CN106932567A - A kind of preparation method of the immune targeted nano gold of recombinant protein A mediation - Google Patents
A kind of preparation method of the immune targeted nano gold of recombinant protein A mediation Download PDFInfo
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Abstract
The preparation method of the immune targeted nano gold of recombinant protein A mediation, a nanometer gold surface is fixed on by the use of Chong ZuProteinA G as affinity ligand by antibody, forms immune nano Au composite.The compound is made up of antibody, recombinant protein A and the part of nm of gold three, contain a C-terminal cysteine after recombinant protein A is engineered, can single site be coupled in nm of gold, reduce steric hindrance, the binding ability with antibody is increased again, Fc sections of antibody is specifically bound by non-covalent bond afterwards, obtains acting on more preferable antibody directional effect than physical absorption and chemical bonding.The method can prepare the multicomponent of complexity, multi-functional compound targeting immune nanometer gold to meet different demands.Immune targeted nano gold prepared by the present invention, not only with atopic and stability, also with stronger antijamming capability and environment resistance, has good application prospect in fields such as target tracing, bio-pharmaceuticals and accurate treatments.
Description
Technical field
The present invention relates to nm of gold and antibody coupling technology, more particularly to using antibody Fc section and self-assembled nanometer gold surface
Recombinant protein A (ProteinA G) specific binding prepare immune targeted nano gold.
Background technology
In recent years, with the development of nanometer technology, applications to nanostructures is increasingly becoming the emphasis of research.Nm of gold conduct
The important member of metal nano material, except itself has unique physicochemical properties, is also equipped with small-size effect, surface effect
Should with the property such as macro quanta tunnel effect, therefore, in the field such as nm of gold is detected in material, biology sensor and immune targeting
Show great potentiality.Targeting field is particularly immunized, as stablizing, can mark, the carrier that biological safety is high, nm of gold
Needs are combined with targeting substance could obtain targeting, can combine to form the anti-of antigen-antibody complexes with antigentic specificity
Body is optimal selection.
The method of the immune targeted nano gold of connection at present mainly includes physical absorption and chemical bonding：
(1) physical absorption
Physical absorption is connection antibody most simple effective method.In physical adsorption process, antibody mainly passes through hydrophobic work
With or Hydrogenbond electrostatic interaction be connected hydrophilic or hydrophobic carrier surface (referring to Fig. 1).
(2) it is chemically bonded
Antibody and carrier surface some specific functional groups (amino, sulfydryl, carboxyl and aldehyde radical etc.) form chemical bond to be connected
Antibody.For example using silane coupler APTES (3-aminopropytruethoxysilane
APTES) amino is produced to be covalently attached in the form of amido link with the carboxyl of antibody in nanometer gold surface；Using glutaraldehyde one end
Aldehyde radical forms covalent bond and is connected nm of gold with amino, and another terminal aldehyde group forms the covalent bond of stabilization with the amino of antibody；Using also
Former agent (dredging base ethanol or dithiothreitol (DTT)) is by the sulfydryl in antibody disulfide bond and the maleimide knot of nm of gold surface modification
Close；In N- hydroxysuccinimides (N-Hydroxysuccinimide, NHS) and 1- (3- dimethylamino-propyls) -3- ethyl carbon
Under conditions of diimmonium salt hydrochlorate (1-Ethyl-3- (3 '-dimethylaminopropyl) carbodiimide, EDC) is present,
Peptide bond is formed using the amino and the carboxyl for being connected to nanometer gold surface of antibody lysine side-chain (referring to Fig. 2).
Above-mentioned common method can synthesize immune targeted nano gold, but have the shortcomings that different degrees of:Physical absorption
Method nm of gold can directly adsorb antibody by electrostatic interaction, but uncontrollable antibody and nm of gold binding site, it may appear that
Connection antibody heterogeneity, bioactivity is low, easily come off from nanometer gold surface；Chemical bonding is because above-mentioned functional group is in antibody
Heavy chain and light chain are distributed, and the chemical bond formed by random incorporation can equally reduce the bioactivity and load-carry duty of antibody.
Therefore the effective load-carry duty for how improving nm of gold in actual applications is the key for improving targeted nano gold efficiency and effect.
The content of the invention
The purpose of the present invention is to overcome deficiencies of the prior art, there is provided a kind of recombinant protein A (ProteinA
G) the preparation method of the immune targeted nano gold of mediation, using antibody Fc section and nanometer gold surface Chong ZuProteinA G specificity
Combine to form targeting immune nanometer gold system, it is ensured that antigenic determinant fully exposes Antibody Fab fragment, substantially increases antibody surface
Load-carry duty, with good reaction stability and specificity.
ProteinA G be respectively aureus cell wall with the composition of the cell surface protein of streptococcus G races into
Point, containing IgG antibody land, can be combined (referring to figure with the Fc fragments in people and various mammalian blood serum IgG molecules
3).Contain a C-terminal cysteine after Chong ZuProteinA G are engineered, can be with single site combination nm of gold, so as to drop
Low steric hindrance, increased the binding ability (referring to Fig. 3) of nm of gold and IgG.Further, since ProteinA lack Guang in G
Propylhomoserin and cysteine, it is impossible to form disulfide bond, it is ensured that ProteinA G can specifically bind Fc sections of antibody and compare physics
Absorption obtains more preferable antibody directional effect with chemical bond and effect, is expected to turn into the immune targeted nano gold antibody load-carry duty of solution
With the key of the low problem of bioactivity.
Technical scheme
The preparation method of the immune targeted nano gold of the recombinant protein A (ProteinA G) mediation is comprised the steps of:
1st step, the preparation of nm of gold
During nm of gold is prepared with trisodium citrate reduction method, by all glass apparatus with the chloroazotic acid of new configuration
(HNO3:HCL,1:3) clean, redistilled water is rinsed, and uses preceding drying.
1.1st step, 0.01% gold chloride 50-100ml is added in conical flask it is placed on heating magnetic stirring apparatus, it is fixed
Reaction temperature is 95-120 DEG C, mixing speed 300-400r/min.
The preparation of the 1.2nd step, 20nm nm of gold or 40nm nm of gold
1.2.1 steps, preparation 20nm nm of gold (referring to Fig. 4)：1% is rapidly added in solution described in above-mentioned 1.1st step
Trisodium citrate 0.75-1.5ml, the solution seethed with excitement after 25s begins to change into micro- blueness, and (about after 70s) treats that blueness suddenly becomes fresh
Red, continues agitating heating and boils 10-15min, and reaction is fully completed when golden, and solution is gradually stable into Chinese red or claret,
Now stop heating continuing to stir 15-20min, distilled water constant volume 50.0-100mL, 2-8 DEG C of preservation are used after naturally cooling to room temperature
(referring to Fig. 6)；
1.2.2 steps, preparation 40nm nm of gold (referring to Fig. 5)：1% trisodium citrate 0.5- is rapidly added in above-mentioned solution
The solution seethed with excitement after 1ml, 25s begins to change into micro- blueness, and (about after 120s) treats that blueness suddenly becomes cerise；Continue agitating heating
10-15min is boiled, reaction is fully completed when golden, solution is gradually stable into Chinese red or claret, now stopped heating and continue to stir
15-20min is mixed, is preserved (referring to Fig. 7) with distilled water constant volume 50.0-100.0mL, 2-8 DEG C after naturally cooling to room temperature；
2nd step, self assembly rPA G
2.1st step, with 0.01M phosphate buffers (pH=7.2-7.4) by rPA G be formulated as concentration and be
1mg/ml solution；
2.2nd step, ensure the 1st step prepare nano-Au solution PH=7.0-8.0 under conditions of, by rPA G
Self assembly on the surface of nanogold particle, by volume 1:The rPA that 10-20 prepares the 2.1st step G solution delay
In the slow nano-Au solution prepared added to the 1st step, mixed liquor is mixed using rotary incubator, and rotating speed keeps as the case may be
10-20 turns/min, and 4 DEG C overnight；
13000 turns/min is centrifuged three times after the completion of 2.3rd step, reaction, each 15min, topples over supernatant removal unassembled
ProteinA G, precipitation with 1ml 0.01M phosphate buffers (pH=7.2-7.4) dissolve, be configured to rPA G repair
The nano-Au solution of decorations simultaneously ensures that solution recovers deep claret；
3rd step, the connection of antibody Fc end
Take the preparation of the 2nd step rPA G modifications nano-Au solution 1ml, be slowly added to appropriate or excessive antibodies
(antibody addition 100-500ug), mixed liquor using rotary incubator mix, as the case may be rotating speed keep 10-20 turn/
Min, 4 DEG C of shaken overnights, 13000 turns/min is centrifuged three times after the completion of reaction, each 15min, topples over supernatant and removes excessive antibodies,
Precipitation is resuspended with 1ml 0.01M phosphate buffers (pH=7.2-7.4), is configured to connect the immune targeted nano gold solution of antibody
And ensure that solution recovers deep claret (referring to Fig. 8-9)；
The storage and detection of the 4th step, immune targeted nano gold
The bovine serum albumin(BSA) that 100ul concentration is 10% is added in immune targeted nano gold solution prepared by the steps of 1ml the 3rd
(BSA), on the one hand closing nanometer gold surface is not associated with site, eliminates immune non-specific binding reaction；On the other hand ensure to exempt from
The antibody of epidemic disease targeted nano gold is not degraded.After 4 DEG C of vibration 1-2h of mixed liquor, can be preserved at 2-8 DEG C long-term；Use preceding 13000
Turn/min centrifugation 15min, precipitation 1ml 0.01M phosphate buffers (pH=7.0) is resuspended after toppling over supernatant, it is ensured that solution recovers
Be can be used after deep claret, detection antibody combination situation is tested using immune impression using preceding (referring to Figure 10).
The advantages of the present invention：
Immune targeted nano gold prepared by the present invention can be coupled in nm of gold with single site, that is, reduce space bit
Resistance, increased the binding ability with antibody again, specifically bind Fc sections of antibody by non-covalent bond afterwards, obtain being inhaled than physics
Echo chemical bonding and act on more preferable antibody directional effect.The preparation method can prepare many of complexity according to concrete application situation
Component, multi-functional compound targeting immune nanometer gold meet different demands.Immune targeted nano gold prepared by the present invention, no
Only there is atopic and stability, also with stronger antijamming capability and environment resistance, in target tracing, biological system
The field such as medicine and accurate treatment has good application prospect.
Brief description of the drawings
Fig. 1 nm of gold physical absorption antibody schematic diagrames.
Under NHS and ECD existence conditions, nm of gold connects antibody schematic diagram to Fig. 2 by carboxyl functional group.
Fig. 3 rPAs G mediations the schematic diagram that is combined with antibody of nm of gold.
Fig. 4 trisodium citrate reduction methods prepare the perspective electron microscope of diameter 20nm nm of gold.
Fig. 5 trisodium citrate reduction methods prepare the perspective electron microscope of diameter 40nm nm of gold.
The measure of Fig. 6 20nm nano-Au solution gradient dilution standard curves, ultraviolet-visible spectrum detection nano-Au solution
Ultraviolet absorption peak in 520nm, with absorbance as ordinate, with wavelength as abscissa, standard curve：Y=-0.083Xc+
0.442(R2=0.993).
The measure of Fig. 7 40nm nano-Au solution gradient dilution standard curves, ultraviolet-visible spectrum detection nano-Au solution
Ultraviolet absorption peak in 520nm, with absorbance as ordinate, with wavelength as abscissa, standard curve Y=-0.068Xc+0.377
(R2=0.989).
Fig. 8 rPAs G mediation 20nm nm of gold connect SPACA3, SPAG8 and OR1D2 Antibody preparation respectively
Immune targeted nano gold, the ultraviolet absorption peak of ultraviolet-visible spectrum detection nano-Au solution is in 528nm, antibody ultraviolet absorption peak
In 277nm.
Fig. 9 rPAs G mediation 40nm nm of gold connect SPACA3, SPAG8 and OR1D2 Antibody preparation respectively
Immune targeted nano gold, the ultraviolet absorption peak of ultraviolet-visible spectrum detection nano-Au solution is in 529nm, antibody ultraviolet absorption peak
In 277nm.
The immune impression experiments of Figure 10 carry situation using the antibody of the immune targeted nano gold of anti-igg antibody detection.1st, 3,5 swimming
Road is successively polyclonal SPACA1, SPAG8 and OR1D2 antibody of rabbit igg, and applied sample amount 25ug, 2,4,6 swimming lanes are respectively 0.5ml knots
Close the protein extract of the immune targeted nano gold solution of polyclonal SPACA3, SPAG8 and OR1D2 antibody of rabbit igg.
Specific embodiment
Embodiment 1：RPA G mediations the 20nm nm of gold immune targeted nano of connection SPACA3 Antibody preparations
Gold
Immune targeted nano gold system preparation method, step is as follows:
Antibody Fc section to form targeting immune nanometer gold system by being specifically bound with nanometer gold surface Chong ZuProteinA G
System, the preparation method of the immune targeted nano gold is comprised the steps of:
1st, the preparation of 20nm nm of gold:All glass apparatus are equal during preparing nm of gold with trisodium citrate reduction method
With the chloroazotic acid (HNO3 of new configuration:HCL,1:3) clean, redistilled water is rinsed, and uses preceding drying.By 0.01% gold chloride 50ml
It is added in conical flask and is placed on heating magnetic stirring apparatus, fixed reaction temperature is 95 DEG C, mixing speed 300r/min.Above-mentioned
1% trisodium citrate 0.75ml is rapidly added in solution, the solution seethed with excitement after 25s begins to change into micro- blueness, it is blue prominent after about 70s
So become cerise.Continue agitating heating and boil 10min, reaction is fully completed when golden, solution is gradually stable into Chinese red or wine
Red, now stops heating and continues to stir 15-20min, and distilled water constant volume 50.0mL, 2-8 DEG C of guarantor are used after naturally cooling to room temperature
Deposit.Using a diameter of 20nm of transmission electron microscope observing nm of gold (referring to Fig. 4)；20nm nano-Au solutions are in ultraviolet-visible spectrum
Absworption peak is 520nm (referring to Fig. 6)；
2nd, self assembly rPA G:With 0.01M phosphate buffers (pH=7.2-7.4) by rPA G
Concentration is formulated as 1mg/ml solution, it is ensured that under conditions of nano-Au solution PH=7.0-8.0, by rPA G from group
Mounted in the surface of nanogold particle, concrete operations are：Take 100ul rPAs G solution be added slowly to 1ml nm of gold
In solution, mixed liquor is mixed using rotary incubator, and rotating speed keeps 10-20 to turn/min as the case may be, and 4 DEG C overnight；Reaction
After the completion of 13000 turns/min be centrifuged three times, each 15min, topple over supernatant remove unassembled ProteinA G, precipitation uses 1ml
0.01M phosphate buffers (pH=7.2-7.4) dissolve, be configured to rPA G modification nano-Au solution and ensure molten
Liquid recovers deep claret；
3rd, antibody Fc end connection:Take rPA G modification nano-Au solution 1ml, be slowly added to appropriate or excessive
SPACA3 antibody (antibody addition 100-500ug), mixed liquor is mixed using rotary incubator, and rotating speed keeps as the case may be
10-20 turns/min, 4 DEG C of shaken overnights, 13000 turns/min is centrifuged three times after the completion of reaction, each 15min, topples over supernatant removing
Excessive antibodies, precipitation is resuspended with 1ml 0.01M phosphate buffers (pH=7.2-7.4), is configured to connect the immune targeting of antibody
Nano-Au solution simultaneously ensures that solution recovers deep claret, and the immune targeted nano gold solution for connecting SPACA3 antibody exists respectively
The visible nm of gold of 528nm and 277nm and antibody ultraviolet absorption peak (referring to Fig. 8)；
4th, the storage and detection of targeted nano gold is immunized:
The storage of 4.1 immune targeted nano gold：It is 10% to add 100ul concentration in the immune targeted nano gold solutions of 1ml
Bovine serum albumin(BSA) (BSA), on the one hand the uncombined site of closing nanometer gold surface, eliminates immune non-specific binding reaction；
On the other hand ensure that the antibody of targeting immune nanometer gold is not degraded.After 4 DEG C of vibration 1-2h of mixed liquor, can be protected at 2-8 DEG C long-term
Deposit；15min is centrifuged using preceding 13000 turns/min, precipitation 1ml 0.01M phosphate buffers (pH=7.0) weight after supernatant is toppled over
It is outstanding, it is ensured that solution can be used after recovering deep claret.
4.2 using protein immunoblot experiment detection antibody joint efficiency (referring to Figure 10).
Protein immunoblot specific experiment method is：Western Blot experimental techniques
4.2.1 the Protein Extraction of targeted nano gold and antibody-solutions is immunized
1) take 0.5ml connect SPACA3 antibody immune targeted nano solution, 13000 turns/min centrifugation 15min, topple on
After clear, precipitation 4 × SDS-PAGE albumen sample-loading buffers 25ul shakes abundant crack protein；25ul SPACA3 antibody is molten
Liquid (concentration is 1mg/ml) and 4 × SDS-PAGE albumen sample-loading buffer by volume (3：1) the abundant crack protein of mixing concussion,
95 DEG C of 5min of heating, -20 DEG C of preservations are stand-by.
4.2.2 main agents are prepared
4.2.2.1 the preparation of separation gel buffer solution 1.5mmol/LTris-HCL (pH8.8)
Weigh 18.15gTris to be dissolved in 40ml deionized waters, mixed with 1mol/LHCL and be adjusted to pH8.8, plus deionized water
It is diluted to 100ml final volumes.4 DEG C of preservations after filtering.
4.2.2.2 the preparation of concentration glue buffer solution 0.5mmol/LTris-HCL (pH6.8)
Weigh 6.05g Tris to be dissolved in 40ml water, mixed with 1mol/LHCL and be adjusted to pH6.8, plus deionized water is diluted to
100ml final volumes.4 DEG C of preservations after filtering.
4.2.2.3 the preparation of 10% separation gel
4.2.2.4 the preparation of 5% concentration glue
4.2.2.5 the preparation of 5 × Tris- glycine running buffers
Tris 30.0g, glycine 140.0g, SDS 5.0g are weighed, plus ddH20 is settled to 1000ml.
4.2.2.6 the preparation of 10 × transferring film buffer solution
Weigh Tris 30.3g, glycine 144.1g, ddH20 and be settled to 1600ml.When using, plus 400ml methyl alcohol is (overall
Product 2L), then it is diluted to 1 × transferring film buffer solution.
4.2.2.7 the preparation (5 ×) of sample-loading buffer：4 DEG C of preservations
Finally plus ddH2O is settled to 200ml.
4.2.2.8 the preparation of TBST
10×TBS：Tris 24.2g, NaCl 80g, add water and are settled to 1L, pH 7.6.10 × TBS is diluted to 1 ×
TBS, 1000mlTBS+0.5mlTween-20=TBST
4.2.2.9 5% skim milk：(- 20 DEG C of preservations)
General every time to prepare 30ml, 1.5g skim milk+30mlTBST, stirring fully can be used for sealer.
4.2.3 Western Blot experimental procedures
4.2.3.1 the configuration of PAGE gel
PAGE gel need to be prepared protein electrophoresis the previous day is carried out, prepare 8% or 10% separation gel, 8 milliliters of encapsulating
Left and right, absolute ethyl alcohol sealing.It is gelled after about one hour and (can solidify whether glue during situation determine plate coagulates according to remaining separation gel admittedly
Gu), ethanol is removed, 5 milliliters of 5% spacer gel is prepared, encapsulating to plate is filled, and inserts 1.5mm Teflon combs, is gathered within 30-40 minutes
(can solidify whether glue during situation determines plate solidifies according to remaining separation gel) after conjunction, be placed in deionized water and be put in 4 degrees Celsius
Refrigerator；
4.2.3.2 prefabricated gel is placed on electrophoresis apparatus, adds 1 × electrophoretic buffer, used after careful extraction comb
Suction pipe blows away collodion silk, and 3ul marker, 33.3ul SPACA3 antibody lysate and 20ul connection are separately added into swimming lane
The protein lysate of the immune targeted nano gold of SPACA3 antibody, constant pressure 60V carries out electrophoresis, after albumen marker separates band
High voltage stops electrophoresis to 90V when observation Loading buffer are close to plate bottom；
4.2.3.3 pvdf membrane is cut according to electrophoresis road number, makes film size slightly larger compared with protein sample road, cut
Pvdf membrane need to be put in methyl alcohol to activate the positive charged group above film, it is easier combined with electronegative protein, activation
After be put into during electricity turns liquid, cut 6 3M filter paper of 8 × 6cm sizes；
4.2.3.4 gel slab is taken out, Protein transfer is carried out using the Trans-Blot Semi-Dry of BIO-RAD companies,
Electric flap is put in 1 × electrophoresis liquid, white one side it is placed below, be sequentially placed sponge, the 3M filter paper 3 of 8 × 6cm, gel,
Pvdf membrane, the 3M filter paper 3 of 8 × 6cm, sponge, need to ensure bubble-free, the electric flap of upset black one side between gel and pvdf membrane
And it is fixed, electric turn trough, constant current 250mA 120-150 minutes are placed in, whole device need to be put in frozen water mixed liquor to ensure
Temperature is not too high；
4.2.3.5 after electricity turns to terminate, take out pvdf membrane, be close to gel one faces up, the upper left side of film cut one it is small
Film is washed 3 times in angle to distinguish positive and negative with TBST, 10 minutes every time；
4.2.3.6 pvdf membrane is incubated one hour under 5% milk normal temperature, to reduce the non-specific binding of antigen-antibody；
4.2.3.7 TBST washes film 3 times, 10 minutes every time；
4.2.3.8 with TBST according to 1:The goat-anti rabbit secondary antibody of 10000 dilution horseradish peroxidase HRP marks, at room temperature
It is gentle to rock incubation one hour；
4.2.3.9 TBST washes film 3 times, 10 minutes every time, when washing film for the last time, opens Bio-Rad gel imaging systems
Make machine precooling, preparing developer liquid takes ECL Western blotting detection reagents, by Solution A
With Solution B according to 1:1 prepare, wash film it is complete after, pvdf membrane is put on glass dish and is placed in exposure instrument, be added dropwise development
Liquid, proceeds by and takes pictures.The polyclonal SPACA3 antibody 25ug of 1st swimming lane detection rabbit igg；6th swimming lane detection 0.5ml rabbit iggs are more
The total protein extract of the immune targeted nano gold solutions of clonal antibody SPACA3, two visible clearly protein bands of swimming lane,
Exposure results refer to Figure 10.
Embodiment 2：RPA G mediations the 40nm nm of gold immune targeted nano of connection SPAG8 Antibody preparations
Gold；
Immune targeted nano gold system preparation method, step is as follows:
Antibody Fc section to form targeting immune nanometer gold system by being specifically bound with nanometer gold surface Chong ZuProteinA G
System, the preparation method of the immune targeted nano gold is comprised the steps of:
1st, the preparation of 40nm nm of gold:All glass apparatus are equal during preparing nm of gold with trisodium citrate reduction method
With the chloroazotic acid (HNO3 of new configuration:HCL,1:3) clean, redistilled water is rinsed, and uses preceding drying.By 0.01% gold chloride
100ml is placed on heating magnetic stirring apparatus in being added to conical flask, and fixed reaction temperature is 120 DEG C, mixing speed 400r/min.
1% trisodium citrate 1ml is rapidly added in above-mentioned solution, the solution seethed with excitement after 25s begins to change into micro- blueness, it is blue after about 120s
Color suddenly becomes cerise.Continue agitating heating and boil 10min, reaction is fully completed when golden, solution is gradually stable into Chinese red
Or claret, now stop heating and continue to stir 15-20min, use distilled water constant volume 100.0mL, 2-8 after naturally cooling to room temperature
DEG C preserve.Using a diameter of 40nm of transmission electron microscope observing nm of gold (referring to Fig. 5)；40nm nano-Au solutions are in ultraviolet-visible light
The absworption peak of spectrum is 520nm (referring to Fig. 7)；
2nd, self assembly rPA G:With 0.01M phosphate buffers (pH=7.2-7.4) by rPA G
Concentration is formulated as 1mg/ml solution, it is ensured that under conditions of nano-Au solution PH=7.0-8.0, by rPA G from group
Mounted in the surface of nanogold particle, concrete operations are：Take 100ul rPAs G solution be added slowly to 1ml nm of gold
In solution, mixed liquor is mixed using rotary incubator, and rotating speed keeps 10-20 to turn/min as the case may be, and 1-2 is small for room temperature concussion
When；13000 turns/min is centrifuged three times after the completion of reaction, each 15min, topple over supernatant remove unassembled ProteinA G, precipitation
With 1ml 0.01M phosphate buffers (pH=7.2-7.4) dissolve, be configured to rPA G modification nano-Au solution simultaneously
Ensure that solution recovers deep claret；
3rd, antibody Fc end connection:Take rPA G modification nano-Au solution 1ml, be slowly added to appropriate or excessive
OR1D2 antibody (antibody addition 100-500ug), mixed liquor is mixed using rotary incubator, and rotating speed keeps as the case may be
10-20 turns/min, and room temperature is shaken 2-3 hours, and 13000 turns/min is centrifuged three times after the completion of reaction, and each 15min topples over supernatant
Excessive antibodies are removed, precipitation is resuspended with 1ml 0.01M phosphate buffers (pH=7.2-7.4), be configured to immune targeted nano gold
Solution simultaneously ensures that solution recovers deep claret, connect the immune targeted nano gold solution of OR1D2 antibody respectively in 529nm and
The visible nm of gold of 277nm and antibody protein ultraviolet absorption peak (referring to Fig. 9)；
4th, the storage and detection of targeted nano gold is immunized:
4.1st, the storage of targeted nano gold is immunized：It is 10% to add 100ul concentration in the immune targeted nano gold solutions of 1ml
Bovine serum albumin(BSA) (BSA), on the one hand the uncombined site of closing nanometer gold surface, eliminates immune non-specific binding reaction；
On the other hand ensure that the antibody of targeting immune nanometer gold is not degraded.After 4 DEG C of vibration 1-2h of mixed liquor, can be protected at 2-8 DEG C long-term
Deposit；15min is centrifuged using preceding 13000 turns/min, precipitation 1ml 0.01M phosphate buffers (pH=7.0) weight after supernatant is toppled over
It is outstanding, it is ensured that solution can be used after recovering deep claret.
4.2nd, using protein immunoblot experiment detection antibody joint efficiency
Protein immunoblot specific experiment method is：Western Blot experimental techniques
4.2.1 the Protein Extraction of targeted nano gold and antibody-solutions, is immunized
Take the immune targeted nano solution that 0.5ml connects the polyclonal OR1D2 antibody of rabbit igg, 13000 turns/min centrifugations
15min, after toppling over supernatant, precipitation 4 × SDS-PAGE albumen sample-loading buffers 25ul shakes abundant crack protein；By 25ul
OR1D2 antibody-solutions (concentration is 1mg/ml) and 4 × SDS-PAGE albumen sample-loading buffer by volume (3：1) mixing concussion is filled
Divide crack protein, heat 95 DEG C of 5min, -20 DEG C of preservations are stand-by.
4.2.2, main agents are prepared
Main agents and its preparation are omited herein with embodiment 1.
4.2.3, Western Blot experimental procedures
Experimental procedure is omited herein with embodiment 1.
Experimental result：The polyclonal OR1D2 antibody 25ug of 5th swimming lane detection rabbit igg；6th swimming lane detection 0.5ml rabbit iggs are more
The total protein extract of the immune targeted nano gold solutions of clonal antibody OR1D2, two visible clearly protein bands of swimming lane expose
Light result refers to Figure 10.
Claims (1)
1. a kind of preparation method of the immune targeted nano gold that recombinant protein A is mediated, the method utilizes antibody Fc section and nm of gold
Surface recombinant protein A (ProteinA G) specific binding forms targeting immune nanometer gold system, it is ensured that Antibody Fab fragment antigen is determined
Determine cluster fully to expose, substantially increase antibody surface load-carry duty, with good reaction stability and specificity；It is specific include with
Lower step:：
1st step, the preparation of nm of gold
1.1st step, 0.01% gold chloride 50-100ml is added in conical flask it is placed on heating magnetic stirring apparatus, fixed reaction
Temperature is 95-120 DEG C, mixing speed 300-400r/min；
The preparation of the 1.2nd step, 20nm nm of gold or 40nm nm of gold
1.2.1 steps, preparation 20nm nm of gold：1% trisodium citrate 0.75- is rapidly added in solution described in the 1.1st step
The solution seethed with excitement after 1.5ml, 25s begins to change into micro- blueness, treats that blueness becomes cerise, continues agitating heating and boils 10-
15min, reaction is fully completed when golden, and solution is gradually stable into Chinese red or claret, is now stopped heating and is continued to stir 15-
20min, is preserved after naturally cooling to room temperature with distilled water constant volume 50.0-100.0mL, 2-8 DEG C；
1.2.2 steps, preparation 40nm nm of gold：1% trisodium citrate 0.5-1ml is rapidly added in solution described in the 1.1st step,
The solution seethed with excitement after 25s begins to change into micro- blueness, treats that blueness becomes cerise；Continue agitating heating and boil 10-15min, work as gold
Reaction is fully completed, solution is gradually stable into Chinese red or claret, now stop heating and continue to stir 15-20min, it is naturally cold
But to after room temperature with distilled water constant volume 50.0-100mL, 2-8 DEG C of preservation；
2nd step, the 1.2nd step reaction on the basis of, self assembly Chong ZuProteinA G
2.1st step, with the phosphate buffer of 0.01M, pH=7.2-7.4 by rPA G to be configured to concentration be 1mg/ml
Solution；
2.2nd step, ensure the 1st step prepare nano-Au solution PH=7.0-8.0 under conditions of, by rPA G from
The surface of nanogold particle is assembled in, concrete operations are：By volume 1:The restructuring that 10-20 prepares the 2.1st step
ProteinA G solution be added slowly to the 1st step preparation nano-Au solution in, mixed liquor using rotary incubator mix, rotating speed
It is maintained at 10-20 and turns/min, 4 DEG C overnight；
Be centrifuged after the completion of 2.3rd step, reaction, topple over supernatant, remove unassembled rPA G, precipitation 1ml 0.01M,
PH=7.2-7.4 phosphate buffer dissolving, be configured to rPA G modification nano-Au solution and ensure that solution is extensive
Multiple depth claret；
3rd step, the connection of antibody Fc end
Take the 2nd step preparation rPA G modification nano-Au solution 1ml, be slowly added to 100-500ug antibody, mix
Liquid is mixed using rotary incubator, and rotating speed is maintained at 10-20 and turns/min, 4 DEG C of shaken overnights；After the completion of reaction be centrifuged, topple on
Excessive antibodies are gone in removing, and precipitation is resuspended with the phosphate buffer of 1ml 0.01M, pH=7.2-7.4, are configured to connect exempting from for antibody
Epidemic disease targeted nano gold solution simultaneously ensures that solution recovers deep claret；
The storage and detection of the 4th step, immune targeted nano gold
The bovine serum albumin(BSA) that 100ul concentration is 10% is added in immune targeted nano gold solution prepared by the steps of 1ml the 3rd
(BSA), on the one hand closing nanometer gold surface is not associated with site, eliminates immune non-specific binding reaction；On the other hand ensure to exempt from
The antibody of epidemic disease targeted nano gold is not degraded；After 4 DEG C of vibration 1-2h of mixed liquor, preserved at 2-8 DEG C long-term；Using first 13000 turns/
Min is centrifuged 15min, and the phosphate buffer for toppling over precipitation 1ml 0.01M, pH=7.0 after supernatant is resuspended, it is ensured that solution recovery depth
Be can be used after claret, detection antibody combination situation is tested using immune impression using preceding.
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| CN114395028A (en) * | 2022-02-09 | 2022-04-26 | 天津市泌尿外科研究所 | Preparation method of human seminal membrane protein SPACA1 specific antigen coupling protein and antibody |
| CN114395028B (en) * | 2022-02-09 | 2024-01-30 | 天津市泌尿外科研究所 | Preparation method of human sperm membrane protein SPACA1 specific antigen coupling protein and antibody |
| CN114778812A (en) * | 2022-04-19 | 2022-07-22 | 武汉轻工大学 | Antibody directional labeling colloidal gold immune probe, preparation method thereof and lead ion test paper based on antibody directional labeling |
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