WO2006047928A1

WO2006047928A1 - Preparation and application of the type of cell detecting immunoassaying chips for cancer and their use

Info

Publication number: WO2006047928A1
Application number: PCT/CN2005/001693
Authority: WO
Inventors: Zuming Tang; Zuhong Lu; Yuzhi Yang; Chunxiu Zhang; Yinglei Yu
Original assignee: Southeast University
Priority date: 2004-11-03
Filing date: 2005-10-17
Publication date: 2006-05-11
Also published as: CN1619310A

Abstract

A preparation of the type of cell detecting immunoassaying chip for cancer, comprising: modifies the carrier with aldehyde groups, amino groups; fixes the corresponding antibody on the modified carrier; then leaves the modified carrier in moist condition for a period of time; washes it with buffer after taking out. The detecting steps employing such chip comprising: blood from which red cells is removed is prepared into suspending liquid; then drops them onto such chip; and observes them with microscope or CCD or stains the cells and observes the binding situation of each site.

Description

Preparation and detection method of tumor immune cell detection chip technology

The invention relates to a method for preparing and detecting a nucleated cell immune chip in a peripheral blood of a human tumor patient, and belongs to the technical field of manufacture and detection of a biochip. Background technique

Cancer is one of the common diseases that endanger human health. Since the pathogenesis has not been thoroughly explored, there are no specific treatments, especially in the advanced stage. The prognosis of cancer patients is often related to the discovery of cancer sooner or later. Early detection of the use of immunotherapy or surgery can cure. If it is in the middle and late, it is very unfavorable for the prognosis of cancer patients. This shows the importance of early diagnosis of cancer patients. If cancer can be detected early, early diagnosis, most patients get radical cure. However, most of the patients seen in the clinic are not early cancers. Because there are often no special symptoms in the early stages of cancer. Therefore, patients generally do not take the initiative to go to the hospital for examination, and once they feel obvious, the condition is often in the middle and late stages. Therefore, many cancer patients, once found in the middle or late stage, have brought great difficulties to the treatment of patients. If there is a way to diagnose in the early stages of cancer, it will bring hope to patient treatment. It is not an illusion to cure cancer patients in this way.

There are many methods for the diagnosis of tumors. For example, biochemical, immunological and imaging methods have made great progress in recent years, but the pathological method is still the most reliable. Of course, there are some problems in pathological diagnosis. First, pathological diagnosis requires tumor tissue, so it is generally not possible to obtain tumors from early patients. Because it is a biopsy specimen, the specific material and the sliced part are all sampled. Under the light microscope, only a small part of the lesion is seen, and sometimes it cannot fully represent the whole lesion. In addition, whether the pathological diagnosis is reliable or not is also related to the selection of pathological specimens. Sometimes there are false negative results. The use of tumor-specific antigens and tumor-associated antibody diagnosis is also a more effective early diagnosis method for the diagnosis of tumors. However, some tumors cannot be diagnosed at an early stage because of their limited expression. A more effective early diagnosis method for tumor diagnosis using tumor marker tumor-specific antigen and tumor-associated antigen. Specific for tumor type (direct tumor); high sensitivity, can be detected at low tumor load; and the concentration of markers in blood or other body fluids is directly related to tumor cell load, tumor markers appear in this type of tumor All patients. Most tumors can release antigenic macromolecular substances into the blood circulation and can be detected by immunological methods.

However, it is still lack of specificity or sensitivity for early diagnosis of tumors or screening of large numbers of tumors. Tumor markers.

Cancer is caused by genetic, viral infection or some other physicochemical factors. First, it causes normal cells to undergo gene mutation or activation of normal resting genes to express new protein molecules. These proteins can act on cell membranes, and the body has nuclear cell membranes. Corresponding changes will also occur, especially in the blood, the life cycle of nucleated cells is short, the cell membrane is affected, and it is easily recognized by some immune cells in the body. After the new protein is released into the body fluid, these proteins may be some immune cells of the body. Identification, specific antigen presenting cells recognize and present to lymphocytes to produce antibodies. It can be seen that the nucleated cell membrane in the blood changes early in the early stage of cancer. How to find and grasp this change in time is a key to the early diagnosis of tumor. We applied antibodies that are currently identified to be associated with tumor genes and expressed proteins to detect differences between patients and normal organisms. After clinical trials, it was found that tumors can be detected earlier and can be diagnosed before the formation of solid tumors. It provides a reliable diagnostic method for patients with cancer to be detected early and treated promptly. At present, no detection of nucleated cells in peripheral blood has been used to judge and detect tumors earlier. Invented content

technical problem

It is an object of the present invention to provide a technique for preparing a tumor immunocyte chip and a method for performing tumor detection using the immune cell chip. The method has the advantages of high accuracy and convenient use, and has great significance for early diagnosis of tumors and timely treatment of tumor patients.

Technical solutions

The preparation method of the tumor immune cell detecting chip of the invention is:

First, chemical modification is carried out on the carrier, that is, the amino group and the aldehyde group are modified to ensure that the chemical group on the carrier can react with the antibody to bind to the carrier, and can maintain the immunological activity of the antibody bound to the carrier; The above-mentioned antibodies on the slides modified with chemical groups, such as: lung cancer, liver cancer, gastric cancer and other tumors, and new protein antibodies expressed by gene mutations such as skeletal tumor leukemia, are placed at 0 ° C—

Store at 55 °C in a humid environment. Remove the slide after 0.1–72 hours, using pH 4.0_ 10, ion concentration 0.00

-0.75 m/L buffer cleaning.

The chemical modification on the carrier, that is, the modification of the amino group, the aldehyde group is as follows:

a. Slide cleaning: Take the slide glass and wash it with water, soak it in a solution consisting of hydrogen peroxide and concentrated sulfuric acid, then rinse it with deionized water or distilled water, boil it, dry it under nitrogen flow, and keep it for use; b. Aminosiliconization on the surface of the slide: The cleaned slide is immersed in a solution containing 0.1-10.0% aminopropyltrimethoxysilane "3-aminopropyltriethoxysilane" in 95% acetone/water for 3-30 minutes. After taking out, the slides are washed successively with acetone and deionized distilled water, and finally dried and stored;

c. Aldehyde modification on the surface of the slide: Place the siliconized wafer into a PB solution (phosphate buffer) containing 1%-10% glutaraldehyde, soak at room temperature, and use PB after removal. The solution is washed, then rinsed with deionized distilled water, dried with nitrogen, and stored for later use;

d. Poly-lysine modification on the surface of the slide: The cleaned slide is placed in a PBS solution (phosphate sodium chloride buffer) containing 2% to 5% polylysine, and the PB is used first. Wash, rinse with deionized distilled water, blow dry, and store:

e, preparation of agarose film on the surface of the slide: according to the ratio of 0.5-3.0g agarose to 100ml of double distilled water to form agarose solution, completely mixed and boil; cover the agarose solution in preheated aminosilicon垸 modified glass slide; after the agarose solidifies, the slide is dried and stored;

f, calf serum albumin-N-hydroxysuccinimide (BSA-NHS) modification on the surface of the slide: 1.76 g of N-N'-disuccinimidyl carbonate, with 1 to 5 ml of NN, -2 The ratio of isopropylethylamine was dissolved in 65-69 ml of anhydrous dimethylformamide "DMF", and the slide was placed in the solution; rinsed with ethanol; the slide was immersed in 0.2-5 % calf serum albumin in PBS solution at room temperature; rinse with deionized water and then with ethanol; remove residual solvent, then immerse in anhydrous dimethylformamide "DMF" solution, leave at room temperature, dry and preserve :

g. Preparation of antibodies using related oncogenes and their expressed proteins, and immobilizing these antibodies on a vector. The detection method is: adding the white blood cells or lymphocyte separation liquid to the peripheral blood containing the anticoagulant, removing the red blood cells to obtain the nucleated cells in the blood, and suspending the nucleated cells in the PBS or other isotonic solution with the human body, Then, the suspended cells in the solution are added to the tumor immune cell detection chip, placed in an environment of 2 ° C - 55 ° C for 0.1 _ 72 h; the carrier is taken out, with a pH of 4.0-12, the ion concentration of 0.001-1.100 M solution will not The free nucleated cells bound by the antibody on the carrier are washed away; then, the cells are stained on a microscope or CCD or the cells are stained to observe the binding of the cells at each point, that is, the detection is performed.

The immune cell chip of the present invention is characterized in that a chemical group is modified on a carrier, and a tumor-associated marker antibody is modified on the carrier, and a specific protein on the surface of the nuclear cell membrane in the blood of the cancer is combined with an antibody on the chip, according to The presence or absence of cells at each point can be used to determine the presence or absence of tumors and tumors.

The carrier of the present invention may be a high molecular polymer, or may be a slide glass or the like, and may be repaired on the carrier. Decorated with amino and aldehyde groups.

Advantageous Effects; Since the method of the present invention is in vitro detection, it does not have any other harm to the human body except for collecting peripheral blood, and can be detected before the solid tumor is formed, and the accuracy is high. Easy to use, it is of great significance for the early diagnosis of tumors and for the timely treatment of cancer patients. detailed description

The preparation method of the tumor immune cell detecting chip of the invention is as follows: First, chemical modification is performed on the carrier, that is, the amino group and the aldehyde group are modified to ensure that the chemical gene on the carrier can react with the antibody to bind to the carrier, and can maintain The immunological activity of the antibody bound to the carrier; and then the corresponding antibody is spotted on the slide with the chemical gene modified above, such as: lung cancer, liver cancer, gastric cancer and the like, and new protein antibodies expressed by gene mutations such as skeletal tumor leukemia Store in O'C—55Ό in a humid environment for 0.1-72 hours, remove the slide, and wash with a buffer of pH 4.0-10, ion concentration 0.00-0.75m/L. The specific steps are as follows:

1. Chip production:

1) Slide cleaning

The slides were washed with water, placed in a solution consisting of 1/3 hydrogen peroxide and 2/3 concentrated sulfuric acid, and immersed for 0.5-3.0 hours. Then rinse with ionic or distilled water for 2-4 times, then boil with deionized water or distilled water for 2-30 minutes; dry under nitrogen flow and store in a dry place.

(1) Aminosiliconization on the surface of the slide

The cleaned slide is immersed in a solution containing 0.1-10.0% of aminopropyltriethoxysilane in 95% acetone/water for 30-30 minutes. After taking out, the slide is washed with acetone for 2-4 times. Each time for 1 to 5 minutes, rinse with deionized distilled water for 1 to 5 times, each time for 1 to 5 minutes, and finally dry at 60-180 ° C, and store in a dry place.

(2) Aldehyde modification on the surface of the slide

The siliconized wafer is placed in a PB solution (0.001 - 0.150M PH4.5 - 11.0) containing 1% - 10% glutaraldehyde, soaked at room temperature for 0.1-10 hours, and then taken out with PB ( 0.001 - 0.150 M pH 4.5 - 11.0) Wash 2-6 times, then rinse 3 times with deionized distilled water, blow dry with nitrogen, and store at 4 ° C for later use. (3) Poly-lysine modification on the surface of the slide

Place the cleaned slide into a PBS solution containing 3% polylysine (0.01 -0.14M pH405-11.0) for 0.1-10 hours, remove it and wash it with PB 2-6 times, then use deionized Rinse with distilled water for 2-4 times, blow dry, and store at 4 °C.

2) Preparation of agarose film on the surface of the slide

(1) 0.5-3.0g of agarose is added to 100ml of double distilled water to form agarose solution, completely mixed in a microwave oven and boiled for 2-10 minutes;

(2) Covering a 1 _5 ml agarose solution on a pre-warmed aminosilicone-modified glass slide at about 60 ° C;

(3) After the agarose is solidified, the slide is dried overnight at 10 to 120 °C. It can be stored under room temperature and dry conditions;

(4) Before use, the agarose membrane should be immersed in 0.1-100 mM NaI04 solution, left at room temperature for 0.05-6.00 hours, rinsed thoroughly with double distilled water, dried with nitrogen, and dried at room temperature or low temperature.

3) . Calf serum albumin-N-hydroxysuccinimide (BSA-NHS) modification on the surface of the slide

(1) 1.76 g of N-N^-disuccinimidyl carbonate, with 1 - 5 ml of N-N, -diisopropylethylamine, dissolved in 65-69 ml of anhydrous dimethylformamide (DMF). Remove the slide into the solution at room temperature for 1 to 6 hours.

(2) Rinse several times with 95% ethanol.

(3) The slide was immersed in a solution containing 0.2-5% BSA in PBS (0.01M pH 7.4) and reacted at room temperature for 05-24 hours.

(4) Rinse several times with deionized water, then rinse twice with 95% ethanol.

(5) Remove the residual solvent, immerse in DMF solution, and let stand for 0.5-6 hours at room temperature. The solvent was removed and the gas was dried and stored at 4 °C.

On the above-described vector modified with a chemical gene, the corresponding antibody (a new protein antibody expressed by a gene mutation such as a lung cancer, a liver cancer, a gastric cancer, or a skeletal tumor leukemia) is manually or by a spotting instrument. Place in a humidified environment (put the carrier in a humid box) for 0.1-72 hours, remove the carrier, and wash it for 1 to 5 times with a buffer solution of pH 4.0-10, ion concentration 0.00-0.75 m/L. . To remove unbound antibody on the carrier. The prepared chip is dried and stored in the dark.

2, detection method:

Human peripheral blood and leukocyte separation fluid or lymphocytes containing anticoagulant at a ratio of 1 to 1 by volume Separate the solution, centrifuge 500-3000 rpm to remove red blood cells in the blood, then suspend the nucleated cells in the buffer, and centrifuge the cells at 500-4000 rpm to remove the leukocyte separation solution or lymphocyte separation solution from the supernatant. The precipitated nucleated cells were suspended in a buffer to prepare a nucleated cell suspension.

Take out the prepared chip, add the appropriate amount of the separated nucleated cell suspension, and place it at 2Ό—60°C for 0.1-72 hours. The cell suspension should not be dry. Always keep the cells in the liquid and remove them. Rinse with buffer to remove unbound free cells. The cells are then observed or stained on a microscope or CCD to observe the binding of the cells to each point to determine if the patient has a tumor and what kind of tumor.

Claims

Claim

A method for preparing a tumor immune cell detecting chip, which is characterized in that first chemical modification is carried out on a carrier, that is, an amino group and an aldehyde group are modified to ensure that a chemical group on the carrier can react with the antibody to bind to the carrier. And can maintain the immunological activity of the antibody bound to the carrier; and then point the corresponding antibody on the above-mentioned slide with the chemical group modified, such as: lung cancer, liver cancer, gastric cancer and other tumors and skeletal tumor leukemia and other gene mutation expression The new protein antibody, etc., was stored in a humid environment at 0Ό-55 °C for 0.1-72 hours, and then the slide was taken out and washed with a buffer of pH 4.0_10 and an ion concentration of 0.00-0.75 m/L.

A method for preparing a tumor immune cell detecting chip according to claim 1, characterized in that the chemical modification on the carrier, that is, the modification of the amino group and the aldehyde group, is as follows:

a. Slide cleaning: Take the slide glass and wash it with water, soak it in a solution consisting of hydrogen peroxide and concentrated sulfuric acid, then rinse it with deionized water or distilled water, boil it, dry it under nitrogen flow, and keep it for use; b Aminosiliconization on the surface of the slide: The cleaned slide is immersed in a solution containing 0.1-10.0% aminopropyltrimethoxysilane "3-aminopropyltriethoxysilane" in 95% acetone/water for 3-30 minutes. After taking out, the slides are washed successively with acetone and deionized distilled water, and finally dried and stored;

c. Aldehyde modification on the surface of the slide: The siliconized wafer is placed in a phosphate buffer containing 1%-10% glutaraldehyde, soaked at room temperature, and then washed with PB solution, then Rinse with deionized distilled water, blow dry with nitrogen, and store for future use;

d. Poly-lysine modification on the surface of the slide: The cleaned slide is placed in a phosphate sodium chloride buffer containing 2% to 5% polylysine, and then washed out with PB and then used. Rinse with deionized distilled water, blow dry, and store:

f, calf serum albumin-N-hydroxysuccinimide (BSA-NHS) modification on the surface of the slide: 1.76 g of N-N'-disuccinimidyl carbonate, with 1-5 ml of Ν-Ν' -diisopropylethylamine ratio, the above two materials were dissolved in 65-69 ml of anhydrous dimethylformamide "DMF", and the slide was placed in the solution; rinsed with ethanol; the slide was immersed in 0.2 — 5% calf serum albumin in PBS at room temperature; rinse with deionized water and then with ethanol; remove residual solvent, then immerse in anhydrous Methylformamide "DMF" solution, placed at room temperature, dried and stored;

g. Preparation of antibodies using related oncogenes and their expressed proteins, and immobilizing these antibodies on a vector.

3. A method for detecting a chip prepared by the method for preparing a tumor immune cell detecting chip according to claim 1, wherein the peripheral blood containing the anticoagulant is added to the leukocyte or lymphocyte separating solution, Remove red blood cells to obtain nucleated cells in the blood, suspend the nucleated cells in PBS or other isotonic solution with human body, and then add the suspension cells in the solution to the tumor immune cell detection chip, and place them at 2 ° C - 55 0.1-72h in °C environment; remove the carrier, wash away the free nucleated cells not bound by the antibody on the carrier with pH 4.0-12, ion concentration 0.001-U00M solution; then observe or stain the cells on the microscope or CCD Observe the binding of cells at each point, that is, perform detection.