CN101294903B - Method for detecting identical antigen expression on identical sample - Google Patents
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- CN101294903B CN101294903B CN2008100479148A CN200810047914A CN101294903B CN 101294903 B CN101294903 B CN 101294903B CN 2008100479148 A CN2008100479148 A CN 2008100479148A CN 200810047914 A CN200810047914 A CN 200810047914A CN 101294903 B CN101294903 B CN 101294903B
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Abstract
The invention discloses a method for detecting the same antigen expression on the same specimen. A fluorescent quantum dot marked streptavidin compound can be directly combined with a biotin conjugate second antibody, while the biotin conjugate second antibody can also be combined with horseradish peroxidase marked streptavidin in immunoenzyme technique, so that the immunofluorescence and immunoenzyme techniques can be perfectly combined, thereby detecting the same antigen expression on the same specimen. The method can be used for detecting the antigen expression state of the fresh organizations of human bodies and animals, paraffin embedding organizations, and the states of antigen expression in different cell lines. The method has the advantages of higher sensitivity, more persuasive results, low experiment cost, and moderate requirements for experimental conditions. The whole experiment process does not need to avoid light, and can be completed in an ordinary immunohistochemical room. The method is suitable for wide popularization and application.
Description
Technical field
The invention belongs to the immunopathology detection range, more specifically relate to two kinds of technology of utilization detect same antigen presentation on same sample method, the range of application of this method is suitable for the interior antigen presentation situation of flesh tissue, paraffin-embedded tissue and different clones of human body and animal.
Background technology
Immunohistochemistry (immunohistochemistry) claims immunocytochemistry (immunocytochemistry) again, it is applied immunology ultimate principle---antigen-antibody reaction, promptly refer to specific antibody with developer (fluorescein, enzyme, metallic ion, isotope) mark in the histocyte original position by antigen-antibody reaction and histochemical color reaction, corresponding antigens is carried out a technology of qualitative, location, quantitative measurement.It combines immunoreactive specificity, histochemical observability dexterously, by the microscope video picture and the amplification of (comprising fluorescent microscope, electron microscope), detect various antigenic substances (as protein, polypeptide, enzyme, hormone, pathogen and acceptor etc.) at cell, subcellsular level.Immunohistochemistry technology can be divided into immunofluorescence, immuno-enzymatic, immunoferritin, immune gold and radio-immunity from shadow technology etc. according to the kind of label.What be used for pathological diagnosis mainly contains immunofluorescence and immunoenzyme technics.
Immunofluorescence technique: be the immunohistochemistry technology that sets up the earliest.It utilizes the principle of antigen and antibody specific combination, earlier known antibodies is put on fluorescein,, observes under fluorescent microscope as the corresponding antigens in the pin check cell or tissue with this.After the irradiation of the fluorescein stimulated luminescence in the antigen antibody complex, promptly can send the fluorescence of certain wavelength, thus the location of certain antigen in can determining to organize, and then also can carry out quantitative test.Because immunofluorescence technique high specificity, highly sensitive, fast and convenient, so in clinical pathology diagnosis, check, use relatively wider model.But, because immunofluorescence technique must have fluorescent microscope, fluorescence intensity in time prolongation and disappear gradually, the result is difficult for shortcomings such as long preservation, is subjected to certain limitation in popularization and application, and is replaced by immunoenzyme gradually.
Immunoenzyme technics: be after immunofluorescence, the technology that grows up the sixties in 20th century.Ultimate principle is antibody and tissue or the cytosis of elder generation with enzyme labeling, the substrate that adds enzyme then, generate coloured insoluble product or the particle with certain electron density, by light microscopic or Electronic Speculum, pair cell surface and intracellular various antigenic component position research.The major advantage that this method is compared with immunofluorescence technique is: accurate positioning, good contrast, but stained preparation long preservation are suitable for light, electron microscopy study etc.
Immunoenzyme technics is present the most frequently used method.The development of immunoenzyme technics is very fast, has derived multiple labeling method, and along with the updating and innovating of method, its specificity and sensitivity are all improving constantly, and it is also more and more convenient to use.That widely uses in pathological diagnosis at present surely belongs to PAP method, ABC method, SP method etc.
Be called as quantum dot (quantum dots according to relevant report fluorescence semiconductor nano particle serendipitous recent years, QDs) provide a kind of potential method to remove to overcome very narrow, the fluorescent lifetime weak point of excitation wavelength range of traditional fluorescein such as organic fluorophor, shortcomings such as easy quencher, beginning is Preliminary Applications in immunofluorescence analysis, and has obtained gratifying effect.Quantum dot is the semiconductor nano microcrystal again, and using more at present is the nanoparticle that II-VI family or III-V family element are formed, and (Y is S, and Se Te), also has some composite structures and sandwich construction to mainly contain CdY.Compare with traditional labelled reagent, quantum dot has following characteristic: 1. have scale effect and good luminescent properties.Quantum dot is the polyelectron system, and luminescence efficiency is far above individual molecule, and launch organic fluorescent dye only 20 times, stability is strong more than 100 times, can stand repeated multiple times and excite, unlike the easy quencher of the fluorescence of organic fluorescent dye and lanthanide series.Quantum dot is strong to the resistivity of photobleaching, and fluorescence lifetime is long.2. quantum dot has good bio-compatibility.With the quantum dot thing that makes marks the activity of biomacromolecule sample there is not injury, and the coupling linked method of quantum dot and biomacromolecule and mode are relatively single, comprise and utilize chemical bond and electrostatic force to carry out coupling that do not resemble traditional labelled reagent, every kind of reagent all needs specific coupling linked method.
At present, nearly all utilization immunofluorescence technique and immunoenzyme technics detect in the bibliographical information of antigen presentation, all on two samples, carry out respectively, can make experiment condition inconsistent like this, also can make use respectively these two kinds of technology for detection to the result deviation is arranged.Simultaneously, more human and material resources have been expended.Yet, up to the present, also do not find similar we on same sample, successively use immunofluorescence technique and immunoenzyme technics to detect the report of same antigen presentation.
Summary of the invention
The objective of the invention is to be to provide a kind of method that on same sample, detects same antigen presentation, adopt immunofluorescence technique and immunoenzyme technics to detect with the expression of antigen again.This method is utilized the quantum dot labelled streptavidin, applicability is wider, thereby the detection that can also proceed immunoenzyme technics after immunofluorescence technique detects antigen presentation and analysis result also also can show the position of same antigen presentation, and is consistent with the testing result of immunofluorescence technique.
Because traditional fluorescein such as fluorescein isothiocynate (FITC), tetramethyl isocyanic acid Luo Daming (TRITC), Texas are red only can mark one anti-or two anti-, in the ban after carrying out the immunofluorescence detection on the sample, owing to do not need to add biotinylated IgG or IgM, and the Streptavidin that makes horseradish peroxidase-labeled in the follow-up immunoenzyme does not have binding site, event can not successively carry out immunofluorescence technique on same sample and immunoenzyme detects same antigen, and detects the poor effect of antigen presentation on paraffin-embedded tissue.After the recent findings quantum dot, research to its biological characteristic is advanced by leaps and bounds, quantum dot has good bio-compatibility, except can be directly with one anti-, two anti-couplings, can also directly be coupled with Streptavidin, this just makes its range of application wider, and also the detection for immunoenzyme provides opportunity.The present invention mainly utilizes commercial quantum dot-labeled Streptavidin compound (available from an ancient woman's ornament source, Wuhan technology of quantum dots Development Co., Ltd) to carry out the detection of immunofluorescence technique earlier, because added one is anti-in this method, particularly two anti-be biotinylation IgG or IgM, can combine with quantum dot-labeled Streptavidin compound also and can combine, so just help having combined these two kinds of technological perfectionisms with the Streptavidin (available from Bioisystech Co., Ltd of China fir Golden Bridge in Beijing) of horseradish peroxidase-labeled.Whom can be regardless of earlier after whom by these two kinds of technology of principle, but consider mounting and store method is different as a result, the detection of carrying out earlier carrying out immunoenzyme technics again after the detection of immunofluorescence is to all not influences each other, so adopted this order.Its concrete steps are as follows:
1. at first be paraffin-embedded tissue dewaxing, aquation, TBS (be 0.01M pH7.4 TBS solution: trihydroxy aminomethane 1.21g, sodium chloride 7.6g, adding distil water transfers to 1000ml, the concentrated hydrochloric acid adjust pH is 7.4) flushing 2～4 times, each 3min; Next is that flesh tissue is directly since the 3rd step; The 3rd be cultured cell fixing after, add TBS flushing 2～4 times, each 3min adds penetrating liquid (0.1%Triton-X100) room temperature (20-25 ℃, below identical) then and hatches 5-10min, last TBS flushing 2～4 times, each 3min carried out for the 3rd step.
2.0.01M the pH6.0 citrate buffer solution [A liquid: the 0.1M/L citric acid (citric acid 21.0g, adding distil water transfers to 1000ml, 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, get A liquid 1.9ml, B liquid 8.1ml, adding distil water 90ml gets final product], microwave or high pressure (1.01 * 10
5-2.02 * 10
5Handkerchief) carries out antigen retrieval, naturally cool to room temperature, TBS flushing 2～4 times, each 3min;
3. drip 37 ℃ of wet boxes of 2%BSA (bovine serum albumin(BSA)) sealing damping fluid (being that 2g BSA is dissolved in the 100ml TBS solution) and hatch 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 add Tween 20 in TBS solution, the final concentration that makes the latter is 0.05%) 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. (working concentration: 1: 50～200), 37 ℃ of wet boxes are hatched 30～60min, and TBS-T rinsing 3min/ time * (2～4) are inferior to drip quantum dot-labeled Streptavidin compound with the dilution of 2%BSA sealing damping fluid;
9. Dropwise 5 0% glycerine mounting, last fluorescent microscope is with different wave length (400-550nm) excitation quantum point, and is positive to occur orange-red fluorescent grain in the cell;
10. sample autofluorescence contrast: sample only adds TBS (50-200 μ l) or buffering glycerine mounting, if in the fluorescence microscope tissue fluorescence is arranged, is called 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 ℃ preservation, 2-4 finds after week that fluorescence signal weakens, and sample to be checked places TBS to soak 5～15min, comes off naturally until cover glass;
12. repeating step 7,8,9 respectively once, can recover the fluorescence signal of antigen again.
13. also can be after step 11, add the Streptavidin (50-200 μ l) of horseradish peroxidase-labeled, 37 ℃ of wet boxes are hatched 15～20min, and TBS rinsing 3min/ time * (2～4) are inferior;
14.DAB colour developing, observations under the optical microscope.Can be observed the location and the immunofluorescence technique basically identical of antigen to be checked.
The present invention can labelled streptavidin form under the inspiration of compound at quantum dot, successfully on same sample, carried out immunofluorescence technique first and immunoenzyme detects same antigen, compare with classic method, the advantage of this invention: 1. sensitivity is higher, traditional immunofluorescence label thing is difficult on the paraffin-embedded sample expression that detects antigen, and is that the immunofluorescence technique of mark can be carried out detection of antigens well with the quantum dot on paraffin specimen; 2. the result is more convincing, and because of immunofluorescence technique and immunoenzyme detect the term harmonization of antigen presentation, the location of antigen is more clear, and background is lower, and the positive rate goodness of fit of same antigen presentation can reach more than 95%, sees accompanying drawing 1-3; 3. the result can long preservation, and the result of immunofluorescence can preserve for 2～4 weeks even longer among the present invention, thereby helped carrying out better qualitative, quantitative analysis results; 4. experimental cost is lower, particularly utilize organization chip (every chip price is at 600～2000 yuans) to study, classic method needs two chips, and the present invention can carry out on a chip, can save experiment fees and valuable specimen resource, also save experimental period; 5. experiment condition is not harsh, and whole experiment does not all need lucifuge, all can finish at general Immune-Histochemical Lab, is suitable for wide popularization and application.
Description of drawings
Present the positive expression fluorescence of orange-red fluorescent grain * 40 in the kytoplasm of the cancer cell of Fig. 1 Caveolin-1 albumen in lung squamous cell carcinoma cancers and an after birth and the matter
After Fig. 2 fluorescence signal weakened, the fluorescence signal of Caveolin-1 albumen in lung squamous cell carcinoma cancers recovered fluorescence * 100 again
After Fig. 3 fluorescence signal weakens, present the general light of the positive expression of brown yellow granule * 40 in the kytoplasm of the cancer cell of Caveolin-1 albumen in lung squamous cell carcinoma cancers and after birth and the matter
The expression that is positive in lung cancer cell line A549 of Fig. 4 Caveolin-1 albumen is positioned cell membrane fluorescence * 100
Embodiment
Below by specific embodiment the present invention is described in further details, but content of the present invention be not limited to for embodiment.
Example 1 detects the expression of Caveolin-1 albumen on the cancerous lung tissue chip, step is:
1.4 the dewaxing of the thick cancerous lung tissue chip of μ m, aquation, TBS (pH7.4) wash 2～4 times, each 3min;
2.0.01M the PH6.0 citrate buffer solution, the microwave antigen retrieval naturally cools to room temperature (20-25 ℃, below identical), TBS flushing 2～4 times, each 3min;
37 ℃ of wet boxes of 3.2%BSA (bovine serum albumin(BSA)) sealing damping fluid (being that 2g BSA is dissolved in the 100ml TBS solution) are hatched 30～35min;
4. drip Caveolin-1 (1: 100) antibody, 37 ℃ of wet boxes were hatched 1.5～2 hours or 4 ℃ of refrigerator overnight, and TBS-T (pH7.4) rinsing 3min/ time * (2～4) are inferior;
37 ℃ of wet boxes of 5.2%BSA sealing damping fluid are hatched 10～15min;
6. drip the biotinylation goat anti-rabbit igg, 37 ℃ of wet boxes are hatched 30～35min, and TBS-T rinsing 3min/ time * (2～4) are inferior;
37 ℃ of wet boxes of 7.2%BSA sealing damping fluid are hatched 10～15min;
8. drip the quantum dot-labeled Streptavidin compound (working concentration 1: 100) with the dilution of 2%BSA sealing damping fluid, 37 ℃ of wet boxes are hatched 30～60min, and TBS-T rinsing 3min/ time * (2～4) are inferior;
9. Dropwise 5 0% glycerine mounting, the blue-light excited quantum dot of last fluorescent microscope, Caveolin-1 albumen is the positive (see figure 1) of orange-red fluorescent grain to occur in the kytoplasm of lung carcinoma cell and an after birth and the matter;
10. sample autofluorescence contrast: sample only adds TBS (50-200 μ l) or buffering glycerine mounting, if in the fluorescence microscope tissue fluorescence is arranged, is called 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 ℃ preservation, 2-4 finds after week that fluorescence signal weakens, and organization chip places TBS to soak 5～15min, comes off naturally until cover glass;
12. repeating step 7,8,9 respectively once, can recover the fluorescence signal (see figure 2) of Caveolin-1 albumen again.
13. also can be after step 11, the 37 ℃ of wet boxes of Streptavidin (50-200 μ l) that add horseradish peroxidase-labeled are hatched 15～20min, and TBS rinsing 3min/ time * (2～4) are inferior;
14.DAB colour developing, observations under the optical microscope.Observe pale brown look appears in Caveolin-1 albumen in the kytoplasm of lung carcinoma cell and after birth positive signal (see figure 3).
Example 2 detects the expression of Caveolin-1 albumen on lung cancer cell line A549 cell, step is:
1.A549 after the cell fixation, add TBS (pH7.4) flushing 2～4 times, each 3min adds penetrating liquid (0.1%Triton-X 100) incubated at room 5-10min then, last TBS flushing 2～4 times, each 3min;
37 ℃ of wet boxes of 2.2%BSA sealing damping fluid are hatched 30～35min;
3. drip Caveolin-1 (1: 100) antibody, hatched 1.5～2 hours or 4 ℃ of refrigerator overnight for 37 ℃, TBS-T (pH7.4) rinsing 3min/ time * (2～4) are inferior;
37 ℃ of wet boxes of 4.2%BSA sealing damping fluid are hatched 10～15min;
5. drip biotinylation goat anti-rabbit igg (50-200 μ l), 37 ℃ of wet boxes are hatched 40～45min, and TBS-T rinsing 3min/ time * (2～4) are inferior;
37 ℃ of wet boxes of 6.2%BSA sealing damping fluid are hatched 10～20min;
7. drip the quantum dot-labeled Streptavidin compound (working concentration: 1: 100) with the dilution of 2%BSA sealing damping fluid, 37 ℃ of wet boxes are hatched 45～60min, and TBS-T rinsing 3min/ time * (2～4) are inferior;
8. Dropwise 5 0% glycerine mounting, the blue-light excited quantum dot of last fluorescent microscope, Caveolin-1 albumen is the positive (see figure 4) of orange-red fluorescent grain to occur in the kytoplasm of lung carcinoma cell and the after birth;
9. sample autofluorescence contrast: sample only adds TBS (50-200 μ l) or buffering glycerine mounting, if in the fluorescence microscope tissue fluorescence is arranged, is called autofluorescence.Positive control: do the histochemical stain of direct method immunofluorescence with known positive sample, result's fluorescence that should be positive.
10.2-8 ℃ preservation, 2-4 finds after week that fluorescence signal weakens, and cell climbing sheet places TBS to soak 5～15min, comes off naturally until cover glass;
11. repeating step 6,7,8 respectively once, can recover the fluorescence signal of Caveolin-1 albumen again.
12. also can be after step 10, the 37 ℃ of wet boxes of Streptavidin (50-200 μ l) that add horseradish peroxidase-labeled are hatched 15～20min, and TBS rinsing 3min/ time * (2～4) are inferior;
13.DAB colour developing, observations under the optical microscope.Observe pale brown look appears in Caveolin-1 albumen in the kytoplasm of lung carcinoma cell and after birth positive signal.
More than comprehensive, this method is simple as can be seen, and repeatability is high, and cost is lower, all can finish at general Immune-Histochemical Lab, for wide popularization and application is laid a good foundation.
Claims (1)
1. a method that detects same antigen presentation on same sample the steps include:
(1) paraffin-embedded tissue dewaxing, aquation, TBS wash 2～4 times, and each 3min carried out for (2) step then, wherein TBS is a 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; After cultured cell is fixing, add TBS flushing 2～4 times, each 3min adds penetrating liquid: 0.1%Triton-X 100 then, incubated at room 5-10min, and last TBS flushing 2～4 times, each 3min carried out for (3) step;
(2) 0.01M pH6.0 citrate buffer solution, microwave or high pressure carry out antigen retrieval, be cooled to room temperature, TBS flushing 2～4 times, each 3min, wherein 0.01M pH6.0 citrate buffer solution is prepared as follows, A liquid: the 0.1M 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 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;
(3) drip 2%BSA bovine serum albumin(BSA) sealing damping fluid, promptly 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 rinsing 3min/ time * (2～4) are inferior, and wherein TBS-T is adding Tween 20 in TBS solution, and the final concentration that makes the latter is 0.05%;
(5) drip 2%BSA sealing damping fluid, 37 ℃ of wet boxes are hatched 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) add the Streptavidin of horseradish peroxidase-labeled, 37 ℃ of wet boxes are hatched 15～20min, and TBS rinsing 3min/ time * (2～4) are inferior;
(13) DAB colour developing, observations, the location of observing antigen to be checked is consistent with immunofluorescence technique.
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| CN101782573B (en) * | 2010-01-11 | 2014-09-10 | 天津师范大学 | Immunofluorescence method for detecting nucleolus C23 protein positioning under heavy metal stress |
| CN102072959B (en) * | 2010-11-16 | 2013-08-14 | 武汉大学 | Method for simultaneously labelling collagen type IV, macrophage and neovascularisation of tumors |
| KR20120128440A (en) * | 2011-05-17 | 2012-11-27 | 삼성전자주식회사 | Kit and method for detecting target material |
| CN103063849A (en) * | 2012-12-28 | 2013-04-24 | 武汉大学 | A method for simultaneous detection of cancer-associated fibroblasts and expressed proteins thereof |
| CN105699155B (en) * | 2016-01-29 | 2019-09-24 | 山东省千佛山医院 | The dyeing chemistry detection method of intestinal mucosa brain-derived neurotrophic factor BDNF sample |
| CN107907396B (en) * | 2017-11-07 | 2020-11-10 | 山西大学 | Automatic immunohistochemical staining system for biological tissues |
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