent pad is filter paper or glass cellulose membrane. Chromatographic membrane is nitrocellulose membrane or cellulose acetate membrane. Gold pads are glass cellulose membranes coated with gold-labeled antibodies. The sample absorbent pad is non-woven fabric or glass cellulose membrane. 4. a kind of semi-quantitative detection trace oxidized low-density lipoprotein colloidal gold test strip according to claim 1 is characterized in that this test strip can semi-quantitatively detect human serum or blood plasma oxidized low-density lipoprotein content, thereby application Early auxiliary clinical diagnosis of atherosclerotic cardiovascular disease.

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