CN102375057A - Method for simultaneously detecting a plurality of serum markers by homogeneous fluorescence - Google Patents

Abstract

The invention belongs to the field of biomedicine and detection, and relates to a method for simultaneously detecting multiple serum markers with homogeneous fluorescence. In this method, after the carcinoembryonic antigen CEA and neuron-specific enolase NSE biotinylated antibodies are reacted with standard samples or samples to be tested, rabbit anti-CEA antibodies and FITC-labeled NSE-labeled antibodies are directly added to react to form antigen antibodies. After the complex is combined with streptavidin immobilized on polystyrene microspheres, two kinds of fluorescent quantum dots are added after washing, the quantum dots are dissociated after the reaction is completed, and the carcinoembryonic antigen CEA and neuron specificity are measured simultaneously. Enolase NSE concentration, this method requires a small amount of serum, multiple indicators can be detected simultaneously, the detection time is fast, the diagnosis cost is low, the operation is simple, it is suitable for promotion, and it can provide the correct reference direction for the early diagnosis and treatment of clinical tumors and other major diseases.

Description

A method for the simultaneous detection of multiple serum markers by homogeneous fluorescence

technical field

The invention belongs to the field of biomedicine and detection, relates to a method for detecting serum markers, in particular to a method for simultaneously detecting multiple serum markers with homogeneous fluorescence. This method can provide a variety of serum markers for the early diagnosis of clinical tumors and other major diseases.

Background technique

According to the incidence of cancer in my country and the ranking of the incidence of top ten malignant tumors provided by the Cancer Prevention and Control Office of the Ministry of Health, there are about 2.2 million new cancer cases in China every year, and 1.6 million deaths due to cancer. In the past 20 years, one out of every 4-5 deaths in China died of cancer, ranking first among the causes of death. Among them, lung cancer and breast cancer rank first in the incidence of malignant tumors in men and women, and lung cancer has the highest mortality rate of malignant tumors in men and women. It is predicted that by 2020, there will be 5.5 million new cancer cases in China, of which the death toll will reach 4 million. According to the latest cancer monitoring data from the Shanghai Center for Disease Control and Prevention, there were 46,000 new cancer cases in Shanghai in 2009, of which 54% were male and 46% were female; cancer is the second cause of death after cardiovascular and cerebrovascular diseases In Shanghai, about 30,000 people die of cancer every year, of which 60% are males and 40% are females. More than 2 people per 1,000 Shanghainese die from cancer; currently there are about 180,000 surviving cancer patients in Shanghai , more than 1 in every 100 people is a cancer patient. The survey shows that the related cancer incidence, death and the number of existing patients are still on the rise.

At present, the clinical diagnosis methods for major diseases such as lung cancer mainly include: Chest X-ray examination is the first choice, and the limit of finding pulmonary nodules is larger than 1cm in diameter, and at this time the tumor may have invaded the bronchial epithelium and vascular epithelium. Some scholars suggest that patients over 60 years old Smokers need to do low-dose spiral CT screening every year; percutaneous fine-needle aspiration biopsy is more accurate in diagnosing malignant pulmonary nodules, but this method is invasive and can cause certain complications. Such as pneumothorax and hemoptysis, etc.; sputum cytology examination uses sputum examination to find cancer cells, especially multiple sputum examinations, which is helpful for the diagnosis of central tumors originating in the large air duct, such as squamous cell carcinoma and small cell carcinoma. Peripheral tumors of the small airways, such as adenocarcinomas, especially those less than 2 cm in diameter, are only occasionally detected by sputum examination, but they are of great significance. The biggest advantage of sputum cytology is that it is non-invasive. Fiberoptic bronchoscopy is the most commonly used diagnostic tool for obtaining histological evidence of lung cancer, but it has limitations in diagnosing early-stage lung cancer because these lesions are difficult to detect with the naked eye. Fluorescence endoscopy can significantly increase the detection rate of precancerous lesions and carcinoma in situ, and can play an important role in the screening and follow-up of lung cancer high-risk groups, but the inspection is expensive. Rapid development, low cost, high sensitivity, and good specificity non-invasive early diagnosis technology, especially serological diagnostic technology, has become a hot research field in clinical practice in recent years.

The importance of joint detection of multiple serum tumor markers has gradually been recognized by clinicians. In the diagnosis of lung cancer, serum tumor markers mainly include carcinoembryonic antigen (CEA), neuronal enolase (NSE), p53 antibody, cytokeratin fragment 19 (CYFRA21-1), vascular endothelial growth factor, etc. CEA is an acidic glycoprotein with human embryonic antigenicity. It is a good tumor marker for judging the efficacy of lung cancer, detecting the development of the disease and evaluating the prognosis. A large number of experimental results show that 2/3 of non-small cell lung cancer (NSCLC ) and 1/3 of small cell lung cancer (SCLC) patients, the CEA level was elevated, the positive rate of CEA in lung cancer was 62.85%, and the positive rate of CEA in lung adenocarcinoma was 81.4%. NSE is a sugar-cleaving enzyme that is elevated in tumors of neuroendoderm and neuroendocrine tissue origin. SCLC is most often a neurosecretory tumor, so NSE is a sensitive and specific tumor marker for SCLC.

However, although CEA and NSE in serum have been used in the diagnosis of major diseases such as lung cancer, a large number of clinical data show that a single tumor marker has the disadvantage of low specificity, especially for early tumors with a low detection rate. In clinical practice, it is often necessary to use separate detection and joint analysis of multiple markers to improve the sensitivity of tumor detection. However, each marker requires a radiation or enzyme-linked immunosorbent kit, which directly leads to the ubiquity of these multi-index detection methods, such as the large amount of serum required, long detection time, high diagnostic costs, and complicated operations. , Financial resources are unrealistic, thus limiting their clinical application. Biochip is a high-tech developed rapidly in the field of life sciences in recent years. It has the characteristics of high throughput, miniaturization and automation. distance.

Contents of the invention

The purpose of the present invention is to overcome the defects of the prior art, such as a single detection marker, a long experiment period, a large amount of serum, high diagnostic costs, etc., to provide a method for simultaneously detecting multiple serum markers, especially a uniform A method for the simultaneous detection of multiple serum markers by phase fluorescence. This method can provide a variety of serum markers for the early diagnosis of clinical tumors and other major diseases.

For realizing the purpose of the present invention, the present invention adopts following technical scheme:

Provides a method for simultaneous detection of multiple serum markers, especially a method for simultaneous detection of multiple serum markers with homogeneous fluorescence. This method reacts with standard samples or samples to be tested by CEA and NSE biotinylated coated antibodies , then directly add rabbit anti-CEA antibody and FITC-labeled NSE-labeled antibody, react to form an antigen-antibody complex and combine with streptavidin immobilized on polystyrene microspheres, add 525nm quantum dots and 655nm quantum dots after washing, The concentration of CEA and NSE was obtained by measuring the dissociated quantum dots after the reaction was finished and washed.

The method of the invention can simultaneously identify and detect two cancer indicators: carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE).

Specifically, the method of the present invention is a method for simultaneous detection of multiple serum markers by homogeneous fluorescence, which is characterized in that it comprises the steps of:

1) preparing a streptavidin-polystyrene microsphere complex;

2) Prepare FITC-labeled NSE-labeled antibody;

3) Prepare biotinylated CEA and NSE labeled antibodies;

4) Fluorescence detection.

In the method of the present invention, EDC reaction is used to combine streptavidin on polystyrene microspheres, and the streptavidin-polystyrene microspheres formed by it are used as enrichment and separation carriers for two kinds of tumor markers object detection.

In the method of the present invention, biotinylated coated antibodies of CEA and NSE tumor markers and FITC-labeled NSE-labeled antibodies are prepared respectively.

In the method of the present invention, at first mix the biotinylated coating antibody of CEA and NSE tumor marker, CEA, NSE in the standard solution or serum sample, the rabbit anti-CEA labeled antibody and the NSE labeled antibody of FITC label prepared respectively; Add Enrichment and separation of carrier streptavidin-polystyrene microspheres; then adding anti-rabbit 525nm quantum dots and anti-FITC 655nm quantum dots, after washing and dissociation, measure the fluorescence intensity of the supernatant at 525nm and 655nm excited by 355nm.

In the method of the present invention, after mixing the two biotinylated coated antibodies, two tumor marker standard solutions or serum samples containing CEA and NSE were added, and reacted at 37° C. for 1 h; Combine rabbit anti-CEA-labeled antibody and FITC-labeled NSE-labeled antibody, and react at 37°C for 1 h; Reaction for 1 h; after this step, anti-rabbit 525nm quantum dots and anti-FITC 655nm quantum dots were added after washing and reacted at 37°C for 1h.

In the method of the present invention, the quantum dots are dissociated using a 0.1M PBS solution containing 8M urea and 10% SDS.

In the method of the present invention, two kinds of fluorescent quantum dots are used to simultaneously measure two kinds of tumor markers CEA and NSE.

The method of the present invention is not limited to the detection of serum markers CEA and NSE, and can also detect other serum markers or more than two serum markers.

In the method of the present invention, the determination of tumor markers is not limited to using two kinds of fluorescent quantum dots, and other nanoparticles with similar fluorescent properties can also be used to determine tumor markers.

The measuring steps of the method of the present invention are measured on the same instrument.

The beneficial effect of the present invention is that, the advantage of the inventive method has:

1. Serum carcinoembryonic antigen (CEA), neuron-specific enolase (NSE) and cytokeratin fragment 19 (CYFRA21-1) were tested for primary lung cancer, benign lung disease and healthy people. Its significance in the early diagnosis, pathological classification and curative effect judgment of lung cancer;

2. This method can simultaneously identify and detect two cancer indicators: carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE);

3. This method requires a small amount of serum, simultaneous detection of multiple indicators, fast detection time, low diagnostic cost, simple operation, and is suitable for promotion. It provides the correct reference direction for major breakthroughs in the early diagnosis technology of lung cancer and other major diseases.

For ease of understanding, the method of the present invention will be described in detail below through specific drawings and embodiments. It should be pointed out that the specific examples and accompanying drawings are only for illustration. Obviously, those skilled in the art can make various amendments and changes within the scope of the present invention according to the description herein. These amendments and Modifications are also included within the scope of the present invention.

Description of drawings

Fig. 1 is a schematic diagram of the method of the present invention.

Detailed ways

Example 1

1) Preparation of streptavidin-polystyrene microsphere complex

The typical steps are as follows: First, transfer 500 μL of carboxypolystyrene microspheres into a 1.5 mL centrifuge tube, centrifuge at 12,000 rpm with pH=6.0 imidazole solution (0.1M) and wash three times, then add 100 μL of imidazole solution containing 30 mg of EDC to activate at 37°C 30min; then centrifuge and wash the activated polystyrene microspheres with imidazole solution three times, and finally disperse in 500μL imidazole solution; then add 200μL (0.2mg) of streptavidin and react at 37°C for 2h; wash with PBSTW washing solution three times Afterwards, 500 μL of PBS solution containing 2% BSA was added, and blocked at 37°C for 1 h; after the streptavidin-polystyrene microsphere complex was washed, it was stored in 500 μL of PBS solution containing 2% BSA until use.

2) Preparation of FITC-labeled NSE-labeled antibody

A typical procedure is as follows: Concentrate 150 μL of NSE antibody to 90 μL of 0.1 M sodium carbonate/sodium bicarbonate solution at pH 9.2, then add 10 μL of fluorescein isothiocyanate (FITC) in 0.1 M sodium carbonate/sodium bicarbonate solution at 0.1 M sodium carbonate/sodium bicarbonate solution was dialyzed for 12 hours, and then dialyzed with PBS solution for 12 hours, then the solution was concentrated to 150 μL for use, and the protein concentration was measured by ultraviolet spectrophotometry to be 2 mg/mL.

3) Preparation of biotinylated CEA and NSE labeled antibodies

The typical steps are as follows: After removing impurities, 100 μL LCEA or NSE-labeled antibody was dissolved in 100 μL PBS, and 5 μL of 0.1 M NHS-biotin (N-hydroxysuccinimide biotin) dissolved in DMF was added to react at 4 °C for 2.5 h, and then It was dialyzed overnight with PBS, concentrated to 100 μL for use, measured by UV spectrophotometry and adjusted to a protein concentration of 2 mg/mL.

4) Fluorescence detection

Typical steps are as follows: Add 0.0625 μL of CEA and NSE biotinylated coated antibodies to 25 μL of 0.1 MPBS containing 2% BSA and 0.5% Triton-X 100 in a 1.5 mL centrifuge tube, add 25 μL of standard samples or samples to be tested in React at 37°C for 1 hour, then add 25 μL containing 0.5 μL rabbit anti-CEA antibody and 0.5 μL FITC-labeled NSE-labeled antibody, and react at 37°C for 1 hour; take 10 μL streptavidin-polystyrene microsphere complex and wash once, Dilute to 50 μL with 2% BSA, 0.5% Triton-X 100 in 0.1 MPBS, then add 50 μL of the above antibody-antigen-labeled antibody solution, and react at 37°C for 1 hour; wash with 0.1M PBS containing 0.5% Tween 20 for 3 times, Add 100 μL of anti-rabbit 525nm quantum dots and anti-FITC 655nm quantum dots diluted 1∶500 times, and react at 37°C for 1 h; The 0.1MPBS solution of %SDS was used to dissociate the quantum dots, and the washing solution was filtered after 30 min; excited at 355nm, and the fluorescence intensity of the supernatant was measured at 525nm and 655nm to quantify the content of CEA and NSE in serum.

Claims (12)

1. A method for the simultaneous detection of multiple serum markers by homogeneous fluorescence, characterized in that, after the biotinylation coated antibody is reacted with a standard sample or a test sample by carcinoembryonic antigen CEA and neuron-specific enolase NSE , directly add rabbit anti-CEA antibody and FITC-labeled NSE-labeled antibody, react to form an antigen-antibody complex and combine with streptavidin immobilized on polystyrene microspheres, add 525nm fluorescent quantum dots and 655nm fluorescent quantum dots after washing After the reaction is finished and washed, the quantum dots are dissociated, and the concentrations of carcinoembryonic antigen CEA and neuron-specific enolase NSE are measured simultaneously, which includes the steps of: 1) preparing a streptavidin-polystyrene microsphere complex; 2) Prepare FITC-labeled NSE-labeled antibody; 3) Prepare biotinylated CEA and NSE labeled antibodies; 4) Fluorescence detection. 2. by the method for the homogeneous fluorescence of claim 1 to detect multiple serum markers simultaneously, it is characterized in that, in described step 1), utilize EDC reaction to bind streptavidin on polystyrene microspheres , which formed streptavidin-polystyrene microspheres as enrichment and separation carriers. 3. The method according to claim 1, characterized in that, in the method, biotinylated coated antibodies of CEA and NSE tumor markers and FITC-labeled NSE-labeled antibodies are prepared respectively. 4. by the described method of claim 1, it is characterized in that, in described method, after the biotinylated coating antibody of CEA, NSE tumor marker is mixed, add two kinds of tumor markers containing CEA, NSE Standard solutions or serum samples were reacted at 37°C for 1h. 5. The method according to claim 4, characterized in that, after the step is completed, the rabbit anti-CEA-labeled antibody and the FITC-labeled NSE-labeled antibody are directly combined without washing, and reacted at 37° C. for 1 h. 6 . The method according to claim 5 , characterized in that, after the step is finished, directly add to the prepared streptavidin-polystyrene microspheres without washing, and react at 37° C. for 1 h. 7. The method according to claim 6, characterized in that, after the step is finished, anti-rabbit 525nm quantum dots and anti-FITC 655nm quantum dots are added after washing, and reacted at 37°C for 1h. 8. by the described method of claim 1 or 7, it is characterized in that, with the 0.1M PBS solution containing 8M urea, 10%SDS dissociation quantum dot. 9. The method according to claim 1, characterized in that two kinds of tumor markers CEA and NSE are measured simultaneously with two kinds of fluorescent quantum dots. 10. The method according to claim 1, wherein said serum markers also include other serum markers or two or more serum markers. 11. The method of claim 1, wherein the fluorescent light further comprises other nanoparticles having similar fluorescent properties. 12. The method according to claim 1, characterized in that, the same measuring instrument is used in the measuring step.

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