CN110244041A - It is a kind of detect albumen kit and its application - Google Patents
It is a kind of detect albumen kit and its application Download PDFInfo
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- CN110244041A CN110244041A CN201910559911.0A CN201910559911A CN110244041A CN 110244041 A CN110244041 A CN 110244041A CN 201910559911 A CN201910559911 A CN 201910559911A CN 110244041 A CN110244041 A CN 110244041A
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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Abstract
The present invention provide it is a kind of detect albumen kit and its application, be related to technical field of biological chemistry detection, kit of the present invention includes: the conjugate of 1) goat anti-rabbit igg and PAMAM；2) conjugate of sheep anti-mouse igg and quantum dot；3) the anti-A of the mouse of testing protein and rabbit-anti B；The anti-A of mouse and rabbit-anti B need to meet the same antigen of detection, and not have cross reaction.Kit of the present invention is applied widely and can be able to detect most of albumen, including cell factor, secreted protein, intracellular protein and cell surface protein with flexible combination.
Description
Technical field
The present invention relates to technical field of biological chemistry detection more particularly to a kind of kit for detecting albumen and its applications.
Background technique
Flow cytometry (Flow Cytometry, FCM) is the cell or other biologies of defiled in a kind of pair of liquid stream
Particle (such as microballoon, bacterium, small scale mode biology) carries out the technology of fast quantitative analysis and sorting one by one.As using streaming
The technology platform that cell art is detected, modern flow cytometer result from six the seventies of last century.By nearly 40 years
Develop and perfect, the flow cytometer of today is very mature, and is widely applied to from basic research to clinical practice
Various aspects, cover cell biology, immunology, hematology, oncology, pharmacology, science of heredity and clinical examination etc. neck
Domain plays an important role in each subject.
In conjunction with immunofluorescence method, the cell with different surfaces specific antigen can be recognized and be counted to flow cytometry,
Such as identifies T and bone-marrow-derived lymphocyte with fluorescein-labeled immunoglobulin and further divided according to the difference of cell surface antigen
Discern different T and bone-marrow-derived lymphocyte subgroup, and each cell of measurement the quantity with antigen, density and its kinetic parameter
Deng.It can also will be received with "+" and without the cell colony classification of certain species specific antigen of "-" with streaming cell sorting techniques
Collection, for studying its functional characteristic.
Immunohistochemistry is that applied immunology basic principle --- antigen-antibody reaction, i.e., antigen is in conjunction with antibody specificity
Principle, make the color developing agent (fluorescein, enzyme, metal ion, isotope) of labelled antibody develop the color to determine group by chemically reacting
Intracellular antigen (peptide and protein) is knitted, it is positioned, qualitative and relative quantification research, referred to as immunohistochemistry
Technology (immunohistochemistry) or immunocytochemical technique (immunocytochemistry).
In clinical diagnosis, protein molecular is common Testing index.In clinic common testing protein include cell because
Son, intracellular protein, cell surface protein, the diversified forms such as secreted protein.It can for cell surface protein or intracellular protein
To be detected with the method for immunohistochemistry；For cell factor, secreted protein and part intracellular protein immunohistochemistry
Mode is difficult to detect, therefore streaming is preferable detection method.But streaming, which can not directly detect protein molecular, can only detect cell,
It is conventional to use CBA method to overcome the problems, such as this, i.e., substitute cell using artificial synthesized microballoon, microsphere surface coating to
The antibody of albumen is surveyed, when sample to be tested contains corresponding testing protein, the antibody on artificial microballoon can capture it, add PE
The anti-protein antibodies of dye-coupling, the fluorescence antibody can be with albumen, and microballoon forms sandwich structure, finally in streaming
In detected.But the conventional method scope of application is small, can not achieve the detection of multiple types albumen.
Summary of the invention
The present invention is in order to overcome existing flow cytometry and immunohistochemical reaction that can not directly detect secreted protein
Defect, provide it is a kind of detect albumen kit and its application, can be used for the detection of multiple types albumen, examination of the present invention
Agent box is applied widely and can be able to detect the most cells factor with flexible combination, secreted protein, intracellular protein and thin
Cellular surface albumen.
The present invention provides a kind of kit for detecting albumen, the kit includes:
1) conjugate of goat anti-rabbit igg and PAMAM；
2) conjugate of sheep anti-mouse igg and quantum dot；
3) the anti-A of the mouse of testing protein and rabbit-anti B；
The anti-A of mouse and rabbit-anti B need to meet the same antigen of detection, and not have cross reaction.
Preferably, the testing protein includes cell factor, intracellular protein, cell surface protein and secreted protein.
Preferably, the preparation method of the conjugate of the sheep anti-mouse igg and quantum dot, comprising the following steps:
S1, quantum dot is dissolved in activating solution, sequentially adds Sulfo-NHS and EDC solution, activate 3~8min, from
The heart discards supernatant, and obtained precipitating is dissolved with activating solution, obtains activation quantum dot solution；
The activating solution is the 5mM BS buffer that the pH value of 0.01% polysorbas20 containing mass concentration is 7.4；
S2, sheep anti-mouse igg is mixed with the activation quantum dot solution that step S1 is obtained, is protected from light 10~16h at 4 DEG C,
The BSA of mass concentration 8~15% is added, reacts 20~40min at 37 DEG C, adds washing lotion, supernatant is removed in centrifugation, precipitates with washing lotion
Dissolution, supernatant is removed in centrifugation again, will be deposited in save and dissolve in liquid, and low-speed centrifugal collects supernatant, obtains sheep anti-mouse igg and amount
The conjugate of son point；
The washing lotion is the BS buffer of the 5mM pH 8.0 of 0.01% polysorbas20 containing mass concentration；
It is described to save the washing lotion that liquid is the BSA containing mass concentration 10%.
Preferably, the quantum dot includes the hud typed carboxyl water-soluble quantum dot of CdSe/ZnS that concentration is 5mg/mL；
The quantum dot is dissolved in 5 times of volume activating solutions；The activating solution of 3 times of volumes of the precipitating dissolves；The quantum dot and goat-anti
The mass ratio of mouse IgG is 2.25mg:0.1mg；The molar ratio of the Sulfo-NHS and EDC is 1:1.
Preferably, the preparation method of the conjugate of the goat anti-rabbit igg and PAMAM, comprising the following steps:
60mg PAMAM is dissolved in 2~5mL DMSO, succinic anhydride is added to after being completely dissolved in stirring, is protected from light
3~5h dialyses, and freeze-drying obtains modified PAMAM freeze-dried powder；
By modified PAMAM freeze-dried powder MES buffer solution, NHS and EDC is added, is added goat anti-rabbit igg antibody, 4
DEG C 12~16h of low-temp reaction is dialysed with the bag filter of 3500~5000MWCO, after dialysed again with 8000MWCO bag filter, freeze-drying
Obtain goat anti-rabbit igg and the conjugate of PAMAM.
Preferably, the charge ratio of the PAMAM and succinic anhydride is in 1:(10~50)；The molar ratio of NHS and EDC is
1:1；The ratio of described EDC, NHS and goat anti-rabbit igg antibody is 40umol:40umol:1ug.
The present invention also provides application of the kit described in above-mentioned technical proposal in the Flow cytometry of albumen.
Preferably, the Flow cytometry the following steps are included:
(1) the mouse anti-A and rabbit-anti B that testing protein is added carry out the incubation of first antibody, obtain in conjunction with first antibody to
Survey albumen；
(2) conjugates of the testing protein for the combination first antibody for obtaining step (1) and quantum dot and sheep anti-mouse igg and
PAMAM is mixed with the conjugate of goat anti-rabbit igg, and the second anti-incubation, washing are carried out under the conditions of being protected from light for 37 DEG C；
(3) flow type analyzer is splined on to be analyzed.
The present invention also provides application of the kit described in above-mentioned technical proposal in the detection of protein immunization groupization.
Preferably, immunohistochemistry detection the following steps are included:
N1, testing protein and the anti-A of mouse are subjected to first antibody incubation；
N2, the conjugates that quantum dot and sheep anti-mouse igg is added, secondary antibody incubation is carried out under the conditions of being protected from light for 37 DEG C, is washed
It washs；
N3, nuclear targeting, washing, microscopy are carried out.
The present invention provides a kind of kits for detecting albumen, comprising: 1) conjugate of goat anti-rabbit igg and PAMAM；2) sheep
The conjugate of anti-mouse IgG and quantum dot；3) the anti-A of the mouse of testing protein and rabbit-anti B；It is same that the anti-A of mouse and rabbit-anti B need to meet detection
One antigen, and there is no cross reaction.The goat anti-rabbit igg of kit of the present invention and the conjugate of PAMAM and sheep anti-mouse igg with
When the conjugate of quantum dot is applied in combination, it is added with the sheep anti-mouse igg anti-A of the mouse in conjunction with the conjugate of quantum dot, and with sheep
After rabbit-anti B of the anti-rabbit IgG in conjunction with the conjugate of PAMAM, in the presence of testing protein, it is capable of forming ' PAMAM coupling
Goat anti-rabbit igg-rabbit-anti testing protein-anti-the testing protein of testing protein-mouse-quantum point coupling sheep anti-mouse igg ' compound, only
There is rabbit-anti B corresponding with testing protein with the anti-A of mouse, so that it may detect corresponding testing protein, application range is very wide.With
When the flow cytometer detection of immunohistochemical experiment or cell surface protein, kit of the present invention can flexibly be split, it is only necessary to be used
Sheep anti-mouse igg albumen corresponding with the conjugate of quantum dot and the anti-A of mouse this two parts capture, without the use of PAMAM and antibody
The part of coupling.Kit of the present invention can be used in the detection of multiple types albumen, including cell factor, intracellular protein,
Cell surface protein, the diversified forms such as secreted protein；And kit of the present invention is of wide application, as long as having corresponding
The anti-A of mouse can detect corresponding testing protein with rabbit-anti B.
Detailed description of the invention
Fig. 1 is the composite junction composition that kit of the present invention detects that albumen is formed；
Fig. 2-A is the potential diagram in PAMAM (G5 generation)；
Fig. 2-B is the potential diagram of cPAMAM (i.e. modified PAMAM)；
Fig. 3 is the fluorescence pattern of the couplet of PAMAM and goat anti-rabbit igg；
Fig. 4 is the ELISA Resistance detecting result of the couplet of PAMAM and goat anti-rabbit igg；
Fig. 5 is the sheep anti-mouse igg of ELISA method detection and the fluorescence observation result of quantum point coupling object；
Fig. 6-A is the grain-size graph of quantum dot；
Fig. 6-B is the grain-size graph of the conjugate of sheep anti-mouse igg and quantum dot；
Fig. 7 is the fluorescence pattern of the conjugate of sheep anti-mouse igg and quantum dot；
Fig. 8 is the standard curve for improveing the detection of BCA method；
Fig. 9-A is the streaming figure of L02 cell, and left figure is that scatter plot right figure is unimodal figure；
Fig. 9-B is the streaming figure of MCF-7 cell, and left figure is that scatter plot right figure is unimodal figure；
Figure 10-A is the hela Cell sheet glass under the natural light visual field；
Figure 10-B is the hela Cell sheet glass under the ultraviolet light visual field (blue is nucleus, and red is β-actin)；
Figure 10-C is A, the merge figure of B i.e. mixing cyclogram.
Specific embodiment
The present invention provides a kind of kit for detecting albumen, the kit includes:
1) conjugate of goat anti-rabbit igg and PAMAM；
2) conjugate of sheep anti-mouse igg and quantum dot；
3) the anti-A of the mouse of testing protein and rabbit-anti B；
The anti-A of mouse and rabbit-anti B need to meet the same antigen of detection, and not have cross reaction.In the present invention, the mouse is anti-
Interconnection will not occur for A and rabbit-anti B, and the sandwich of " the anti-A- albumen-rabbit-anti B of mouse " is capable of forming in the presence of testing protein
Interlayer structure, without being to skip testing protein from connection.In the present invention, the anti-A of the mouse and rabbit-anti B preferably remain natural structure
To meeting the requirement of experiment such as FCM or IHC, to be applied to streaming.
In the present invention, the testing protein includes cell factor, intracellular protein, cell surface protein and secreting type egg
It is white.Specifically, kit albumen that can be detected of the present invention includes Caspase3, caspase8, caspase9, hsp27,
AKT, hsp70 etc..Kit of the present invention being capable of efficient detection cell factor, intracellular protein, cell surface protein and secretion
Type albumen, and these albumen are difficult directly to be detected in streaming detection method.
In the present invention, the conjugate of the goat anti-rabbit igg and PAMAM can be specifically bound with rabbit-anti B, the goat-anti
The conjugate of mouse IgG and quantum dot can be specifically bound with the anti-A of mouse, and the two captures testing protein jointly, forms " PAMAM- sheep
The compound of the anti-rabbit IgG- rabbit-anti B- testing protein-anti-A- sheep anti-mouse igg-quantum dot of mouse ", as shown in Figure 1.Quantum dot is in this hair
Bright effect is luminous agent, and the solution of the present invention, such as specific embodiment party of the present invention can be achieved in quantum dot known in the art
The CdSe/ZnS core-shell type carboxyl water-soluble quantum dot that case uses.The effect of PAMAM is similar to ' artificial cell ', to make to combine
It is detected in streaming." conjugate of sheep anti-mouse igg and quantum dot " of the present invention can also be individually used in immunohistochemical reaction.
In the present invention, the preparation method of the conjugate of the sheep anti-mouse igg and quantum dot, comprising the following steps:
S1, quantum dot is dissolved in activating solution, sequentially adds Sulfo-NHS and EDC solution, activate 3~8min, from
The heart discards supernatant, and obtained precipitating is dissolved with activating solution, obtains activation quantum dot solution；
The activating solution is the 5mM BS buffer that the pH value of 0.01% polysorbas20 containing mass concentration is 7.4；
S2, sheep anti-mouse igg is mixed with the activation quantum dot solution that step S1 is obtained, is protected from light 10~16h at 4 DEG C,
The BSA of mass concentration 8~15% is added, reacts 20~40min at 37 DEG C, adds washing lotion, supernatant is removed in centrifugation, precipitates with washing lotion
Dissolution, supernatant is removed in centrifugation again, will be deposited in save and dissolve in liquid, and low-speed centrifugal collects supernatant, obtains sheep anti-mouse igg and amount
The conjugate of son point；
The washing lotion is the BS buffer of the 5mM pH 8.0 of 0.01% polysorbas20 containing mass concentration；
It is described to save the washing lotion that liquid is the BSA containing mass concentration 10%.
Quantum dot is dissolved in activating solution by the present invention, sequentially adds Sulfo-NHS and EDC solution, and activation 3~
8min, centrifugation discard supernatant, and obtained precipitating is dissolved with activating solution, obtain activation quantum dot solution；The activating solution be containing
The BS buffer for the 5mM that the pH value of 0.01% polysorbas20 of mass concentration is 7.4.In the present invention, the activating solution is as buffering
Liquid can provide reaction environment for the activation of Sulfo-NHS and EDC, it may have the effect of washing.In the present invention, the amount
Son is selected preferably to be mixed according to the volume ratio of 0.5~0.6:2~3 with activating solution, more preferably 0.45:2.25.In the present invention,
It is preferably stirred by ultrasonic when the activation.In the present invention, the ultrasonic time is preferably 5min.In the present invention, ultrasonic
Partial activation liquid is added after reaction, preferably according to the volume ratio of quantum dot initial volume and activating solution 40~60:8~15, to
System after ultrasound adds activating solution.In the present invention, the revolving speed of the centrifugation is preferably 10000~15000rpm, more preferably
For 12000rpm.In the present invention, the time of the centrifugation is preferably 1~4min, more preferably 2min.In the present invention, institute
The interval time for stating Sulfo-NHS and EDC addition is preferably no less than 3min.In the present invention, the Sulfo-NHS and quantum
The volume ratio of point is preferably 40~60:10~25, more preferably 45:15.In the present invention, the volume of the EDC and quantum dot
Than being preferably 40~60:10~25, more preferably 45:15.In the present invention, the Sulfo-NHS is used to activate amino, EDC
For activated carboxyl.The present invention is not particularly limited the type of the quantum dot, using well known to those skilled in the art normal
Advise commercially available quantum dot.Present invention preferably uses the hud typed carboxyl water-soluble quantum dots of CdSe/ZnS, and EDC can be to quantum dot
Activation is played, so that the carboxyl of quantum dot surface is activated to be coupled with the antibody that amino is activated.
After obtaining activation quantum dot solution, the present invention mixes sheep anti-mouse igg with activation quantum dot solution, is protected from light at 4 DEG C
10~16h is reacted, the BSA of mass concentration 8~15% is added, reacts 20~40min at 37 DEG C, add washing lotion, centrifugation is gone
Clearly, precipitating is dissolved with washing lotion, and supernatant is removed in centrifugation again, will be deposited in save and be dissolved in liquid, low-speed centrifugal is collected supernatant, obtained
The conjugate of sheep anti-mouse igg and quantum dot；The BS that the washing lotion is the 5mM pH 8.0 of 0.01% polysorbas20 containing mass concentration is slow
Fliud flushing；It is described to save the washing lotion that liquid is the BSA containing mass concentration 10%.In the present invention, the preservation liquid can play long Kubo
The effect deposited.In the present invention, the ratio between quality volume of the sheep anti-mouse igg and quantum dot solution is preferably 60~150 μ g:
400~600 μ L mix sheep anti-mouse igg with the quantum dot solution；The ratio between the quality and volume are more preferably 100 μ g:
450μL.In the present invention, the time being protected from light is preferably 12~14h.In the present invention, described excellent when being protected from light
The adjoint oscillation and temperature control of choosing are at 4 DEG C, to keep the activity of antibody.In the present invention, after being protected from light, it is preferably added to matter
Measure the BSA of concentration 10%.The purpose of present invention addition BSA is the quantum dot that chelating falls non-coupled antibody.In the present invention, it is added
The time that BSA is incubated for is preferably 30min.In the present invention, the revolving speed of the centrifugation is preferably 10000~15000rpm, more excellent
It is selected as 12000rpm.In the present invention, the time of the centrifugation is preferably 1~4min, more preferably 2min.The present invention obtains sheep
After the conjugate of anti-mouse IgG and quantum dot, preferably the conjugate of sheep anti-mouse igg and quantum dot is protected from light in refrigerator it is stored refrigerated,
Stability can be kept 3 months or so.
Highest " conjugate of sheep anti-mouse igg and quantum dot " in order to obtain concentration, BSA incubation reaction will be added in the present invention
Gained liquid is centrifuged 3 times afterwards, and the supernatant being centrifuged twice before giving up takes the supernatant of last time low-speed centrifugal, is protected from light cryo-conservation.This
To show that the preceding supernatant being centrifuged twice also contains resistant for invention experiment, but the supernatant resistance that be centrifuged for the third time when collects
Most strong, concentration highest, refrigerator, which saves, at 4 DEG C can guarantee in 3 months effectively, be it is a kind of more preferably " sheep anti-mouse igg with
The conjugate of quantum dot " preparation method.In the present invention, quantum dot concentration is preferably 5mg/ml, preferably CdSe/ZnS nucleocapsid
Type carboxyl water-soluble quantum dot, quantum dot of the present invention and activating solution be added for the first time volume ratio be preferably 1:5 (450ul:
2.25ml), cleaning step activating solution additional amount is preferably all 3 times of quantum dot volume (1.35ml) later.IgG additional amount is
100ug, i.e. quantum dot and IgG mass ratio are preferably that 2.25mg:0.1mg, EDC and NHS follow the addition of equimolar ratio, EDC:
NHS=1:1mol.
In the present invention, the preparation method of the conjugate of the goat anti-rabbit igg and PAMAM, comprising the following steps:
60mg PAMAM is dissolved in 2~5mLDMSO, succinic anhydride is added to after being completely dissolved in stirring, is protected from light 3
~5h dialyses, and freeze-drying obtains modified PAMAM freeze-dried powder；
By modified PAMAM freeze-dried powder MES buffer solution, NHS and EDC is added, is added goat anti-rabbit igg antibody, 4
DEG C 12~16h of low-temp reaction is dialysed with the bag filter of 3500~5000MWCO, after dialysed again with 8000MWCO bag filter, freeze-drying
Obtain goat anti-rabbit igg and the conjugate of PAMAM.
60mg PAMAM is dissolved in 2~5mLDMSO by the present invention, and succinic anhydride is added to after being completely dissolved in stirring, is kept away
3~5h of light reaction dialyses, and freeze-drying obtains modified PAMAM freeze-dried powder.In the present invention, described to be modified as PAMAM table
The amino chelating in face falls to reduce its bio-toxicity.In the present invention, according to the non-electroneutral of PAMAM, then it is preferred in advance
PAMAM is corrected, to reduce its cytotoxicity.As used in the specific embodiment of the invention, G5 is for PAMAM and surface contains
There are a large amount of amino, the present invention carries out chelatropic reaction for PAMAM by succinic anhydride and G5, the strong positive charge of neutralization of ammonia base bring,
To prevent the PAMAM cytotoxicity of forceful electric power.In the present invention, the additional proportion of the PAMAM and succinic anhydride should ensure that
Charge ratio is in 1:10~1:50, and because PAMAM used in the embodiment of the present invention is G5 generation, every mole contains 64 moles of amino, because
It is added that PAMAM 0.96 is micro- rubs in this embodiment of the present invention, the succinic acid of addition is 2.456 mmoles, makes amino carboxyl ratio in system
In 1:40.
By modified PAMAM freeze-dried powder MES buffer solution, NHS and EDC is added, is added goat anti-rabbit igg antibody, 4
DEG C 12~16h of low-temp reaction is dialysed with the bag filter of 3500~5000MWCO, after dialysed again with 8000MWCO bag filter, freeze-drying
Obtain goat anti-rabbit igg and the conjugate of PAMAM.In the present invention, the molar ratio of NHS and EDC optimization is 1:1, MES buffer
Formula is preferably that the MES of 4.26g is dissolved in 200ml water.In the present invention, it is preferred to, EDC:NHS: goat anti-rabbit igg antibody=
40umol:40umol:1ug.In embodiments of the present invention, preferably modified PAMAM freeze-dried powder 0.019g is delayed with 4mL MES
Fliud flushing dissolution, is added NHS0.023g and EDC 0.385g later.In the present invention, low-temp reaction is preferably protected from light at described 4 DEG C
Reaction.In the present invention, described to be protected from light preferred adjoint oscillation, the revolving speed of oscillation is preferably 150~250rpm, more preferably
For 180rpm.In the present invention, in the presence of EDC, NHS, amino carboxyl is activated independently to be filled PAMAM with antibody.?
In the present invention, the bag filter is using preceding preferably in the sodium bicarbonate containing mass-volume concentration 2%, 1mmol/L EDTA
10min is boiled in the solution of disodium, distilled water reuses after cleaning.Purpose using bag filter is in order to which molecular size to be lower than
Block the small molecule compound removal in aperture.In the present invention, the time of the dialysis is preferably 1~3d, more preferably 2d；Institute
Stating the time dialysed again is preferably 1.5~2.5d, more preferably 2d.In the present invention, the drying is preferably that vacuum refrigeration is dry
It is dry.
In the present invention, it is also preferable to include cleaning solution, dilution, confining liquid, fixer and nuclei dyeings for the kit
One of color liquid is a variety of.It is selected according to reagent needed for Flow cytometry or immunohistochemistry detection.
The present invention also provides application of the kit described in above-mentioned technical proposal in the Flow cytometry of albumen.
In the present invention, the Flow cytometry the following steps are included:
(1) the mouse anti-A and rabbit-anti B that testing protein is added carry out the incubation of first antibody, obtain in conjunction with first antibody to
Survey albumen；
(2) conjugates of the testing protein for the combination first antibody for obtaining step (1) and quantum dot and sheep anti-mouse igg and
PAMAM is mixed with the conjugate of goat anti-rabbit igg, and the second anti-incubation, washing are carried out under the conditions of being protected from light for 37 DEG C；
(3) flow type analyzer is splined on to be analyzed.
Pretreatment to testing protein, preferably includes following steps:
It needs to collect cell culture medium supernatant for secreted protein, carries out the processing such as centrifugal filtration.
For intracellular protein and cell surface protein, then need to carry out cell cracking acquisition.Specific method is: 1) will
The cell of 10cm ware culture removes culture medium, rinses cell surface with cold PBS 2ml, goes washing lotion, appropriate trypsin solution is added and disappears
Change appropriate time, then rinse cell, 2000 turns of centrifugations remove supernatant in 3 minutes
2) cell precipitation is dissolved with 1ml PBS buffer solution, counts cell density
3) appropriate T-PER histone extracts reagent is added in above-mentioned cell PBS solution according to cell density on ice
Albumen 30min is extracted in cracking, is mixed within every 10 minutes primary.
4) solution 14000rpm after above-mentioned cracking is centrifuged 10min, collects supernatant.Supernatant is gained protein mixture.
The present invention also provides application of the kit described in above-mentioned technical proposal in the detection of protein immunization groupization.
In the present invention, immunohistochemistry detection the following steps are included:
N1, testing protein and the anti-A of mouse are subjected to first antibody incubation；
N2, the conjugates that quantum dot and sheep anti-mouse igg is added, secondary antibody incubation is carried out under the conditions of being protected from light for 37 DEG C, is washed
It washs；
N3, nuclear targeting, washing, microscopy are carried out.
Pretreatment to testing protein, preferably are as follows: cell is fixed and is closed.In the present invention, the fixation of cell
It is preferred that are as follows: intracellular protein needs to carry out methanol permeable membrane process, and cell surface protein is then directly fixed.Closing is preferably used
10%BSA is incubated for closing cell 1 hour.Specifically, the fixation and closed process preferably include following steps: 10cm's
A slide is added in Tissue Culture Dish, allows cell climbing sheet (slide is wiped repeatedly in advance with ethyl alcohol and ultraviolet irradiation 4h is air-dried)；
Cell sheet glass taking-up is put into sky ware ,+5% glacial acetic acid of 95% ethyl alcohol of 100-200ul is added, incubation at room temperature 5min is fixed thin
Born of the same parents, after washed three times with PBS；With 1%BSA, PBST (0.1%Tween20) the incubated cell 30min of 22.52mg/ml glycine；
Antibody is diluted to suitable concentration, incubated cell with the PBST containing 1%BSA later.
In the present invention, the dyeing of nuclei dyeing toner, in the present invention, the nuclei dyeing is preferably added in the dyeing
Toner preferably includes DAPI, Hoechst33342 or acridine orange.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
In the following embodiment of the present invention, it is all made of the material in following sources:
PAMAM: morning source molecule brand G5 (amino-type), article No. CYD-150A；
Goat anti-rabbit igg: abcam brand, article No. ab6702；
Quantum dot: the hud typed carboxyl water-soluble quantum dot of CdSe/ZnS, amount point science and technology, 011406-1；
Sheep anti-mouse igg: bioss brand, article No. bs0296G；
Potentiometric detection: Malvern company, Britain NANO ZS90 nano particle size and ZETA potentiometer；
Fluorescence detection: RF-5301PC type sepectrophotofluorometer；
Embodiment 1
1, the coupling of PAMAM and goat anti-rabbit igg:
(1) the strong positive charge of the amino terminal of chelating PAMAM G5
60mg PAMAM is dissolved in the DMSO of 2ml, is stirred at room temperature to dissolution, the succinic anhydride of 0.246g is added, shakes
180 turns of 4h of bed, whole process are protected from light operation, obtain the modified solution of PAMAM.
(2) prepare the bag filter of 3500MWCO, the sodium bicarbonate for being 2% with 1L concentration, 1mmol/LEDTA.Na2 solution boils
Boiling 10 minutes, after it is clean with distilled water diafiltration.
(3) the modified solution of above-mentioned PAMAM is transferred in 3500MWCO bag filter, dialysis for 24 hours, after dialyzate is frozen
It is dry, it weighs (for 0.057g), lyophilized products is denoted as cPAMAM.
(4) MES buffer is configured, 4.26gMES is taken to be dissolved in 200ml water；
The MES solution for configuring NHS, takes 0.023gNHS to be dissolved in the MES buffer of 4ml；
The MES solution for configuring EDC, takes 0.385gEDC to be dissolved in the MES buffer of 4ml；
The MES solution of 100ul above-mentioned NHS and EDC is respectively added in 2ml MES buffer, adds the IgG of 35.7 μ L,
CPAMAM 0.019g is added after reaction 15 minutes, 4 DEG C of shaking tables are stayed overnight, react 16h, obtain the coupled product of cPAMAM and IgG.
(5) product dialysis freeze-drying:
Above-mentioned coupled product is transferred in the bag filter of 3500MWCO, dialyse 2 days, after be transferred to 8000MWCO bag filter
In dialyse again 2 days, will again dialyzate be lyophilized, obtain the couplet of PAMAM and goat anti-rabbit igg.
A, potential change
Each 1ml of cPAMAM for taking PAMAM respectively and being prepared is added in partial size current potential cup, is detected, is sentenced with potentiometer
It reads, as a result as shown in Fig. 2-A and Fig. 2-B.
Fig. 2-A is the potential diagram of PAMAM, and current potential is-3.02mV；Fig. 2-B is the potential diagram of cPAMAM, by succinic anhydride
The current potential of cPAMAM after chelating is -0.465mV.It can be seen that the cPAMAM obtained after chelating is chelated successfully, cytotoxicity drop
It is low, it can be used for the connection with sheep anti-mouse igg antibody.G5 has a large amount of amino, powerful positive charge meeting for PAMAM surface modification
Lead to higher cytotoxicity, influence follow-up test and easily leads to antibody activity decline or even inactivation.
B, fluorescence detection
Modified cPAMAM, cPAMAM coupling goat anti-rabbit igg and goat anti-rabbit igg are taken respectively, it is molten with PBS buffer solution
Solution, respectively takes 1ml, is added in cuvette, is detected with RF-5301PC type sepectrophotofluorometer, interpretation, as a result as shown in Figure 3.
Fig. 3 is that 548nm emits fluorescence pattern under light, schemes upper 3 curves, be respectively from top to bottom goat anti-rabbit igg,
The couplet of cPAMAM, cPAMAM and goat anti-rabbit igg.As seen from Figure 3, after cPAMAM and sheep anti-mouse igg being coupled, fluorescence
Position does not obviously deviate, but fluorescence intensity reduces.
C, ELISA surveys resistance
S1, by the couplet of the PAMAM of synthesis and goat anti-rabbit igg respectively with 2mg/ml, 1mg/ml, 0.5mg/ml,
The concentration of 0.25mg/ml, 0.125mg/ml and 0.0625mg/ml, are layered in elisa plate with 100ul, the control of 2, every hole, simultaneously
10%BSA, 100ul bed board, 2 controls are added in lower row.Plate is sealed, 4 DEG C are incubated overnight
S2, with the hole PBS buffer solution 200ul/ board-washing, 3 minutes every time, by plank in being patted dry on blotting paper after washing 3 times
S3, by rabbit igg-HRP, be diluted to 10ug/ml, the hole 100ul/ is added in above-mentioned 14 holes；
S4, configuration TMB developing solution, are protected from light mixing after configuration method A, B liquid 1:1 mixing, then by 100ulTMB developing solution
It is added in above-mentioned 14 holes, 37 DEG C are incubated for 30 minutes
S5, the sulfuric acid 50ul that 2M is added terminate reaction in every hole.In 450nm survey fluorescence intensity, as a result as shown in figure 4,
The goat anti-rabbit igg of PAMAM coupling is still containing the resistance of goat anti-rabbit igg, i.e. the coupling of PAMAM and goat anti-rabbit igg antibody is not
The missing for causing antibody resistance remains to capture rabbit igg-HRP and completes ELISA reaction.
2, the coupling of quantum dot and sheep anti-mouse igg:
The configuration of reaction reagent
1. 50mM borax: 1.907g borax+100ml water
2. 200mM boric acid: 1.237g boric acid+100ml water
3. PH8.0BS solution: 1.+7ml is 2. by 3ml
4. PH7.4BS solution: 1.+9ml is 2. by 1ml
5. washing lotion: 3. diluting 40 times of+0.01%Tween20
6. activating solution: 4. diluting 40 times of+0.01%Tween20
7. EDC reaction solution: 6. 5ml+0.27gEDC
8. Sulfo.NHS reaction solution: 6. 5ml+0.378gSulfo.NHS
1) 450 μ L quantum dots are dissolved in activating solution 6. 2.25ml, and 8. 150 μ L are added and mix, 7. 150 μ L is added after 3 minutes,
Ultrasound 5min is mixed later；
2) 6. 1.5ml is added, 12000rpm is centrifuged 5min, removes supernatant, dissolves and precipitates with 5. 1.2ml, and ultrasound mixes；
3) antibody 100ug is added, 4 DEG C of shaking tables are stayed overnight, and whole process is protected from light.After taking out ultrasound 30s mixing later, it is added 10%
BSA 150 μ L, 37 DEG C of reaction 30min.
4) 100 μ L are added 5., are mixed, 12000rpm is centrifuged 2min, collects supernatant and is fitted into (1) number pipe；
5) 5. 1ml being added in the precipitating that step 4) obtains, being mixed, 12000rpm is centrifuged 2min, collects supernatant in (2) number
Guan Zhong；
6) 5. 1ml is added in the precipitating that step 5) obtains and 10%BSA, ultrasonic 1h, 820g/min are centrifuged 2 minutes,
It goes to precipitate, supernatant is collected in (3) pipe；
The supernatant of (1) of collection, (2), (3) number pipe is marked according to the method described above, and carries out ELISA detection:
A, natural air drying after pvdf membrane is activated 10 seconds with methanol is distinguished with Pencil marks front and dividing 5 areas above
1-5 is marked, the concentration of source of mouse AKT antibody 200ug/ml takes antibody point 4th area before label of 1ul, in No. 5 areas with 10%BSA
1ul draws point.Pvdf membrane is kept to lay flat, 4 DEG C are incubated overnight.
2) use 10%BSA3ml, whole pvdf membrane impregnated into 2h and closes whole film, after with PBS buffer solution flushing membrane item 2 times,
3 minutes every time.
3) each 50ul of supernatant of above-mentioned (1), (2), (3) number pipe is added to 1,2,3 areas, pure QD is added as control in 4th area
(3) number pipe supernatant is added as control 2 in 1st, 5 area.37 DEG C of incubation 1h, after washed 2 times, every time 3 minutes with PBS
4) above-mentioned pvdf membrane is irradiated to observation fluorescence under ultraviolet lamp.The sheep anti-mouse igg and quantum of ELISA method detection
The fluorescence observation result of point conjugate is as shown in figure 5, as shown in Figure 5,1,2,3 Qu Junyou signals show (1), (2), (3) number pipe
Supernatant it is resistant；But (3) number pipe is most strong, (1) number pipe takes second place, and (2) number pipe is most weak.Product is saved in 4 DEG C of refrigerators, can be protected
Card had comparatively ideal resistance in 3 months.
4th area are to compare 1 (the anti-AKT+QD of mouse) because the not addition of sheep anti-mouse igg, hardly residual after washed
There are the markings that antibody is added.
5th area are 2 (BSA+ Yang KangshuIgG &QD) of control because BSA and Yang KangshuIgG &QD will not be coupled, in cleaning 2
After secondary, only dim light can be stayed not occur the brightness in range as 1st~3 area, as a result as shown in Figure 5.
A, partial size current potential
" conjugate of sheep anti-mouse igg and quantum dot " for taking 500 times of diluted quantum dots respectively and being prepared respectively takes 1ml
It is added in partial size current potential cup, is detected with potentiometer, interpretation, as a result as shown in Fig. 6-A and Fig. 6-B.
Fig. 6-A is the grain-size graph of 500 times of diluted quantum dots, particle size 79.17；Fig. 6-B is sheep anti-mouse igg and quantum
The grain-size graph of the conjugate of point, particle size is 36.49 and 268.3, inhomogenous.Occur bimodal may be that could not be coupled institute completely
It causes, but 268.3 partial size product proof is coupled successfully, the inhomogenous use effect for having no effect on the conjugate of particle size
Fruit.
B, fluorescence detection
" conjugate of sheep anti-mouse igg and quantum dot " and the preservation for taking 500 times of diluted quantum dots respectively, being prepared
Liquid (pH8.0,2.5mM BS, 0.1%BSA) respectively takes 1ml, is added in cuvette, with RF-5301PC type sepectrophotofluorometer
Detection, interpretation, as a result as shown in Figure 7.
The scanning of 515~628nm fluorescence intensity is carried out with 360nm exciting light, three curves are respectively from top to bottom in figure
The peak figure of the conjugate of sheep anti-mouse igg and quantum dot, preservation liquid peak figure, pure quantum dot peak figure,
C, BCA method surveys protein concentration
Because the quantum point coupling antibody of synthesis has quantum dot, BCA cannot be applied completely and surveys protein method, therefore in former BCA
Some changes are done in method, specific change method is then the multiple control groups of setting, and control 1: dilution fluid apertures is as negative hole, control
2: quantum dot is compareed the BCA albumen of 3,0.25mg/ml, dilution is replaced with 10 times of diluted amounts with 10 times of diluted
Sub-, No. 4 holes of sample to be tested are the quantum point coupling IgG for diluting 10 times, finally bring BCA standard curve into and measure coupled product
Concentration is 1.45mg/ml.
Embodiment 2
For detecting the kit of cell factor Hsp27
Illustrate: using MCF-7 breast cancer cell as positive cell, (corresponding result such as Fig. 9-A, Fig. 9-A are L02 cell
Streaming figure, left figure are that scatter plot right figure is unimodal figure).
Using L02 Human normal hepatocyte as negative cells, (corresponding result such as Fig. 9-B, Fig. 9-B are the stream of MCF-7 cell
Formula figure, left figure are that scatter plot right figure is unimodal figure).
Antigen Hsp27 is captured as primary antibody with rabbit-anti so that the mouse of Hsp27 is anti-.
PAMAM is added and is coupled goat anti-rabbit igg, 37 DEG C are incubated for 30 minutes, and the product of incubation is divided into two；
In the product of above-mentioned bisection, will take it is a be added quantum point coupling sheep anti-mouse igg, 37 DEG C are incubated for 30 minutes, separately
Portion is not dealt with, it is as negative control in streaming, and it is sample to be tested that quantum point coupling sheep anti-mouse igg, which is added,.
It can see by Fig. 9-A and Fig. 9-B, negative L02 cell, the not offset of curve, but MCF-7 breast cancer cell
Offset is obvious, can prove that Hsp27 protein content is more than L02 cell in MCF-7 cell, secretes in tumour cell with it and increases
Characteristic be identical.Therefore the combination can be used for the detection of tumour cell differential protein.
Embodiment 3
For detecting intracellular protein β-actin
Illustrate: this experiment is immunohistochemical experiment, only using this part of quantum point coupling sheep anti-mouse igg.
A slide progress cell climbing sheet is added in hela cell ware in culture, and (slide is wiped repeatedly in advance with ethyl alcohol
And ultraviolet irradiation 4h is air-dried).
Slide taking-up is put into sky ware ,+5% glacial acetic acid of 95% ethyl alcohol of 100~200ul is added, is incubated at room temperature 5min
Fixed cell, after washed three times with PBS.
With 1%BSA, PBST (0.1%Tween20) the incubated cell 30min of 22.52mg/ml glycine
Abcam brand mouse anti β-actin antibody (ab6276) is diluted to 5ug/ with the PBST solution of 1%BSA
The concentration of ml is incubated at room temperature 1h or 4 DEG C overnight in wet ware, pours out Incubating Solution later, washed three times with PBS, every time plus after washing lotion
It is cleaned within 5 minutes in 110 turns on shaking table.
Quantum point coupling sheep anti-mouse igg is diluted to the concentration of 10ug/ml with the PBST solution of 1%BSA, is protected from light in 37 DEG C
Be incubated for 1h, after pour out secondary antibody Incubating Solution, washed three times with PBS, every time plus washing lotion after in 110 turns 5 minutes on shaking table.
It redyes: being incubated for slide 5 minutes with the DAPI of 1ug/ml, with PBS rinsing cell 2 times.
Coverslip is closed with nail polish, observes slide with ultraviolet light under the microscope.The results are shown in Figure 10, wherein figure
10-A is the hela cell observed under the natural light visual field, and Figure 10-B is the hela cell observed under ultraviolet light, Figure 10-
C is A, the merge of two figures of B, i.e. visual field stacking chart.Using Hela cell as research object, with the anti-β-of mouse after being fixed with ethyl alcohol
Actin antibody incubation cell, rear sheep anti-mouse igg-quantum dot with synthesis is incubated for, in fluorescence microscopy microscopic observation β-
Actin (i.e. cytoskeletal protein is shown in figure RED sector) under the ultraviolet visual field from the figure, it can be seen that need to can only allow DAPI
The nucleus of dyeing shines simultaneously with the fluorescence that quantum point coupling object issues, and overcomes other probe dyes and needs to switch visual field sight
Examine the shortcomings that nucleus is with testing protein.Wherein red is in β-actin (cytoskeletal protein) red circle detected
It is the obvious visual field, it can be seen that just portion is distributed testing protein around nucleus in the cell.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of kit for detecting albumen, which is characterized in that the kit includes:
1) conjugate of goat anti-rabbit igg and PAMAM；
2) conjugate of sheep anti-mouse igg and quantum dot；
3) the anti-A of the mouse of testing protein and rabbit-anti B；
The anti-A of mouse and rabbit-anti B need to meet the same antigen of detection, and not have cross reaction.
2. kit according to claim 1, which is characterized in that the testing protein include cell factor, intracellular protein,
Cell surface protein and secreted protein.
3. kit according to claim 1, which is characterized in that the system of the conjugate of the sheep anti-mouse igg and quantum dot
Preparation Method, comprising the following steps:
S1, quantum dot is dissolved in activating solution, sequentially adds Sulfo-NHS and EDC solution, activate 3~8min, centrifugation is abandoned
Supernatant is removed, obtained precipitating is dissolved with activating solution, obtains activation quantum dot solution；
The activating solution is the 5mM BS buffer that the pH value of 0.01% polysorbas20 containing mass concentration is 7.4；
S2, sheep anti-mouse igg is mixed with the activation quantum dot solution that step S1 is obtained, is protected from light 10~16h at 4 DEG C, then plus
Enter the BSA of mass concentration 8~15%, react 20~40min at 37 DEG C, add washing lotion, supernatant is removed in centrifugation, precipitates molten with washing lotion
Solution, supernatant is removed in centrifugation again, will be deposited in save and dissolve in liquid, low-speed centrifugal collects supernatant, obtains sheep anti-mouse igg and quantum
The conjugate of point；
The washing lotion is the BS buffer of the 5mM pH 8.0 of 0.01% polysorbas20 containing mass concentration；
It is described to save the washing lotion that liquid is the BSA containing mass concentration 10%.
4. kit according to claim 3, which is characterized in that the quantum dot includes the CdSe/ that concentration is 5mg/mL
ZnS core shell mould carboxyl water-soluble quantum dot；The quantum dot is dissolved in 5 times of volume activating solutions；The work of 3 times of volumes of the precipitating
Change liquid dissolution；The mass ratio of the quantum dot and sheep anti-mouse igg is 2.25mg:0.1mg；Mole of the Sulfo-NHS and EDC
Than for 1:1.
5. kit according to claim 1, which is characterized in that the preparation of the conjugate of the goat anti-rabbit igg and PAMAM
Method, comprising the following steps:
60mg PAMAM is dissolved in 2~5mL DMSO, succinic anhydride is added to after being completely dissolved in stirring, it is protected from light 3~
5h dialyses, and freeze-drying obtains modified PAMAM freeze-dried powder；
By modified PAMAM freeze-dried powder MES buffer solution, NHS and EDC is added, goat anti-rabbit igg antibody is added, 4 DEG C low
Temperature 12~16h of reaction, is dialysed with the bag filter of 3500~5000MWCO, after dialysed again with 8000MWCO bag filter, freeze-drying obtains
The conjugate of goat anti-rabbit igg and PAMAM.
6. kit according to claim 5, which is characterized in that the charge ratio of the PAMAM and succinic anhydride is 1:
(10~50)；The molar ratio of NHS and EDC is 1:1；The ratio of described EDC, NHS and goat anti-rabbit igg antibody is 40umol:
40umol:1ug。
7. application of any one of claim 1~6 kit in the Flow cytometry of albumen.
8. application according to claim 7, which is characterized in that the Flow cytometry the following steps are included:
(1) the mouse anti-A and rabbit-anti B that testing protein is added carry out the incubation of first antibody, obtain the egg to be measured in conjunction with first antibody
It is white；
(2) conjugates of the testing protein for the combination first antibody for obtaining step (1) and quantum dot and sheep anti-mouse igg and
PAMAM is mixed with the conjugate of goat anti-rabbit igg, and the second anti-incubation, washing are carried out under the conditions of being protected from light for 37 DEG C；
(3) flow type analyzer is splined on to be analyzed.
9. application of any one of claim 1~6 kit in the detection of protein immunization groupization.
10. application according to claim 9, which is characterized in that immunohistochemistry detection the following steps are included:
N1, testing protein and the anti-A of mouse are subjected to first antibody incubation；
N2, the conjugates that quantum dot and sheep anti-mouse igg is added carry out secondary antibody incubation, washing under the conditions of being protected from light for 37 DEG C；
N3, nuclear targeting, washing, microscopy are carried out.
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