CN102707069A - Immunohistochemical double-antibody kit - Google Patents
Immunohistochemical double-antibody kit Download PDFInfo
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- CN102707069A CN102707069A CN201210181365XA CN201210181365A CN102707069A CN 102707069 A CN102707069 A CN 102707069A CN 201210181365X A CN201210181365X A CN 201210181365XA CN 201210181365 A CN201210181365 A CN 201210181365A CN 102707069 A CN102707069 A CN 102707069A
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Abstract
The invention provides an immunohistochemical double-antibody kit. The immunohistochemical double-antibody kit comprises a horseradish peroxidase labeled immune globulin solution activated by cyanuric chloride, namely, CC-HRP-Ig. The CC-HRP-Ig is prepared by the method comprising the steps of: step one: activation: cyanuric chloride and horseradish peroxidase or immune globulin are reacted to obtain activated horseradish peroxidase or activated immune globulin; and step two: coupling: the activated horseradish peroxidase and the immune globulin are in coupling reaction or the activated immune globulin and the horseradish peroxidase are in coupling reaction to obtain the CC-HRP-Ig. The immunohistochemical double-antibody kit provided by the invention has high valence of antibody, high sensitivity and low cost.
Description
Technical field
The present invention relates to a kind of SABC two anti-kits, particularly a kind of SABC two anti-kits that comprise Cyanuric Chloride activation horseradish peroxidase-labeled immunoglobulin (Ig).
Background technology
The forties in 19th century, Coons has at first set up immunohistochemical method, is widely applied to biological a plurality of field subsequently, comprises the research of cell function research, neoplastic process, and the detection of infectious diseases etc.In recent years, along with the development of hybridization technique and monoclonal antibody technique, made the diagnosis antibody of preparation have higher specificity and susceptibility.Immunohistochemistry technique has been brought into play great practical value by the development of these new technologies in clinicopathologia, immunology, microbiology diagnosis.
The enzyme labelled antibody technology is enzyme to be connected on the antibody through proper method through covalent bond; Process enzyme labelled antibody; Relend the special catalytic action of enzyme to substrate; Generate coloured insoluble product or particle, under light microscopic or Electronic Speculum, carry out the location of various antigenic components in cell surface and the cell with certain electron density.The quality of enzymic-labelled antibody depends primarily on good, the active strong and high enzyme affinity antibody of affinity of purity, and it is less important to have good preparation method.Horseradish peroxidase is the most frequently used a kind of enzyme.
The method that antibody is connected with enzyme is a lot, and comparatively commonly used mainly is the sodium periodate method of glutaraldehyde method and improvement at present.
Glutaraldehyde is a kind of homotype bifunctional coupling agent, and its two aldehyde radicals can form schiff bases or Michael addition compound with the amino of basic amino acid, also can take place crosslinked with the phenyl ring and the imidazole ring of sulfydryl, tyrosine and the histidine of cystine.Antibody and horseradish peroxidase or alkaline phosphatase all are the biomolecule that is rich in above-mentioned group, so can lead to glutaraldehyde both are coupled together.
According to the order of the reaction of enzyme in the coupling reaction and antibody, glutaraldehyde method can be divided into single stage method and two-step approach.The glutaraldehyde single stage method is reacted coupling agent glutaraldehyde, antibody and enzyme, and this method is simple, save time, effectively and good reproducibility etc.; Shortcoming is that cross-linking reaction speed is different, and the amino acid no of antibody protein is many far beyond HRP, and is fast, wayward with the glutaraldehyde reaction, the condensate that easy formation is bigger, and with the intermolecular crosslinked corresponding minimizing of zymoprotein, influence the mark of enzyme; Joint efficiency is very low, is about 1%-5%, is difficult to obtain desirable enzyme labelled antibody.The glutaraldehyde two step method is at first reacted coupling agent glutaraldehyde and antibody or enzyme, after waiting to remove unnecessary coupling agent, again with another kind of substance reaction; This is fairly simple, and good reproducibility can reduce the generation of self-polymerization thing, and the utilization factor that shortcoming remains enzyme is low, generally has only 2～4% enzyme and protein bound.In sum, the active efficient of glutaraldehyde method HRP and the efficient that combines with IgG are all very low, the reaction poor controllability.
Simple and easy sodium periodate method is with NaIO
4Earlier the glycan molecule with the HRP surface is oxidized to aldehyde radical, the hydroformylation enzyme that forms through the oxidation formation schiff bases that can link to each other with the amino of antibody molecule, and the latter can further use NaBH
4(or monoethanolamine) reduction generates stable enzymic-labelled antibody.The productive rate of enzymic-labelled antibody that this method obtains is high, and nearly 70% HRP and Ig combine, and 90% IgG combines with enzyme, is present the most frequently used method.This law markers step is fairly simple, good reproducibility.Shortcoming is that sodium periodate is opened the sugar ring that HRP brings into play major function, and HRP is active to be reduced; Need NaBH in the reaction
4The participation of (poisonous), dangerous increasing cuts impurity and is difficult for removing.Reaction equation is following:
SABC method two anti-kits are based on two anti-kits of biotin-Streptavidin protein system labelled antibody exploitation.Biotin is also claimed biotin or biotin, is the coenzyme of many carboxylases in the organism.Biotin mainly forms amido link through carboxyl on the side chain and the amino on the antibody molecule with linking of antibody protein and carries out.Avidin is claimed anti-biotin antibodies again, is a kind of glycoprotein.Streptavidin derives from streptomysin bacterium culturing filtrate, relative molecular mass 60000, and its structure is different from Avidin, but possesses the biological nature similar with Avidin, can combine with biotin.With biotin labeling antibody be, need the carboxyl of biotin be carried out chemical modification earlier, process the biotin of activation.Commercial biotin labeling antibody is generally all arranged at present.The shortcoming of SABC method two anti-kits is:
1) interference of endogenous biotin is arranged, form the unspecific staining background easily; Receive the biological interference of endogenous easily, form the background (maybe need increase extra sealing) that is difficult to remove;
2) need the operation of two steps, promptly add biotin labeled antibody earlier, add the Streptavidin of HRP mark again, the running time is long; The kit that biotin relies on needs Streptavidin to participate in, and has increased course of reaction and reaction time.
Summary of the invention
Goal of the invention: the purpose of this invention is to provide high, highly sensitive SABC two anti-kits of a kind of antibody titer and preparation method thereof.
Technical scheme: a kind of SABC two anti-kits provided by the invention comprise horseradish peroxidase (HRP) marked immunoglobulin (IgG) of Cyanuric Chloride (CC) activation, i.e. CC-HRP-Ig;
Said CC-HRP-Ig is made by following method:
(1) activation: Cyanuric Chloride and horseradish peroxidase or immunoglobulin (Ig) reaction get the horseradish peroxidase of activation or the immunoglobulin (Ig) of activation;
(2) coupling: the horseradish peroxidase of said activation and immunoglobulin (Ig) coupling reaction get CC-HRP-Ig, or the immunoglobulin (Ig) of said activation and horseradish peroxidase react CC-HRP-Ig;
Reaction equation is following:
Wherein, said immunoglobulin (Ig) is immunoglobulin G (IgG), unit price immunoglobulin fragment Fab, two valency immunoglobulin fragment F (ab)
2Or immunoglobulin M (IgM), the mass ratio of said Cyanuric Chloride, horseradish peroxidase and immunoglobulin (Ig) is 1: (0.1-10): (0.1-10).
In the step (1), said temperature of reaction is 0 ℃-8 ℃, and the reaction time is 2h-8h.
In the step (2), said temperature of reaction is 25 ℃-40 ℃, reaction time 12h-24h.
Said CC-HRP-Ig exists in solution, and said solution comprises following component: 6-18mg/L CC-HRP-Ig, 5-20g/L bovine serum albumin(BSA), the 0.01-0.1mol/L phosphate buffer of 0.05-0.1g/L thimerosal and pH 7.0-7.4.
In order better to preserve at a lower temperature, can also add the glycerine that volume fraction is 3-5% in the CC-HRP-Ig solution.
Kit also comprises lowlenthal serum solution, and said lowlenthal serum solution comprises: concentration expressed in percentage by volume is the lowlenthal serum of 1-10%, the 0.01-0.1mol/L phosphate buffer of 0.05-0.1g/L thimerosal and pH 7.0-7.4.
Kit comprises that also concentration expressed in percentage by volume is the aqueous hydrogen peroxide solution of 3-10%.
Kit also comprises diaminobenzidine solid or diaminobenzidine concentrate, and the concentration of said diaminobenzidine concentrate is 30-100g/L.
Kit also comprises the diaminobenzidine reaction substrate, and said diaminobenzidine reaction substrate is a volumetric concentration mark 1-10% aqueous hydrogen peroxide solution.
The present invention also provides a kind of preparation method of SABC two anti-kits, may further comprise the steps:
(1) activation: Cyanuric Chloride and horseradish peroxidase or immunoglobulin (Ig) reaction get the horseradish peroxidase of activation or the immunoglobulin (Ig) of activation;
(2) coupling: the horseradish peroxidase of said activation and immunoglobulin (Ig) coupling reaction get CC-HRP-Ig, or the immunoglobulin (Ig) of said activation and horseradish peroxidase react CC-HRP-Ig;
(3) preparation of SABC two anti-kits: utilize said CC-HRP-Ig to prepare SABC two anti-kits; Reaction equation is following:
Beneficial effect: SABC two anti-kit antibody titers provided by the invention are high, and highly sensitive, cost is low.
Kit provided by the invention does not contain biotin and Streptavidin, both can effectively avoid the interference of biotin and the interference of the big molecule space steric hindrance of streptomysin, greatly reduces cost simultaneously yet; This kit comprises the horseradish peroxidase-labeled immunoglobulin (Ig) that adopts the Cyanuric Chloride activation, and catalytic efficiency is high, and is reactive good.When kit provided by the invention was used for enzyme-linked immunosorbent assay (ELISA) and protein immunoblot experiment (Western Blot), dilution ratio can reach 1:5,000 ~ 1:10, and 000, exceed like product 50%-100%.
The preparation method of SABC two anti-kits provided by the invention; Utilize Cyanuric Chloride functional group can with the reaction of different nucleophilic groups and under different temperatures active different characteristic prepare enzyme labelled antibody; Can effectively control reaction time and response intensity, productive rate is high, subsidiary reaction is few.
Preparation method's mild condition of kit provided by the invention need not peracid or crosses alkali; Adopt the hydroxyl coupling on Cyanuric Chloride and the horseradish peroxidase (HRP), need not to open the sugar ring, do not influence the activity of horseradish peroxidase (HRP); Or adopt Cyanuric Chloride to the immunoglobulin (Ig) activation; Reaction does not receive amino limitation; Can make full use of the nucleophilic group such as hydroxyl, sulfydryl of immunoglobulin (Ig), immunoglobulin (Ig) is tired has no influence, has overcome the shortcoming of glutaraldehyde activation and sodium periodate activation simultaneously; This preparation method need not other materials and participates in, and does not particularly have the participation of biotin, has avoided other materials and the interference of organizing the endogenous biotin; This preparation method is easy and simple to handle, and the time is shorter, is applicable to suitability for industrialized production.
Description of drawings
Fig. 1 is an anti-detection figure who resists the sheep anti mouse two anti-kits that are embodiment 9 for MMP-9, two.
Fig. 2 is an anti-detection figure who resists the goat-anti rabbit/mouse kit that is Thermo company for MMP-9, two.
Fig. 3 is an anti-detection figure who resists the goat-anti rabbit two anti-kits that are embodiment 13 for Histone H2B, two.
Fig. 4 is an anti-detection figure who resists the goat-anti rabbit/mouse kit that is Thermo company for Histone H2B, two.
Fig. 5 is an anti-detection figure who resists the goat-anti rabbit two anti-kits that are embodiment 13 for YAP, two.
Fig. 6 is an anti-detection figure who resists the goat-anti rabbit/mouse kit that is Thermo company for YAP, two.
Fig. 7 is for none resists, the detection figure of the goat-anti rabbit two anti-kit negative controls of the two anti-embodiment 13 of being.
Fig. 8 is anti-for none, the detection figure of two anti-goat-anti rabbit/mouse negative controls for Thermo company.
Fig. 9 is an anti-mouse IgG of being, two anti-enzyme-linked immunosorbent assays (ELISA) for the HRP mark sheep anti-mouse igg of embodiment 9 or the HRP of Millipore company mark sheep anti-mouse igg detect figure.
Figure 10 one anti-is rabbit igg, and two anti-enzyme-linked immunosorbent assays (ELISA) for the HRP mark goat anti-rabbit igg of embodiment 13 or the HRP of Millipore company mark goat anti-rabbit igg detect schemes.
Figure 11 is an anti-GAPGH of being, two anti-protein immunoblot experiments (Western Blot) for the HRP mark sheep anti-mouse igg of embodiment 9 or the HRP of Millipore company mark sheep anti-mouse igg detect figure.
Figure 12 is an anti-GAPGH of being, two anti-protein immunoblot experiments (Western Blot) for the HRP mark goat anti-rabbit igg of embodiment 13 or the HRP of Millipore company mark goat anti-rabbit igg detect figure.
Embodiment
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that the described content of embodiment only is used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
Agents useful for same:
1. sodium carbonate (traditional Chinese medicines)
2. Cyanuric Chloride (sigma company)
3. horseradish peroxidase (traditional Chinese medicines)
4. goat anti-rabbit immunoglobulin (self-control, affinity chromatography)
5. sheep anti mouse immunoglobulin (Ig) (self-control, affinity chromatography)
6. ultrafiltration pipe (Millipore)
7. glycerine (traditional Chinese medicines)
8. sodium chloride (traditional Chinese medicines)
9. sodium dihydrogen phosphate (traditional Chinese medicines)
10. sodium hydrogen phosphate (traditional Chinese medicines)
11. glucosan (GE company)
12. lowlenthal serum (self-control)
13. thimerosal (traditional Chinese medicines)
14. diaminobenzidine (sigma company)
15.SDS-polyacrylamide gel electrophoresis (our company's self-control)
16. skim milk (bright company)
17.PVDF film (Millipore company)
18. rabbit immunoglobulin G (self-control, affinity chromatography)
19. rat immune globulin G (self-control, affinity chromatography)
20. elisa plate (Thermo company)
21.Millipore the HRP of company mark goat anti-rabbit antibody (Millipore company)
22.Millipore the HRP of company mark sheep anti-mouse antibody (Millipore company)
23. sex change HeLa cell albumen (self-control)
24.GAPDH one anti-(Bioworld company)
25. binding buffer liquid (self-control, the 0.1mol/L aqueous sodium carbonate of pH9.4)
26. level pad (self-control, the 0.01mol/L aqueous sodium carbonate of pH9.6)
The preparation of embodiment 1:CC-HRP-IgG.
The activation of A:HRP
1) use 1mL cooling binding buffer liquid to dissolve the horseradish peroxidase (HRP) of 6mg, said cooling binding buffer liquid is the 0.1mol/L aqueous sodium carbonate.
2) the HRP solution with dissolving joins in the test tube that contains 6mg Cyanuric Chloride (CC), and little commotio cordis makes Cyanuric Chloride (CC) be suspended in the liquid environment fully.
3) be put on the horizontal shaking table, 0 ℃-4 ℃ concussion reactions are no less than 4 hours.
4) filter residual solid-state Cyanuric Chloride (CC) in the removal liquid, get reactant liquor.
5) utilize the PD-10 gravity post prepare, add the 1mL reactant liquor at every turn, each milliliter changes collection tube one time, tests the absorbance of every pipe, and when effluent is brown, absorbance met or exceeded 2 o'clock, collected this liquid, was the horseradish peroxidase solution of activation; In addition, can use 1 liter of the binding buffer liquid of cooling, 4 ℃ of dialysis 16 hours, liquid is changed once in the centre, and the solution of this moment is the horseradish peroxidase solution of activation; Yet utilize PD-10 gravity post, desalination is more thorough sooner.
B: the preparation of specific IgG
6) use 1 liter of cold junction to close the IgG 16 hours behind the damping fluid dialysis affinity purification, liquid is changed once in the centre, and concentrates with polyglycol and to make final concentration reach 6mg/mL.
C: the horseradish peroxidase of activation combines with specific IgG
7) the IgG solution 1mL (6mg) that gets step 6 joins in the ultrafiltration pipe (Millipore) with the horseradish peroxidase solution of the activation of 1mL, adds the cooling binding buffer liquid of 1mL simultaneously.
8) 0 ℃-4 ℃; 5000rpm is centrifugal, makes volume be concentrated to approximately
9) repeating step 8; Making volume be concentrated to pact
is concentrate; Make bond purer, concentration is higher.
10) take out concentrate in the EP pipe, put into 25 ℃ of-40 ℃ of incubators and leave standstill 16h.
11) liquid is joined in the ultrafiltration pipe once more; The binding equilibrium damping fluid that adds 1mL, 0 ℃ of-4 ℃ of 5000rpm centrifugal to final volume be
12) repeat 11 steps; Make bond purer; Concentration is higher; Be CC-HRP-IgG solution, preserve in-20 ℃ of packing behind adding
glycerine mixing.
The preparation of embodiment 2:CC-HRP-IgG.
The activation of A:IgG
1) use the IgG of 1mL cooling binding buffer liquid dissolving 6mg affinity purification or use 1 liter of cold junction to close damping fluid dialysis affinity purification after IgG16 hour, liquid is changed once in the centre, and concentrates with polyglycol and to make final concentration reach 6mg/mL.
2) above-mentioned IgG is put into the pipe that contains 6mg Cyanuric Chloride (CC), little commotio cordis makes Cyanuric Chloride (CC) be suspended in the liquid environment fully.
3) be put on the horizontal shaking table, 0 ℃-4 ℃ concussion reactions are no less than 4 hours.
4) use the method for filtering to remove residual solid-state Cyanuric Chloride (CC) in the liquid.
5) utilize the PD-10 gravity post prepare, add the IgG solution of 1mL activation at every turn, each milliliter changes collection tube one time, tests the absorbance of every pipe, and when effluent is brown, absorbance met or exceeded 2 o'clock, collected this liquid, was the immunoglobulin G of activation; Or use cold junction to close 1 liter of damping fluid, and to dialyse 16 hours for 0 ℃-4 ℃, liquid is changed once in the centre, is the immunoglobulin G of activation.
The preparation of B:HRP
6) use 1mL to cool off the HRP that binding buffer liquid dissolves 6mg, concentration 6mg/mL.
C: the immunoglobulin G of activation combines with HRP's
7) immunoglobulin G solution 1mL and the HRP solution (6mg) of 1mL of getting the activation of 5 steps join in the ultrafiltration pipe, add the cooling binding buffer liquid (mass ratio of the immunoglobulin G solution of HRP solution and activation is 1:1) of 1mL simultaneously.
8) 0 ℃-4 ℃; 5000rpm is centrifugal; Make that volume-diminished arrives approximately
and probably need that time of 20 minutes, (centrifugal speed can be decided by actual conditions; Speed is big more, and centrifugation time is short more).
9) repeating step 8 once and makes the volume of end reaction approximately be
10) take out liquid in the EP pipe, put into 25 ℃ of-40 ℃ of incubators and leave standstill 16h.
11) join liquid in the concentration tube once more; The binding equilibrium damping fluid that adds 1mL; 0 ℃-4 ℃, 5000rpm centrifugal to final volume be
12) repeating step 11 once; Add
glycerine simultaneously; Preserve in-20 ℃ of packing behind the mixing, promptly get CC-HRP-IgG.
13) above-mentioned two kinds of operations can enlarge reaction system simultaneously.
The preparation of embodiment 3 CC-HRP-IgM
The preparation process of present embodiment and relevant parameter and embodiment 2 are basic identical, and different is: use immunoglobulin M to prepare CC-HRP-IgM as reactant; The mass ratio of said Cyanuric Chloride, horseradish peroxidase and IgM is 1:0.1:0.1; In the step 3), temperature of reaction is 8 ℃, and the reaction time is 2h; In the step 10), temperature of reaction is 40 ℃, and the reaction time is 12h.
The preparation of embodiment 4 CC-HRP-IgM
The preparation process of present embodiment and relevant parameter and embodiment 1 are basic identical, and different is: use immunoglobulin M to prepare CC-HRP-IgM as reactant; The mass ratio of said Cyanuric Chloride, horseradish peroxidase and IgM is 1:10:1; In the step 3), temperature of reaction is 0 ℃, and the reaction time is 8h; In the step 10), temperature of reaction is 25 ℃, and the reaction time is 24h.
The preparation of embodiment 5 CC-HRP-Fab
The preparation process of present embodiment and relevant parameter and embodiment 1 are basic identical, and different is: use unit price immunoglobulin fragment Fab to prepare CC-HRP-Fab as reactant; The mass ratio of said Cyanuric Chloride, horseradish peroxidase and Fab is 1:5:10; In the step 3), temperature of reaction is 8 ℃, and the reaction time is 2h; In the step 10), temperature of reaction is 40 ℃, and the reaction time is 12h.
The preparation of embodiment 6 CC-HRP-Fab
The preparation process of present embodiment and relevant parameter and embodiment 2 are basic identical, and different is: use unit price immunoglobulin fragment Fab to prepare CC-HRP-Fab as reactant; The mass ratio of said Cyanuric Chloride, horseradish peroxidase and Fab is 1:5:1; In the step 3), temperature of reaction is 0 ℃, and the reaction time is 8h; In the step 10), temperature of reaction is 25 ℃, and the reaction time is 24h.
Embodiment 7 CC-HRP-F (ab)
2Preparation
The preparation process of present embodiment and relevant parameter and embodiment 2 are basic identical, and different is: use two valency immunoglobulin fragment F (ab)
2Prepare CC-HRP-F (ab) as reactant
2Said Cyanuric Chloride, horseradish peroxidase and F (ab)
2Mass ratio be 1:1:5; In the step 3), temperature of reaction is 8 ℃, and the reaction time is 2h; In the step 10), temperature of reaction is 40 ℃, and the reaction time is 12h.
Embodiment 8 CC-HRP-F (ab)
2Preparation
The preparation process of present embodiment and relevant parameter and embodiment 1 are basic identical, and different is: use two valency immunoglobulin fragment F (ab)
2Prepare CC-HRP-F (ab) as reactant
2Said Cyanuric Chloride, horseradish peroxidase and F (ab)
2Mass ratio be 1:10:10; In the step 3), temperature of reaction is 0 ℃, and the reaction time is 8h; In the step 10), temperature of reaction is 25 ℃, and the reaction time is 24h.
Embodiment 9 instant SABCs two anti-kit I.
Composition 1:HRP mark goat-anti IgG (CC-HRP-IgG), 10mL.Get the sheep anti mouse CC-HRP-IgG of labeling method preparation of the present invention; Be mixed with the solution of 6mg/L with the aseptic 0.05mol/L pH7.2 of 10mL phosphate buffer (PBS); Add 0.1g bovine serum albumin(BSA) (BSA); Add
l% thimerosal, mixing, 4-8 ℃ of preservation.
Composition 2: diaminobenzidine (DAB) concentrate, 1mL.Get the DAB solid and be dissolved in autoclaved ultrapure water, process the 50g/L concentrate, 4-8 ℃ keeps in Dark Place.
Composition 3: lowlenthal serum solution, 10mL.Remove normal goats serum after the centrifugal filtration; Be made into 10% working fluid with the aseptic 0.05mol/LpH7.2 phosphate buffer of 10mL (PBS); Add
1% thimerosal, 4-8 ℃ of preservation.
Composition 4:3% oxydol 10mL is with ultrapure water behind the high pressure and the preparation of 30% oxydol.
Composition 5: diaminobenzidine reaction substrate, i.e. 1% oxydol 10mL., with the PBS and the preparation of 30% oxydol of the preparation of the ultrapure water behind the high pressure.
Embodiment 10 instant SABCs two anti-kit II.
Composition 1:HRP mark goat-anti IgM (CC-HRP-IgM), 10mL.Get the sheep anti mouse CC-HRP-IgM of labeling method preparation of the present invention; Be mixed with the solution of 18mg/L with the aseptic 0.1mol/L pH7.4 of 10mL phosphate buffer (PBS); Add 0.05g bovine serum albumin(BSA) (BSA); Add
1% thimerosal, mixing, 4-8 ℃ of preservation.
Composition 2: diaminobenzidine (DAB) concentrate, 1mL.Get the DAB solid and be dissolved in autoclaved ultrapure water, process the 30g/L concentrate, 4-8 ℃ keeps in Dark Place.
Composition 3: lowlenthal serum solution, 10mL.Remove normal goats serum after the centrifugal filtration; Be made into 10% working fluid with the aseptic 0.1mol/LpH7.4 phosphate buffer of 10mL (PBS); Add
1% thimerosal, 4-8 ℃ of preservation.
Composition 4:10% oxydol 10mL is with ultrapure water behind the high pressure and the preparation of 30% oxydol.
Composition 5: the diaminobenzidine reaction substrate, 1% oxydol 10mL,, with the PBS and the preparation of 30% oxydol of the preparation of the ultrapure water behind the high pressure.
Embodiment 11 instant SABCs two anti-kit III.
Composition 1:HRP mark goat-anti Fab (CC-HRP-Fab), 10mL.Get the sheep anti mouse CC-HRP-Fab of labeling method preparation of the present invention; Be mixed with the solution of 12mg/L with the aseptic 0.01mol/L pH7.0 of 10mL phosphate buffer (PBS) dilution; Add 0.2g bovine serum albumin(BSA) (BSA); Add
1% thimerosal; Mixing, 4-8 ℃ of preservation.
Composition 2: diaminobenzidine (DAB) concentrate, 1mL.Get the DAB solid and be dissolved in autoclaved ultrapure water, process the 100g/L concentrate, 4-8 ℃ keeps in Dark Place.
Composition 3: lowlenthal serum solution, 10mL.Remove normal goats serum after the centrifugal filtration; Be made into 5% working fluid with the aseptic 0.01mol/LpH7.0 phosphate buffer of 10mL (PBS); Add
1% thimerosal, 4-8 ℃ of preservation.
Composition 4:7% oxydol 10mL is with ultrapure water behind the high pressure and the preparation of 30% oxydol.
Composition 5: the diaminobenzidine reaction substrate, 8% oxydol 10mL,, with the PBS and the preparation of 30% oxydol of the preparation of the ultrapure water behind the high pressure.
Embodiment 12 instant SABCs two anti-kit IV.
Composition 1:HRP mark goat-anti F (ab)
2(CC-HRP-F (ab)
2), 10mL.The sheep anti mouse CC-HRP-IgM that gets labeling method preparation of the present invention is mixed with the solution of 15mg/L; Dilute with the aseptic 0.08mol/L pH7.2 of 10mL phosphate buffer (PBS); Add 0.1g bovine serum albumin(BSA) (BSA); Add
1% thimerosal; Mixing, 4-8 ℃ of preservation.
Composition 2: diaminobenzidine (DAB) concentrate, 1mL.Get the DAB solid and be dissolved in autoclaved ultrapure water, process the 45g/L concentrate, 4-8 ℃ keeps in Dark Place.
Composition 3: lowlenthal serum solution, 10mL.Remove normal goats serum after the centrifugal filtration; Be made into 1% working fluid with the aseptic 0.08mol/LpH7.2 phosphate buffer of 10mL (PBS); Add
1% thimerosal, 4-8 ℃ of preservation.
Composition 4:7% oxydol 10mL is with ultrapure water behind the high pressure and the preparation of 30% oxydol.
Composition 5: the diaminobenzidine reaction substrate, 10% oxydol 10mL,, with the PBS and the preparation of 30% oxydol of the preparation of the ultrapure water behind the high pressure.
Embodiment 13 concentrated type SABCs two anti-kits.
Composition 1:HRP mark goat anti-rabbit igg (CC-HRP-IgG).Goat anti-rabbit igg-the HRP that gets labeling method preparation of the present invention is mixed with the solution of 12mg/L; Dilute with the aseptic 0.01M pH7.2 of 10mL phosphate buffer (PBS); Add 0.1g bovine serum albumin(BSA) (BSA); Add
1% thimerosal, mixing, 4-8 ℃ of preservation.
Composition 2: diaminobenzidine (DAB) concentrate, 1mL.Get the DAB solid and be dissolved in autoclaved ultrapure water, process the 30-100g/L concentrate, 4-8 ℃ keeps in Dark Place.
Composition 3:10% goat work serum, 10mL.Remove normal goats serum after the centrifugal filtration; (PBS) is made into 10% working fluid with phosphate buffer; Add
1% thimerosal, 4-8 ℃ of preservation.
Composition 4:3% oxydol 10mL is with the PBS and the preparation of 30% oxydol of the preparation of the ultrapure water behind the high pressure.
Composition 5: the diaminobenzidine reaction substrate, 3% oxydol 10mL,, with the PBS and the preparation of 30% oxydol of the preparation of the ultrapure water behind the high pressure.
Embodiment 14 utilizes SABC two anti-kits to detect.
Detection method:
1) gets and organize paraffin specimen, be cut into the 4-8um section, transfer to microslide with paraffin slicing machine;
2) through the xylene dewaxing, again through 100% alcohol, 95% alcohol, 75% alcohol and phosphate buffer (PBS) aquation;
3) repaired antigen in 5 minutes with 0.01M pH6.0 sodium citrate high pressure 3 minutes or micro-wave oven heating;
4) with hydrogen peroxide solution sealing 10 minutes, phosphate buffer (PBS) cleaned;
5) add the anti-of proper proportion dilution, hatch for 37 ℃ and hatched 12-18 hour in 1 hour or 4 ℃, phosphate buffer (PBS) cleans;
6) add goat work serum, sealed 10-20 minute, phosphate buffer (PBS) cleans (this link is used as required, if an anti-antibody diluent of using then need not seal);
7) add HRP mark sheep anti mouse or goat anti-rabbit igg, incubated at room 30 minutes or 37 ℃ 15 minutes, phosphate buffer (PBS) cleans; As use embodiment 13 said concentrated type kits to need 1:50-1:100 dilution back to use.
8) get the DAB concentrate, after 20 times-50 times of phosphate buffer (PBS) dilutions, add two DAB substrates, be added to after the mixing on the slide specimen, color development at room temperature, microscopically is observed the colour developing degree, with phosphate buffer (PBS) color development stopping.
Testing result:
Utilize MMP-9 (Millipore company) mouse source monoclonal antibody and YAP (Bioworld company), many anti-detections the in Histone H2B (Bioworld company) rabbit source.Sample behaviour source paraffin section.Positive control two anti-kits are goat-anti rabbit/mouse kit (Thermo company).
Testing result is seen accompanying drawing 1 to 9, wherein:
Fig. 1 is an anti-MMP-9 of being (1:400, PBS dilution), and two resist the detection figure for the sheep anti mouse two anti-kits of embodiment 9;
Fig. 2 is an anti-MMP-9 of being (1:400, PBS dilution), and two resist the detection figure for the goat-anti rabbit/mouse kit of Thermo company;
Fig. 3 is an anti-Histone H2B of being (1:50, PBS dilution), and two resist the detection figure for the goat-anti rabbit two anti-kits of embodiment 13;
Fig. 4 is an anti-Histone H2B of being (1:50, PBS dilution), and two resist the detection figure for the goat-anti rabbit/mouse kit of Thermo company;
Fig. 5 is an anti-YAP of being (1: 50, the PBS dilution), and two resist the detection figure for the goat-anti rabbit two anti-kits of embodiment 13;
Fig. 6 is an anti-YAP of being (1: 50, the PBS dilution), and two resist the detection figure for the goat-anti rabbit/mouse kit of Thermo company;
Fig. 7 is anti-for none, and two resist the detection figure for the goat-anti rabbit two anti-kit negative controls of embodiment 13;
Fig. 8 is anti-for none, and two resist the detection figure for the goat-anti rabbit/mouse negative control of Thermo company.
The result shows: the present invention does not have significant difference with positive goat-anti rabbit/mouse (Thermo company) two anti-kits in positive detection and negative testing process; And non-specific background is lower than contrast, and specificity is stronger.
Embodiment 15 ELISAs (ELlSA) experiment
1. antigen coated: with encapsulating damping fluid comlete antigen (mouse IgG) is diluted, add ELISA Plate with 100 μ l/ holes, 4 ℃ are spent the night, and general dilute concentration is 2 μ g/mL; Saidly encapsulate the 0.1mol/L aqueous sodium carbonate that damping fluid is pH 9.6.
2. sealing: take out ELISA Plate; Need not discard liquid in the hole; The skimmed milk power of preparation 75%, every hole
37 ℃ of incubation 1.5h:
3. washing: discard liquid in the hole,, drain then with washing from the beginning 12 times;
4. application of sample:
(1) Cyanuric Chloride activation HRP mark sheep anti-mouse antibody of the present invention detect by design add with the serial doubling dilution of PBS (1: 1000,1: 2000,1: 4000; 1: 8000,1: 10000,1: 50000; 1: 100000) HRP mark sheep anti-mouse igg to be checked, negative control is done blank simultaneously; 100 μ l/ holes, 37 ℃ of 1h;
(2) commercially available HRP mark sheep anti-mouse antibody detect by design add with the serial doubling dilution of PBS (1: 1000,1: 2000,1: 4000; 1: 8000,1: 10000,1: 50000; 1: 100000) commercially available HRP mark sheep anti-mouse igg to be checked, negative control is done blank simultaneously; 100 μ l/ holes, 37 ℃ of 1h;
5. wash plate: discard liquid in the hole,, clap and do with washing from the beginning 12 times;
6. chromogenic reaction: add substrate solution (being that commercially available TMB uses liquid), 100 μ l/ holes, 37 ℃ of lucifuges reaction 15-20min;
7. stop: every hole adds stop buffer 50 μ l vibration mixing, and said stop buffer is 2mol/L H
2SO
4The WS;
8. measure: leave standstill the OD value that on ELIASA, reads the 450nm place behind the 3-5min;
Testing result is seen Fig. 9, can be known by Fig. 9, and identical extension rate absorbance is bigger; Dilution ratio can reach l: 5,000～l: 10, and OOO exceeds like product 50%-100%.
Embodiment 15 ELISAs experiments (ELlSA)
1. antigen coated: with encapsulating damping fluid comlete antigen (rabbit igg) is diluted, add ELISA Plate with 100 μ l/ holes, 4 ℃ are spent the night, and general dilute concentration is 2 μ g/mL;
2. sealing: take out ELISA Plate; Need not discard liquid in the hole; The skimmed milk power of preparation 75%, every hole
37 ℃ of incubation 1.5h;
3. washing: discard liquid in the hole,, drain then with washing from the beginning 12 times;
4. application of sample:
(1) the HRP mark goat anti-rabbit antibody of Cyanuric Chloride activation of the present invention detects by design and adds with PBS series doubling dilution (1:1000,1:2000,1:4000; 1:8000,1:10000,1:50000; 1:100000) HRP mark goat anti-rabbit igg to be checked, negative control is done blank simultaneously; 100 μ l/ holes, 37 ℃ of 1h;
(2) commercially available HRP mark goat anti-rabbit antibody detects by design and adds with PBS series doubling dilution (1:1000,1:2000,1:4000; 1:8000,1:10000,1:50000; 1:100000) commercially available HRP mark sheep anti-mouse igg to be checked (specially evading) at this, negative control is done blank simultaneously; 100 μ l/ holes, 37 ℃ of 1h;
5. wash plate: discard liquid in the hole,, clap and do with washing from the beginning 12 times;
6. chromogenic reaction: add substrate solution (being that commercially available TMB uses liquid), 100 μ l/ holes, 37 ℃ of lucifuges reaction 15-20min;
7. stop: every hole adds stop buffer 50 μ l, (2M H
2SO
4) the vibration mixing;
8. measure: leave standstill the OD value that on ELIASA, reads the 450nm place behind the 3-5min;
Testing result is seen Figure 10, can be known by Figure 10, and identical extension rate absorbance is bigger; Dilution ratio can reach 1:5,000 ~ 1:10, and 000, exceed like product 50%-100%.
Embodiment 16 protein immunoblots (Western Blot) experiment
1. get 10-20ug sex change HeLa cell albumen, be added on the SDS-polyacrylate hydrogel for preparing in advance an appearance hole and carry out electrophoresis experiment, 80 volts of voltages continue to change 110 volts after 15 minutes, lasting 35 minutes;
2. will run good protein gel and transfer to the film groove, change the film experiment, the albumen in the gel will be transferred on the pvdf membrane with pvdf membrane; 0.35 ampere in electric current continues 2 hours;
3. after changeing film and accomplishing, sealed 1 hour with the skimmed milk power of massfraction 5%;
4. add GAPDH antibody with the dilution of 5% skimmed milk power to pvdf membrane, hatched 12-16 hour for 4-8 ℃;
5. with PBS lotion three times, each 3 minutes;
6. the sheep anti-mouse igg that adds the HRP mark of Cyanuric Chloride activation of the present invention is (with PBS dilution, dilute concentration 1:1000,1:2000,1:4000; 1:8000,1:10000 1:20000), uses certain the brand HRP of company mark sheep anti-mouse igg (to dilute with PBS simultaneously; Dilute concentration 1:1000,1:2000,1:4000; 1:8000,1:10000,1:20000); Incubated at room 1 hour;
7. it is inferior to give a baby a bath on the third day after its birth with PBS, after each 3 minutes, takes pictures in the observation of protein nucleic acid imager.
Testing result is seen Figure 11, can know by Figure 11, and same dilution ratio, the present invention's imaging is more obvious.
Embodiment 17 protein immunoblots (Western Blot)
1. get 10-20ug sex change HeLa cell albumen, be added on the SDS-polyacrylate hydrogel for preparing in advance an appearance hole and carry out electrophoresis experiment, 80 volts of voltages continue to change 110 volts after 15 minutes, lasting 35 minutes;
2. will run good protein gel and transfer to the film groove, change the film experiment, the albumen in the gel will be transferred on the pvdf membrane with pvdf membrane; 0.35 ampere in electric current continues 2 hours;
3. after changeing film and accomplishing, sealed 1 hour with the skimmed milk power of massfraction 5%;
4. add GAPDH antibody with the dilution of 5% skimmed milk power to pvdf membrane, hatched 12-16 hour for 4-8 ℃;
5. with PBS lotion three times, each 3 minutes;
6. the goat anti-rabbit igg that adds the HRP mark of Cyanuric Chloride activation of the present invention is (with PBS dilution, dilute concentration 1:1000,1:2000,1:4000; 1:8000,1:10000 1:20000), uses certain the brand HRP of company mark goat anti-rabbit igg (to dilute with PBS simultaneously; Dilute concentration 1:1000,1:2000,1:4000; 1:8000,1:10000,1:20000); Incubated at room 1 hour;
7. it is inferior to give a baby a bath on the third day after its birth with PBS, after each 3 minutes, takes pictures in the observation of protein nucleic acid imager.
Testing result is seen Figure 12, can know by Figure 12, and same dilution ratio, the present invention's imaging is more obvious.
Claims (10)
1. SABC two anti-kits is characterized in that: comprise the horseradish peroxidase-labeled immunoglobulin (Ig) of Cyanuric Chloride activation, i.e. CC-HRP-Ig;
Said CC-HRP-Ig is made by following method:
(1) activation: Cyanuric Chloride and horseradish peroxidase or immunoglobulin (Ig) reaction get the horseradish peroxidase of activation or the immunoglobulin (Ig) of activation;
(2) coupling: the horseradish peroxidase of said activation and immunoglobulin (Ig) coupling reaction get CC-HRP-Ig, or the immunoglobulin (Ig) of said activation and horseradish peroxidase react CC-HRP-Ig;
Reaction equation is following:
2. a kind of SABC two anti-kits according to claim 1 is characterized in that: said immunoglobulin (Ig) is immunoglobulin G, unit price immunoglobulin fragment Fab, two valency immunoglobulin fragment F (ab)
2Or immunoglobulin M, the mass ratio of said Cyanuric Chloride, horseradish peroxidase and immunoglobulin (Ig) is 1: (0.1-10): (0.1-10).
3. a kind of SABC two anti-kits according to claim 1 is characterized in that: in the step (1), said temperature of reaction is 0 ℃-8 ℃, and the reaction time is 2-8h.
4. a kind of SABC two anti-kits according to claim 1 is characterized in that: in the step (2), said temperature of reaction is 25 ℃-40 ℃, reaction time 12h-24h.
5. a kind of SABC two anti-kits according to claim 1; It is characterized in that: said CC-HRP-Ig exists in solution; Said solution comprises following component: 6-18mg/L CC-HRP-Ig; The 5-20g/L bovine serum albumin(BSA), the 0.01-0.1mol/L phosphate buffer of 0.05-0.1g/L thimerosal and pH 7.0-7.4.
6. a kind of SABC two anti-kits according to claim 1; It is characterized in that: kit also comprises lowlenthal serum solution; Said lowlenthal serum solution comprises: concentration expressed in percentage by volume is the lowlenthal serum of 1-10%, the 0.01-0.1mol/L phosphate buffer of 0.05-0.1g/L thimerosal and pH 7.0-7.4.
7. a kind of SABC two anti-kits according to claim 1 is characterized in that: kit comprises that also concentration expressed in percentage by volume is the aqueous hydrogen peroxide solution of 3-10%.
8. a kind of SABC two anti-kits according to claim 1, it is characterized in that: kit also comprises diaminobenzidine solid or diaminobenzidine concentrate, the concentration of said diaminobenzidine concentrate is 30-100g/L.
9. a kind of SABC two anti-kits according to claim 1, it is characterized in that: kit also comprises the diaminobenzidine reaction substrate, said diaminobenzidine reaction substrate is that concentration expressed in percentage by volume is the 1-10% aqueous hydrogen peroxide solution.
10. the preparation method of the described SABC two anti-kits of claim 1 is characterized in that: may further comprise the steps:
(1) activation: Cyanuric Chloride and horseradish peroxidase or immunoglobulin (Ig) reaction get the horseradish peroxidase of activation or the immunoglobulin (Ig) of activation;
(2) coupling: the horseradish peroxidase of said activation and immunoglobulin (Ig) coupling reaction get CC-HRP-Ig, or the immunoglobulin (Ig) of said activation and horseradish peroxidase react CC-HRP-Ig;
(3) preparation of SABC two anti-kits: utilize said CC-HRP-Ig to prepare SABC two anti-kits; Reaction equation is following:
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| CN102380347A (en) * | 2010-09-06 | 2012-03-21 | 江南大学 | Cholesterol oxidase affinity matrix, synthesis and large-scale purification method thereof for cholesterol oxidase |
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| CN101081873A (en) * | 2007-07-09 | 2007-12-05 | 中北大学 | Modified starch using cyanuric chloride as linking radical and preparation method thereof |
| CN101424691A (en) * | 2008-12-15 | 2009-05-06 | 北京大学第三医院 | Infiltrative breast carcinoma recurring risk assessment kit |
| CN102380347A (en) * | 2010-09-06 | 2012-03-21 | 江南大学 | Cholesterol oxidase affinity matrix, synthesis and large-scale purification method thereof for cholesterol oxidase |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103116029A (en) * | 2013-01-29 | 2013-05-22 | 福州迈新生物技术开发有限公司 | Determining method for sensitivity and affinity of second antibody color appearance system for immunohistochemistry |
| CN103116029B (en) * | 2013-01-29 | 2015-01-21 | 福州迈新生物技术开发有限公司 | Determining method for sensitivity and affinity of second antibody color appearance system for immunohistochemistry |
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