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Protein chip for simultaneously detecting four bladder cancer markers in urine

A protein chip, bladder cancer technology, applied in measurement devices, biological tests, material inspection products, etc., can solve the problems of a large proportion of false negatives, unsuitable for rapid detection, specificity and sensitivity need to be improved, and achieve a simple preparation process. , The effect of chip detection method is fast and cheap

Inactive Publication Date: 2018-08-17
SHENZHEN LUOHU PEOPLELS HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PCR and ELISA methods require pretreatment of urine to extract nucleic acids and proteins, which are not suitable for rapid detection. Although immunochromatography is fast, it has a large proportion of false negatives and can only be qualitative or semi-quantitative. Its specificity and sensitivity are yet to be determined. promote

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  • Protein chip for simultaneously detecting four bladder cancer markers in urine
  • Protein chip for simultaneously detecting four bladder cancer markers in urine
  • Protein chip for simultaneously detecting four bladder cancer markers in urine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Preparation of colloidal gold-coated microspheres and embedding of quantum dots:

[0041] 1. Preparation of colloidal gold

[0042] The colloidal gold particles prepared according to the following method in the present invention have a particle diameter of about 20 nm, and a gold concentration of about 0.1 mg / mL.

[0043] The specific preparation method is: first chloroauric acid (HAUC 14 ) into a 0.01% aqueous solution, take 100mL to 250mL beaker, pickle the beaker, rinse it with distilled water and siliconize it before use. After heating to boiling, add sodium citrate and observe the color change of the chloroauric acid aqueous solution under the boiling state, it can be seen that the chloroauric acid aqueous solution changes from light yellow to gray, then becomes black, then gradually transitions to red and remains stable. Change, the whole process is about 3 minutes. After the red aqueous solution is cooled to room temperature, restore to the origin...

Embodiment 2

[0049] Example 2: Preparation of monoclonal antibodies and coupling to colloidal gold-coated primary microspheres embedded in quantum dots:

[0050] 1. Cell fusion and preparation of hybridoma cells

[0051] Female Balb / c mice aged 6-8 weeks were selected, and recombinant proteins of NMP22, BTA, CD20, and TERT were injected intraperitoneally as antigens, so that the antigens could enter the peripheral immune organs through blood circulation or lymphatic circulation, and stimulate corresponding B lymphocyte clones. They activate, proliferate, and differentiate into primed B lymphocytes. After three days, adopt CO 2 The mice were killed by gas, and the spleen was removed under aseptic operation to prepare a spleen cell suspension. Take mouse myeloma cells and splenocytes in the logarithmic growth phase at a ratio of 5:1, mix them in a sterile and non-toxic environment in a water bath at 40°C, add 50% polyethylene glycol (PEG) as a fusion-promoting agent, and make Lymphocyte...

Embodiment 3

[0060] Example 3: The protein chip prepared by the present invention detects four bladder cancer markers:

[0061] 1. Acquisition of samples to be tested:

[0062] The protein chip prepared by the invention is suitable for detecting trace markers in urine. The mid-morning urine was collected, immediately transferred to the laboratory, stored at low temperature for future use, and the test was completed within 12 hours.

[0063] 2. Co-incubation of the protein chip and the sample to be tested:

[0064] Take the protein chip stored in PBS buffer, and shake it sufficiently to form a microsphere suspension. Add the urine sample and the protein chip in the suspension state to the petri dish at one time, mix thoroughly, and react in a 37°C incubator in the dark for 45min. After taking out the culture dish, add an appropriate amount of microsphere suspension again, mix well, and react in a 37°C incubator in the dark for 30min.

[0065] 3. Detection of four bladder cancer marker...

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Abstract

The invention relates to a protein chip for simultaneously detecting four bladder cancer markers in urine, belonging to the technical field of testing or analysis of materials by means of measuring the chemical or physical properties of the materials. The protein chip simultaneously contains NMP22 microspheres, BTA microspheres, CD20 microspheres and TERT microspheres, wherein the NMP22 microspheres, BTA microspheres, CD20 microspheres and TERT microspheres are respectively formed by coupling colloid gold-coated microspheres embedded with quantum dots to a NMP22 monoclonal antibody, a BTA monoclonal antibody, a CD20 monoclonal antibody and a TERT monoclonal antibody respectively. The protein chip provided by the invention has the following advantages: the protein chip can realize early detection and recurrence detection of bladder cancer by simultaneously detecting NMP22, BTA, CD20 and TERT with high specificity; and the chip is fast in detection means, saves time and labor, and is suitable for extensive clinical application.

Description

technical field [0001] The invention belongs to the technical field of testing or analyzing materials by means of measuring chemical or physical properties of materials, and in particular relates to a protein chip for simultaneously detecting four bladder cancer markers in urine. Background technique [0002] Bladder cancer is a common malignant tumor of the urinary system. The incidence of bladder cancer in Chinese men is increasing year by year and tends to be younger. Although the diagnosis and treatment of bladder cancer have made great progress in recent years, the mortality rate of bladder cancer is still high. Bladder cancer currently lacks effective therapeutic targets and postoperative evaluation indicators, and about 5% to 50% of patients have postoperative recurrence or metastasis. [0003] At present, the diagnosis of bladder cancer, especially the monitoring of postoperative recurrence, is mainly based on urine exfoliation cytology and urethral cystoscopy. How...

Claims

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Application Information

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IPC IPC(8): G01N33/574G01N33/68G01N33/577G01N33/543G01N33/532
CPCG01N33/532G01N33/54313G01N33/57488G01N33/577G01N33/6875
Inventor 吴松雷崎方王书鹏
Owner SHENZHEN LUOHU PEOPLELS HOSPITAL
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