CN101294903A - Method for detecting the expression of the same antigen on the same specimen - Google Patents

Abstract

The invention discloses a method for detecting the expression of the same antigen on the same specimen. The fluorescent quantum dot-labeled streptavidin complex can be directly combined with the biotinylated secondary antibody, and the biotinylated secondary antibody can also be combined with the immunoenzyme technique In combination with horseradish peroxidase-labeled streptavidin, the two techniques of immunofluorescence and immunoenzyme can be combined perfectly, so that the expression of the same antigen can be detected on the same specimen. This method can be used to detect the expression of antigens in fresh tissues, paraffin-embedded tissues and different cell lines of humans and animals. It does not need to be protected from light, and can be completed in general immunohistochemical chambers, which is suitable for wide application.

Description

Method for detecting the expression of the same antigen on the same specimen

technical field

The invention belongs to the field of immunopathology detection, and more specifically relates to a method for detecting the expression of the same antigen on the same specimen using two techniques, and the scope of application of the method is suitable for detecting fresh tissues, paraffin-embedded tissues and different cell lines of humans and animals Antigen expression within.

Background technique

Immunohistochemistry, also known as immunocytochemistry, is the basic principle of applied immunology - antigen-antibody reaction, that is, specific antibodies labeled with chromogenic agents (fluorescein, enzymes, metal ions, isotopes) It is a technology for qualitative, localization and quantitative determination of the corresponding antigen through antigen-antibody reaction and histochemical color reaction in situ on tissue cells. It skillfully combines the specificity of the immune response and the visibility of histochemistry, and detects various antigenic substances (such as proteins, peptides, enzymes, hormones, pathogens and receptors, etc.). According to the types of markers, immunohistochemical techniques can be divided into immunofluorescence, immunoenzyme, immunoferritin, immunogold, and radioimmunoself-image techniques. Immunofluorescence and immunoenzyme techniques are mainly used for pathological diagnosis.

Immunofluorescence technique: It is the earliest established immunohistochemical technique. It utilizes the principle of antigen-antibody specific binding. Firstly, the known antibody is labeled with fluorescein, which is used as a probe to check the corresponding antigen in cells or tissues, and observed under a fluorescent microscope. When the fluorescein in the antigen-antibody complex is irradiated by the excitation light, it will emit fluorescence of a certain wavelength, so that the location of a certain antigen in the tissue can be determined, and quantitative analysis can also be performed. Due to its strong specificity, high sensitivity, rapidity and simplicity, immunofluorescence technology is widely used in clinical pathological diagnosis and testing. However, because the immunofluorescence method must have a fluorescent microscope, the fluorescence intensity gradually fades with time, and the results are not easy to store for a long time. The popularization and application are limited, and it is gradually replaced by the immunoenzyme method.

Immunoenzyme technology: It is a technology developed in the 1960s after immunofluorescence. The basic principle is to use enzyme-labeled antibodies to interact with tissues or cells, and then add enzyme substrates to generate colored insoluble products or particles with a certain electron density. Antigen components were localized. Compared with the immunofluorescence technique, the main advantages of this method are: accurate positioning, good contrast, stained specimens can be stored for a long time, and are suitable for optical and electron microscopy studies.

Immunoenzyme technique is currently the most commonly used method. The development of immunoenzyme technology is very rapid, and a variety of labeling methods have been derived. With the continuous improvement and innovation of the method, its specificity and sensitivity are constantly improving, and its use is becoming more and more convenient. Currently widely used in pathological diagnosis is the PAP method, ABC method, SP method and so on.

Fluorescent semiconductor nanoparticles accidentally discovered in recent years according to related reports are called quantum dots (quantum dots, QDs) provide a potential method to overcome the narrow excitation wavelength range of traditional fluoresceins such as organic fluorophores, light emission Short time, easy quenching and other shortcomings, began to be initially applied in immunofluorescence analysis, and achieved satisfactory results. Quantum dots are also called semiconductor nanocrystals. At present, nanoparticles composed of II-VI or III-V elements are widely used, mainly including CdY (Y is S, Se, Te), and some composite structures and multi-layer structure. Compared with traditional labeling reagents, quantum dots have the following characteristics: ① scale effect and excellent luminescence performance. Quantum dots are a multi-electron system, the luminous efficiency is much higher than that of a single molecule, the emitted light is 20 times that of organic fluorescent dyes, the stability is more than 100 times stronger, and it can withstand repeated excitations, unlike the fluorescence of organic fluorescent dyes and lanthanides Easily quenched. Quantum dots are highly resistant to photobleaching and have a long fluorescence lifetime. ②Quantum dots have good biocompatibility. The use of quantum dots as markers has no harm to the activity of biological macromolecules, and the coupling methods and methods of quantum dots and biological macromolecules are relatively simple, including coupling using chemical bonds and electrostatic forces, unlike traditional labeling reagents, Each reagent requires a specific coupling method.

At present, almost all literature reports using immunofluorescence technology and immunoenzyme technology to detect antigen expression are carried out on two specimens separately, which will make the experimental conditions inconsistent, and will also make the antigens detected by these two technologies separately The results are biased. At the same time, it consumes more manpower and material resources. However, so far, there is no report similar to ours on the same specimen using immunofluorescence and immunoenzyme techniques to detect the expression of the same antigen.

Contents of the invention

The purpose of the present invention is to provide a method for detecting the expression of the same antigen on the same specimen, which uses immunofluorescence technology and immunoenzyme technology to detect the expression of the same antigen. This method uses quantum dots to label streptavidin, which has wider applicability, so that after the immunofluorescence technology detects the antigen expression and analyzes the results, it can continue to detect the immunoenzyme technology and also display the site of the same antigen expression. It was consistent with the detection results of immunofluorescence technique.

Since traditional fluoresceins such as fluorescein isothiocyanate (FITC), rhodamine tetramethylisocyanate (TRITC) and Texas red can only label primary or secondary antibodies, when immunofluorescence detection is performed on specimens first, due to lack of Biotinylated IgG or IgM needs to be added, so that horseradish peroxidase-labeled streptavidin has no binding site in the subsequent immunoenzyme method, so immunofluorescence and immunoenzyme cannot be performed successively on the same specimen The method detects the same antigen, and the effect of detecting antigen expression on paraffin-embedded tissue is poor. Since the recent discovery of quantum dots, the research on its biological characteristics has advanced by leaps and bounds. Quantum dots have good biocompatibility. In addition to being directly coupled with primary and secondary antibodies, they can also be directly coupled with streptavidin, which makes Its application range is wider, and it also provides an opportunity for the detection of immunoenzyme. The present invention mainly utilizes the commercialized quantum dot-labeled streptavidin complex (purchased from Wuhan Jiayuan Quantum Dot Technology Development Co., Ltd.) to carry out the detection of immunofluorescence technology first, because the primary antibody added in the method is particularly Yes, the secondary antibody is biotinylated IgG or IgM, which can be combined with quantum dot-labeled streptavidin complex or with horseradish peroxidase-labeled streptavidin (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. company), so it is conducive to the perfect combination of these two technologies. In principle, these two techniques can be used regardless of who comes first, but considering the different methods of sealing the slides and storing the results, the detection of immunofluorescence first and then the detection of immunoenzyme technology has no effect on each other, so this method is adopted. kind of sequence. The specific steps are as follows:

1. First, paraffin-embedded tissue dewaxing, hydration, TBS (0.01M pH7.4TBS solution: 1.21g trishydroxyaminomethane, 7.6g sodium chloride, add distilled water to adjust to 1000ml, concentrated hydrochloric acid to adjust the pH value to 7.4 ) for 2-4 times, 3 min each time; followed by fresh tissue directly from step 3; thirdly, after the cultured cells were fixed, add TBS to wash 2-4 times, 3 min each time, and then add permeabilization solution (0.1% Triton-X100) incubate at room temperature (20-25°C, the same below) for 5-10min, and finally wash with TBS 2-4 times, 3min each time, and proceed to step 3.

2.0.01M pH6.0 citric acid buffer solution [A solution: 0.1M/L citric acid (citric acid 21.0g, add distilled water to adjust to 1000ml, concentrated hydrochloric acid to adjust the pH value to 6.0); B solution: 0.1M/L citric acid Sodium solution (sodium citrate 29.41g, add distilled water to adjust to 1000ml), when in use, take 1.9ml of A solution, 8.1ml of B solution, add 90ml of distilled water], microwave or high pressure (1.01×10 ⁵ -2.02×10 ⁵ Pa) for antigen retrieval, naturally cooled to room temperature, washed with TBS 2 to 4 times, 3 minutes each time;

3. Add 2% BSA (bovine serum albumin) blocking buffer dropwise (that is, 2g BSA dissolved in 100ml TBS solution) and incubate in a wet box at 37°C for 30-35min;

4. Add the primary antibody to be detected dropwise, incubate in a wet box at 37°C for 1-2 hours or overnight in a refrigerator at 4°C, and rinse with TBS-T (that is, add Tween 20 to the TBS solution so that the final concentration of the latter is 0.05%) for 3 minutes /time×(2～4)times;

5. Add 2% BSA blocking buffer dropwise and incubate in a wet box at 37°C for 10-15 minutes;

6. Add biotinylated goat anti-rabbit or goat anti-mouse IgG dropwise, incubate in a wet box at 37°C for 30 minutes, rinse with TBS-T for 3 minutes/time × (2-4) times;

7. Add 2% BSA blocking buffer dropwise and incubate in a wet box at 37°C for 10-15 minutes;

8. Add quantum dot-labeled streptavidin complex (working concentration: 1:50-200) diluted with 2% BSA blocking buffer dropwise, incubate in a wet box at 37°C for 30-60min, rinse with TBS-T for 3min/ times × (2 to 4) times;

9. Add 50% glycerol dropwise to mount the slide, and use a fluorescence microscope to excite the quantum dots with different wavelengths (400-550nm), and the orange-red fluorescent particles in the cells are positive;

10. Specimen autofluorescence control: only add TBS (50-200μl) or buffered glycerol to mount the specimen. If there is fluorescence in the tissue under fluorescence microscope observation, it is called autofluorescence. Positive control: use known positive specimens for direct immunofluorescence histochemical staining, and the result should be positive fluorescence.

11. Store at 2-8°C. After 2-4 weeks, the fluorescent signal is found to be weakened, and the specimen to be tested is soaked in TBS for 5-15 minutes until the cover glass falls off naturally;

12. Repeat steps 7, 8, and 9 once each to restore the fluorescent signal of the antigen.

13. After step 11, add horseradish peroxidase-labeled streptavidin (50-200μl), incubate in a wet box at 37°C for 15-20min, rinse with TBS for 3min/time x (2-4) times ;

14.DAB color development, observation results under an optical microscope. It can be observed that the localization of the antigen to be tested is basically consistent with the immunofluorescence method.

Inspired by quantum dots being able to label streptavidin to form a complex, the present invention successfully detects the same antigen by immunofluorescence method and immunoenzyme method on the same specimen for the first time. Compared with traditional methods, this invention Advantages: ① Higher sensitivity, traditional immunofluorescence markers are difficult to detect the expression of antigens on paraffin-embedded specimens, and immunofluorescence labeled with quantum dots can be used to detect antigens on paraffin specimens ;②The results are more convincing, because the immunofluorescence method and the immunoenzyme method have the same conditions for detecting antigen expression, the location of the antigen is clearer, the background is lower, and the positive rate of the same antigen expression can reach more than 95%. Figure 1-3; ③The results can be stored for a long time, and the results of immunofluorescence in the present invention can be stored for 2 to 4 weeks or even longer, which is conducive to better qualitative and quantitative analysis results; ④The experimental cost is lower, especially using Tissue chips (the price of each chip is 600 to 2000 yuan) are used for research. The traditional method requires two chips, but the present invention can be carried out on one chip, which can save experimental funds and precious specimen resources, and also save experimental costs. Time; ⑤ The experimental conditions are not harsh, and the whole experimental process does not need to be protected from light, and can be completed in general immunohistochemical laboratories, which is suitable for wide application.

Description of drawings

Figure 1 Caveolin-1 protein presents orange-red fluorescent particles in the cytoplasm, membrane and interstitium of cancer cells in lung squamous cell carcinoma tissue, indicating positive expression Fluorescence ×40

Figure 2 After the fluorescence signal weakened, the fluorescence signal of Caveolin-1 protein in lung squamous cell carcinoma tissue recovered fluorescence ×100

Figure 3 After the fluorescence signal is weakened, Caveolin-1 protein presents brownish-yellow granules in the cytoplasm, membrane and interstitium of cancer cells in lung squamous cell carcinoma tissue, indicating positive expression Puguang×40

Figure 4 Caveolin-1 protein is positively expressed in the lung cancer cell line A549, localized in the cell membrane fluorescence ×100

Detailed ways

The present invention will be described in further detail below through specific examples, but the content of the present invention is not limited to the given examples.

Example 1 detects the expression of Caveolin-1 protein on a lung cancer tissue chip, and the steps are:

1.4 μm thick lung cancer tissue chips were dewaxed, hydrated, and washed with TBS (pH7.4) for 2 to 4 times, each time for 3 minutes;

2. 0.01M pH6.0 citrate buffer solution, microwave antigen retrieval, naturally cool to room temperature (20-25°C, the same below), wash with TBS 2 to 4 times, 3 minutes each time;

3.2% BSA (bovine serum albumin) blocking buffer (that is, 2g BSA dissolved in 100ml TBS solution) and incubated in a wet box at 37°C for 30-35min;

4. Add Caveolin-1 (1:100) antibody dropwise, incubate in a wet box at 37°C for 1.5-2 hours or in a refrigerator at 4°C overnight, rinse with TBS-T (pH7.4) for 3min/time x (2-4) times;

Incubate with 5.2% BSA blocking buffer in a wet box at 37°C for 10-15 minutes;

6. Add biotinylated goat anti-rabbit IgG dropwise, incubate in a wet box at 37°C for 30-35 minutes, rinse with TBS-T for 3 minutes/time × (2-4) times;

Incubate with 7.2% BSA blocking buffer in a humid box at 37°C for 10-15 minutes;

8. Add quantum dot-labeled streptavidin complex (working concentration 1:100) diluted with 2% BSA blocking buffer dropwise, incubate in a wet box at 37°C for 30-60min, rinse with TBS-T for 3min/time ×( 2 to 4) times;

9. Add 50% glycerin to mount the slide, and use a fluorescent microscope to excite the quantum dots with blue light. Caveolin-1 protein is positive for the presence of orange-red fluorescent particles in the cytoplasm, membrane and interstitium of lung cancer cells (see Figure 1);

10. Specimen autofluorescence control: only add TBS (50-200μl) or buffered glycerol to mount the specimen. If there is fluorescence in the tissue under fluorescence microscope observation, it is called autofluorescence. Positive control: use known positive specimens for direct immunofluorescence histochemical staining, and the result should be positive fluorescence.

11. Store at 2-8°C. After 2-4 weeks, the fluorescent signal is found to be weakened, and the tissue chip is soaked in TBS for 5-15 minutes until the cover glass falls off naturally;

12. Repeat steps 7, 8, and 9 each once to restore the fluorescence signal of Caveolin-1 protein (see Figure 2).

13. After step 11, add horseradish peroxidase-labeled streptavidin (50-200 μl) and incubate in a wet box at 37°C for 15-20 minutes, rinse with TBS for 3 minutes/time × (2-4) times;

14.DAB color development, observation results under an optical microscope. It was observed that Caveolin-1 protein showed brownish yellow positive signals in the cytoplasm and membrane of lung cancer cells (see FIG. 3 ).

Example 2 detects the expression of Caveolin-1 protein on lung cancer cell line A549 cells, and the steps are:

1. After A549 cells are fixed, add TBS (pH7.4) to wash 2 to 4 times, each time for 3 minutes, then add permeabilization solution (0.1% Triton-X 100) and incubate at room temperature for 5-10 minutes, and finally wash with TBS 2 to 4 times , 3 minutes each time;

Incubate with 2.2% BSA blocking buffer in a humid box at 37°C for 30-35 minutes;

3. Add Caveolin-1 (1:100) antibody dropwise, incubate at 37°C for 1.5-2 hours or overnight in a refrigerator at 4°C, rinse with TBS-T (pH7.4) for 3min/time x (2-4) times;

Incubate with 4.2% BSA blocking buffer in a wet box at 37°C for 10-15 minutes;

5. Add biotinylated goat anti-rabbit IgG (50-200μl) dropwise, incubate in a wet box at 37°C for 40-45min, rinse with TBS-T for 3min/time x (2-4) times;

Incubate with 6.2% BSA blocking buffer in a humid box at 37°C for 10-20 minutes;

7. Add quantum dot-labeled streptavidin complex (working concentration: 1:100) diluted with 2% BSA blocking buffer dropwise, incubate in a wet box at 37°C for 45-60min, rinse with TBS-T for 3min/time × (2 to 4) times;

8. Add 50% glycerol dropwise to mount the slide, and use a fluorescent microscope to excite the quantum dots with blue light. The Caveolin-1 protein is positive for the presence of orange-red fluorescent particles in the cytoplasm and membrane of lung cancer cells (see Figure 4);

9. Specimen autofluorescence control: the specimen is only added with TBS (50-200μl) or buffered glycerol to mount the slide, and if there is fluorescence in the tissue under fluorescence microscope observation, it is called autofluorescence. Positive control: use known positive specimens for direct immunofluorescence histochemical staining, and the result should be positive fluorescence.

10. Store at 2-8°C. After 2-4 weeks, the fluorescent signal is found to be weakened, and the cell slides are soaked in TBS for 5-15 minutes until the coverslip falls off naturally;

11. Repeat steps 6, 7, and 8 once each to restore the fluorescence signal of Caveolin-1 protein.

12. After step 10, add horseradish peroxidase-labeled streptavidin (50-200 μl) and incubate in a wet box at 37°C for 15-20 minutes, rinse with TBS for 3 minutes/time × (2-4) times;

13.DAB color development, and observe the results under an optical microscope. It was observed that Caveolin-1 protein appeared brown-yellow positive signal in the cytoplasm and membrane of lung cancer cells.

Based on the above, it can be seen that the method is simple and easy to implement, has high repeatability and low cost, and can be completed in general immunohistochemical laboratories, laying the foundation for widespread application.

Claims (1)

1, a kind of method that detects same antigen presentation on same sample the steps include:

(1) at first be that paraffin-embedded tissue dewaxing, aquation, TBS are 0.01M pH7.4TBS solution: trihydroxy aminomethane 1.21g, sodium chloride 7.6g, adding distil water transfers to 1000ml, and the concentrated hydrochloric acid adjust pH is 7.4, washes 2～4 times, each 3min; Next is that flesh tissue is directly since the 3rd step; The 3rd is after cultured cell is fixed, and adds TBS flushing 2～4 times, and each 3min adds penetrating liquid: 0.1%Triton-X100 then, incubated at room 5-10min, and last TBS washes 2～4 times, and each 3min carried out for (3) step;

(2) 0.01M pH6.0 citrate buffer solution, A liquid: 0.1M/L citric acid: citric acid 21.0g, adding distil water transfers to 1000ml, and the concentrated hydrochloric acid adjust pH is 6.0; B liquid: 0.1M/L sodium citrate liquid, sodium citrate 29.41g, adding distil water transfers to 1000ml, during use, gets A liquid 1.9ml, B liquid 8.1ml, adding distil water 90ml, microwave or high pressure carry out antigen retrieval, are cooled to room temperature, TBS flushing 2～4 times, each 3min;

(3) drip 2%BSA bovine serum albumin(BSA) sealing damping fluid, 2g BSA is dissolved in the 100ml TBS solution, and 37 ℃ of wet boxes are hatched 30～35min;

(4) drip to be detected one anti-ly, 37 ℃ of wet boxes were hatched 1～2 hour or 4 ℃ of refrigerator overnight, and TBS-T promptly adds Tween 20 in TBS solution, and the final concentration that makes the latter is 0.05%, and rinsing 3min/ time * (2～4) are inferior;

(5) drip 37 ℃ of wet boxes of 2%BSA sealing damping fluid and hatch 10～15min;

(6) drip biotinylation goat-anti rabbit or sheep anti-mouse igg, 37 ℃ of wet boxes are hatched 30min, and TBS-T rinsing 3min/ time * (2～4) are inferior;

(7) drip 37 ℃ of wet boxes of 2%BSA sealing damping fluid and hatch 10～15min;

(8) drip quantum dot-labeled Streptavidin compound with the dilution of 2%BSA sealing damping fluid, working concentration: 1: 50～200,37 ℃ wet boxes are hatched 30～60min, and TBS-T rinsing 3min/ time * (2～4) are inferior;

(9) Dropwise 5 0% glycerine mounting, last fluorescent microscope is with different wave length excitation quantum point, and is positive to occur orange-red fluorescent grain in the cell;

(10) sample autofluorescence contrast: sample only adds TBS or buffering glycerine mounting, in the fluorescence microscope tissue fluorescence is arranged, and is autofluorescence, positive control: do the histochemical stain of direct method immunofluorescence with known positive sample, result's fluorescence that should be positive;

(11) 2-8 ℃ of preservation, 2-4 finds after week that fluorescence signal weakens, sample to be checked places TBS to soak 5～15min, comes off naturally until cover glass;

(12) fluorescence signal of antigen is respectively once recovered again in repeating step (7), (8), (9);

(13) afterwards, add the Streptavidin of horseradish peroxidase-labeled, 37 ℃ of wet boxes are hatched 15～20min, and TBS rinsing 3min/ time * (2～4) are inferior in step (11);

(14) DAB colour developing, observations, the location of observing antigen to be checked is consistent with immunofluorescence technique.

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