CN102662053B - Immune biochip regeneration method and regenerated immune biochip - Google Patents
Immune biochip regeneration method and regenerated immune biochip Download PDFInfo
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- CN102662053B CN102662053B CN201210117167.7A CN201210117167A CN102662053B CN 102662053 B CN102662053 B CN 102662053B CN 201210117167 A CN201210117167 A CN 201210117167A CN 102662053 B CN102662053 B CN 102662053B
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Abstract
The invention relates to an immune biochip regeneration method and a regenerated immune biochip. An immune biochip adopted in the immune biochip regeneration method comprises a chip substrate and an Fc segment of a first antibody, wherein the Fc segment of the first antibody is formed on the chip substrate. The method comprises the steps as follows: the immune biochip is subjected to immobilization processing through fixing agent, so as to enable the Fc segment of the first antibody to carry positive charges; and the immune biochip subjected to immobilization processing is mixed with a full-length second antibody, so as to enable the full-length second antibody to be combined with the Fc segment of the first antibody carrying the positive charges through electrostatic interaction, and thus, the immune biochip carrying the second antibody is obtained. By utilization of the method, the immune biochip can be regenerated conveniently and efficiently.
Description
Technical field
The present invention relates to immune biochip renovation process and the immune biochip of regenerating.
Background technology
Biochip, by the biomolecule such as antibody, nucleic acid are fixed in chip basal body, by the power of perceptron detectable biomolecule, heat, the physical message such as optical, electrical, thereby detect the information of biomolecule, it provides approach for the mankind find out the information relevant to life.Immunity biochip is a kind of of biochip, for surveying the biomolecule information relevant with immunological phenomena, and the information detecting is changed into electric signal, to process, is the important tool of immunology research, medical diagnosis on disease, treatment and control.But present stage, immune biochip manufacturing process is complicated, with high costs, and used immune biochip regeneration can be reduced to the cost that uses biochip.
Yet current immune biochip renovation process still haves much room for improvement.
Summary of the invention
The following discovery of the present invention based on inventor completes:
For the chip of detectable antigens type, the principle of regeneration is that antigen is removed from antibody, again detectable antigens; Or antigen and antibody are all removed, retightened antibody.Based on these principles, the immune biochip to detectable antigens type, existing renovation process has electrochemical process, acid-base method, strong oxidizing process and Shift Method.Yet these method technology difficulty are large, efficiency is low, manufacturing cost is high, and the sensitivity of the regeneration obtaining immunity biochip is lower, and serviceable life is limited.
The present invention is intended at least solve one of technical matters existing in prior art.For this reason, the invention provides the immune biochip renovation process of detectable antigens type.
According to an aspect of the present invention, the invention provides a kind of immune biochip renovation process, wherein, immune biochip comprises the Fc fragment of chip basal body and first antibody, and the Fc fragment of this first antibody is formed in chip basal body.According to embodiments of the invention, the method comprises: use fixing agent to process immune being fixed of biochip, to make the Fc fragment of first antibody carry positive charge; And the immune biochip of processing through immobilization is mixed with total length second antibody, to total length second antibody is combined by electrostatic interaction with the Fc fragment of carrying the first antibody of positive charge, thereby form the immune biochip that carries second antibody.
According to concrete example of the present invention, utilize immune biochip renovation process of the present invention, can effectively make the regeneration of immune biochip, and regeneration technology simple, take less, efficiency is high, regeneration cost is low, the regeneration immunity biochip sensitivity obtaining is high, long service life.
According to embodiments of the invention, in immune biochip renovation process of the present invention, the Fc fragment of first antibody is by being used distintegrant that total length first antibody is digested and obtained, wherein, total length first antibody is formed in chip basal body, and total length first antibody is combined with antigen.Thus, total length first antibody on used immune biochip is after the digestion process of distintegrant, the Fc fragment that only retains chip basal body and first antibody formed thereon, thereby the immobilization based on immune biochip renovation process of the present invention is follow-up is processed and mixes with total length second antibody, can effectively obtain the immune biochip of regeneration, carry the immune biochip of second antibody.
According to embodiments of the invention, in immune biochip renovation process of the present invention, the kind of distintegrant is not particularly limited.According to concrete example of the present invention, distintegrant can be proteinase, strong acid or highly basic.Wherein, according to embodiments of the invention, the kind of strong acid is also not particularly limited.According to concrete example of the present invention, strong acid can be for being selected from least one of hydrochloric acid, sulfuric acid and nitric acid.According to embodiments of the invention, the kind of highly basic is also not particularly limited.According to concrete example of the present invention, highly basic can be for being selected from least one of NaOH and potassium hydroxide.In addition,, according to embodiments of the invention, the kind of proteinase is also not particularly limited.According to concrete example of the present invention, proteinase can be for being selected from least one of pepsin, trypsase and papain, preferably pepsin.This is because pepsin is the special a kind of enzyme that decomposes the protein that comprises antibody.When adopting pepsin as distintegrant, total length first antibody to be digested in immune biochip renovation process of the present invention, pepsin can be decomposed in the hinge area of total length first antibody three fragments, i.e. two Fab fragments and a Fc fragment, and the Fc fragment of first antibody is retained in chip basal body, and Fab fragment can be by utilizing deionized water rinsing to remove in subsequent step.Wherein, pepsic concentration, pH and temperature have certain restriction, and those skilled in the art can select according to actual experiment situation.According to concrete example of the present invention, distintegrant is 1～10g/L, the pepsin aqueous solution of pH=0.5～2.0.According to some embodiments of the present invention, the temperature of pepsin aqueous solution is 25～37 ℃.Thus, can fully effectively to total length first antibody, digest.
According to embodiments of the invention, in immune biochip renovation process of the present invention, the time of using distintegrant to digest total length first antibody is not particularly limited, and those skilled in the art can select the suitableeest digestion time according to actual experiment situation.According to concrete example of the present invention, use distintegrant to digest 5～10 minutes total length first antibody.Thus, can fully effectively to total length first antibody, digest.
According to embodiments of the invention, in immune biochip renovation process of the present invention, the Fc fragment of first antibody is by being used eluant, eluent that immune biochip is processed and obtained, wherein, immunity biochip further comprises: total length the 3rd antibody, the Fc fragment of itself and first antibody is by electrostatical binding, and total length the 3rd antibody is combined with antigen.Thus, by immune biochip renovation process of the present invention, regenerated, and the immune biochip reusing is after eluant, eluent is processed, the Fc fragment that only retains chip basal body and first antibody formed thereon, thereby the immobilization based on immune biochip renovation process of the present invention is follow-up is processed and mixes with total length second antibody, can effectively obtain the immune biochip of regeneration, carry the immune biochip of second antibody.
By using eluant, eluent, immune biochip is processed to the Fc fragment that obtains first antibody, its principle is, eluant, eluent is rich in a large amount of negative ion, electronegative, thereby it can be combined in residual antibody Fc fragment with new fixing antibody (being total length the 3rd antibody) competition, so can by new antibodies (being total length the 3rd antibody) with and the antigen of upper combination from immune biochip, remove.According to embodiments of the invention, in immune biochip renovation process of the present invention, the kind of eluant, eluent is not particularly limited.According to concrete example of the present invention, eluant, eluent can be for being selected from any one of strong acid or highly basic.Wherein, according to embodiments of the invention, the kind of strong acid is also not particularly limited.According to concrete example of the present invention, strong acid can be for being selected from least one of hydrochloric acid, sulfuric acid and nitric acid.According to embodiments of the invention, the kind of highly basic is also not particularly limited.According to concrete example of the present invention, highly basic can be for being selected from least one of NaOH and potassium hydroxide, preferably NaOH.Wherein, the concentration of NaOH and pH have certain restriction, and those skilled in the art can select according to actual experiment situation.According to concrete example of the present invention, eluant, eluent is 0.04～4g/L, the sodium hydrate aqueous solution of pH=11.0～13.0.Thus, can effectively total length the 3rd antibody and antigen be removed from chip basal body.
According to embodiments of the invention, in immune biochip renovation process of the present invention, the time of using eluant, eluent to process immune biochip is not particularly limited, and those skilled in the art can select the suitableeest processing time according to actual experiment situation.According to concrete example of the present invention, use eluant, eluent to process 5～10 minutes immune biochip.Thus, can effectively total length the 3rd antibody and antigen be removed from chip basal body.
In addition, coupling agent can impel between antibody and chip basal body and connects with covalent bond.According to embodiments of the invention, in immune biochip renovation process of the present invention, utilize coupling agent that the Fc fragment of first antibody is connected with chip basal body.Wherein, according to embodiments of the invention, in immune biochip renovation process of the present invention, the Fc fragment of first antibody and the kind of the coupling agent between chip basal body are also not particularly limited, and the kind of the coupling agent that adopted does not limit the applicable immune biochip renovation process of the present invention of immune biochip.According to concrete examples more of the present invention, coupling agent can be for being selected from least one of silane reagent, high molecular polymer, sulfydryl alkanol and mercaptan acid.
According to embodiments of the invention, in immune biochip renovation process of the present invention, being fixed processed before and afterwards, further comprised the step that the immune biochip of immune biochip and process immobilization processing is cleaned.According to embodiments of the invention, can utilize deionized water to carry out aforementioned cleaning 5 times.
The kation of fixing agent is positively charged, can be with the Fc fragment of first antibody of carrying negative charge by electrostatic interaction combination, and make the Fc fragment of first antibody carry positive charge, thereby the carrying out that can be conducive to subsequent step, after the immune biochip that is about to process through immobilization mixes with total length second antibody, can easily and effectively total length second antibody be fixed in chip basal body, thereby can successfully obtain the immune biochip that carries second antibody.Therefore,, in immune biochip renovation process of the present invention, use fixing agent to process described immune being fixed of biochip.According to embodiments of the invention, in immune biochip renovation process of the present invention, the kind of fixing agent is not particularly limited.According to concrete example of the present invention, fixing agent can be ammoniacal liquor (NH
4oH), metal chlorate, metal sulfate or metal cyanide salt, wherein, metal chlorate can be for being selected from least one of ferric trichloride, cupric chloride, aluminum chloride and manganese chloride, metal sulfate can be for being selected from least one of iron sulfate, ferrous sulphate, aluminium sulphate and manganese sulfate, and metal cyanide salt can be for being selected from least one of ferricyanide, copper cyanider and cyaniding manganese.According to some embodiments of the present invention, preferably, fixing agent is ammoniacal liquor or ferric trichloride, more preferably the aqueous solution of the ammonia spirit of 1% volumetric concentration or the ferric trichloride of 10～30g/L.According to embodiments of the invention, the time that being fixed processed is not particularly limited.According to concrete example of the present invention, can being fixed process 5～10 minutes.Thus, the second antibody of total length or the 3rd antibody can be fixed in the Fc fragment of first antibody fully.
According to embodiments of the invention, in immune biochip renovation process of the present invention, total length second antibody provides with the form of the damping fluid of total length second antibody.The expression way used in this manual " damping fluid of total length second antibody ", refers to the mixed solution of total length second antibody and damping fluid.Wherein, the kind of the damping fluid that adopted is here not particularly limited, for example, can adopt phosphate buffer.Thus, according to concrete examples more of the present invention, total length second antibody can provide with the form of the phosphate buffer of total length second antibody, and the form with the mixed solution of total length second antibody and damping fluid provides.Particularly, according to embodiments of the invention, the phosphate buffer of total length second antibody is the mixed solution of the phosphate buffer of total length second antibody and pH=7.0～7.5, and in the phosphate buffer of total length second antibody, the concentration of total length second antibody is 50～100mg/L, and phosphatic concentration is 0.03～0.5mol/L.Thus, total length second antibody is electrically charged in phosphate buffer, the Fc fragment electrostatical binding of energy and first antibody.
According to embodiments of the invention, in immune biochip renovation process of the present invention, the time that the immune biochip of processing through immobilization is mixed with total length second antibody is not particularly limited, and those skilled in the art can select suitable incorporation time according to actual experiment situation.According to concrete example of the present invention, the immune biochip of processing can be mixed 5～10 minutes with total length second antibody through immobilization.Thus, total length second antibody can be combined on the immune biochip of processing through immobilization fully.
According to embodiments of the invention, immune biochip renovation process of the present invention, may further include and utilize sealer the immune biochip that carries second antibody to be sealed to the step of processing.According to embodiments of the invention, sealer can be for being selected from least one of bovine serum albumin(BSA), skimmed milk power, calf serum, sheep blood serum and network albumen, preferably bovine serum albumin(BSA) phosphate buffer.Wherein, according to embodiments of the invention, bovine serum albumin(BSA) phosphate buffer is to obtain by the bovine serum albumin(BSA) of 1～5g/L being dissolved in the phosphate buffer of pH=7.4.According to embodiments of the invention, in immune biochip renovation process of the present invention, the time of sealing processing is not particularly limited.According to concrete examples more of the present invention, can seal and process 5～10 minutes.Thus, the binding site of the Fc fragment of the first antibody not captured by total length second antibody or total length the 3rd antibody, can be closed agent and capture, thereby can avoid antigen to be detected to be incorporated in the Fc fragment of first antibody, causes false positive to disturb.
It should be noted that, the term that used in this article " first ", " second ", " the 3rd " are only for describing object, to can distinguish easily the different types of antibody being fixed in chip basal body, and can not be interpreted as indication or hint relative importance, or the implicit quantity that indicates indicated technical characterictic.Thus, one or more these features can be expressed or impliedly be comprised to the feature that is limited with " first ", " second ".
According to a further aspect in the invention, the invention provides the immune biochip of a kind of regeneration.According to embodiments of the invention, this immune biochip of regenerating is obtained by immune biochip renovation process of the present invention.Inventor's discovery, regeneration immuno-chip sensitivity of the present invention is high, and cost is low, and long service life can be effectively for surveying the biomolecule information relevant with immunological phenomena.
It should be noted that, immune biochip renovation process of the present invention and the immune biochip of regenerating, the work that is present inventor by arduous creative work and optimization completes, and, compare with traditional immune biochip renovation process and the immune biochip of regenerating, its tool has the following advantages:
(1) regeneration cost can be controlled.In immune biochip renovation process of the present invention, except antibody, distintegrant used, eluant, eluent, fixing agent and sealer are common chemical reagent, and cost can be controlled.Take manual operation as example, with 100 calculating of 1L system solution regeneration chip, (1L system solution refers to, in the method for the invention, while using the solution such as distintegrant, eluant, eluent, fixing agent and sealer to carry out each step, all adopt the container of 1L volume, be about to regenerate in each reagent solution that chip is immersed in 1L successively), utilize immune biochip renovation process of the present invention to carry out immune biochip regeneration, need to use pepsin 1～10g, the ammoniacal liquor (NH of 30% concentration
4oH) 3.3L, NaOH 0.1～10g, (wherein, concrete computation process is as follows: detergent solution decomposition need be used 1 time, needs 1L for bovine serum albumin(BSA) 100～500g; Eluent solution need to be used 99 times, needs 99L; Fixing agent and sealer all need to use 100 times, respectively need 100L.If each reagent solution all adopts preferably, according to the concentration of each reagent solution (pepsin 1～10g/L, NaOH 0.04～4g/L, ammoniacal liquor volumetric concentration 1%, bovine serum albumin(BSA) 1～5g/L) calculate, need altogether pepsin 1～10g, NaOH 3.96～396g, the ammoniacal liquor 3.33L of 30% concentration, bovine serum albumin(BSA) 100～500g).And the volume of general immune biochip is 1～10cm
3, the core number that 1L solution system can be regenerated is simultaneously 100～1000, and the amount of each reagent is to calculate for 100 times and obtain with regeneration chip above, therefore, the average regeneration cost of every chip can be controlled in lower level.And if adopt the micro-fluid chip of robotization, the average regeneration cost of every chip more can reduce by one to two order of magnitude.
(2) recovery time can be controlled.While adopting manual operation to implement immune biochip renovation process of the present invention, the recovery time is 30～50 minutes; When the micro-fluid chip of use robotization carries out immune biochip renovation process of the present invention, the time of each regeneration can be down in 20 minutes.
(3) to reclaim equiment, require low.Can in the water-bath of 25～37 ℃ or constant temperature oven, implement immune biochip renovation process of the present invention.
(4) regeneration times can reach more than 100 times.The regeneration immunity biochip obtaining by immune biochip renovation process of the present invention, because the Fc chip of initial fixing antibody (first antibody) is retained, new fixing antibody (total length second antibody) can be combined in residual antibody Fc fragment, the sensitivity of chip (antigen concentration of minimum detectable) can not decline along with the increase of regeneration times, and therefore the immune biochip of regeneration of the present invention can be reproduced more than 100 times.
(5) in immune biochip renovation process of the present invention, can fix different types of antibody, be that total length second antibody can be the combination of several different types of antibody (mostly being 10 kinds of different types of antibody most), thus, a chip can detect at most 10 kinds of antigen indexs, when needs detect 20 kinds of antigen indexs, after detecting 10 kinds of indexs, chip is regenerated once, on chip, fix 10 kinds of new antibody, to detect 10 kinds of antigen indexs of residue, thereby based on immune biochip renovation process of the present invention, regenerate at twice and detect, can effectively realize the index that chip detects plurality of antigens in batch.
(6) the chip kind that can utilize immune biochip renovation process of the present invention to regenerate is very abundant.Immune biochips such as array chip, chip lab (Lab-on-chip), micro-fluid chip all can be regenerated by immune biochip renovation process of the present invention.
(7) immune biochip renovation process of the present invention to the material of chip surface without specific (special) requirements.Surface is the chip of the materials such as metal, semiconductor, organism, metal oxide, inorganic non-metallic, all can utilize immune biochip renovation process of the present invention to regenerate.
(8) immune biochip renovation process of the present invention to the structure of chip surface without specific (special) requirements.Because the region directly contacting with antibody does not need to draw metal wire, form conductive structure, chip surface can be the micro-fluid chip containing groove structure, can be also other chips without groove structure.
(9) immune biochip renovation process of the present invention to the coupling agent (being the coupling agent between first antibody and chip basal body) of antibody before chip regeneration without specific (special) requirements, can be silane reagent, for example can be for being selected from least one of 3-diglycidyl hydroxypropyl trimethoxy silane, 3-aminopropyl dimethyl monosubstituted ethoxy silane and 3-aminopropyl triethoxysilane; Can be high molecular polymer, for example can be for being selected from least one of glycosaminoglycan, carboxyl glucosan, two amino polyglycol, two carboxy polyethylene glycol, shitosan and poly-piperazine; Also can be sulfydryl alkanol or mercaptan acid, can be for example selected from mercapto methyl alcohol, 2 mercapto ethanol, 3-mercaprol, at least one in 4-Mercaptobutanol, 5-sulfydryl amylalcohol, 6-sulfydryl season alcohol, 7-sulfydryl enanthol, 8-sulfydryl octanol, 9-sulfydryl nonyl alcohol, 10-sulfydryl decyl alcohol, 11-mercaptoundecanol, 12-sulfydryl dodecanol, 13-sulfydryl tridecanol, 14-sulfydryl tetradecanol, 15-sulfydryl pentadecanol, 16-sulfydryl cetyl alcohol, 17-sulfydryl heptadecanol and 18-sulfydryl octadecanol or its derived acids.
(10) the biologically active holding time of the immune biochip of regeneration of the present invention can reach 50 days.Antibody on the immune biochip of regeneration of the present invention, total length second antibody can stably be preserved in PBS solution, can oxidized or decomposition.
Additional aspect of the present invention and advantage in the following description part provide, and part will become obviously from the following description, or recognize by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage accompanying drawing below combination obviously and is easily understood becoming the description of embodiment, wherein:
Fig. 1 has shown the schematic flow sheet of immune according to an embodiment of the invention biochip renovation process;
Fig. 2 has shown the schematic flow sheet of immune in accordance with another embodiment of the present invention biochip renovation process; And
Fig. 3 has shown the schematic flow sheet of immune in accordance with another embodiment of the present invention biochip renovation process.
Embodiment
Describe embodiments of the invention below in detail, it should be noted that the embodiment the following describes is exemplary, only for explaining the present invention, and can not be interpreted as limitation of the present invention.
Conventional method:
With reference to Fig. 1, immune biochip renovation process of the present invention can comprise following steps:
First, the immune biochip of the Fc fragment that comprises first antibody and chip basal body is placed in to fixative solution and soaks 5～10min, to use fixing agent to process immune being fixed of biochip, make the Fc fragment of first antibody carry positive charge;
Secondly, utilize deionized water that the immune biochip of processing through immobilization is rinsed 5 times;
Then, immune biochip through rinsing is mixed with total length second antibody, so that making total length second antibody is combined by electrostatic interaction with the Fc fragment of carrying the first antibody of positive charge, thereby form the immune biochip that carries second antibody, obtain the immune biochip of regeneration, particularly, the phosphate buffer (PBS) that the immune biochip through rinsing is placed in to total length second antibody soaks 15～20min.Wherein, the phosphate buffer of total length second antibody is the mixed solution of the phosphate buffer of total length second antibody and pH=7.0～7.5, and in the phosphate buffer of total length second antibody, the concentration of total length second antibody is 50～100mg/L, and phosphatic concentration is 0.03～0.5mol/L.
Embodiment 1
With reference to Fig. 2, immune biochip renovation process of the present invention can also comprise following steps:
First, by comprising, be combined with the total length first antibody of antigen and the chip of chip basal body and be placed in detergent solution decomposition and soak 5～10min, to use distintegrant to digest total length first antibody, be decomposed into two Fab fragments and a Fc fragment, this Fc fragment is called the Fc fragment of first antibody, and wherein, distintegrant used is 1～10g/L, the pepsin aqueous solution of pH=0.5～2.0, solution temperature is 25～37 ℃;
Secondly, utilize deionized water that the immune biochip of the digestion process through distintegrant is rinsed 5 times, to remove two Fab fragments;
Then, immune biochip through rinsing is placed in to fixative solution and soaks 5～10min, to use fixing agent to process immune being fixed of biochip, make the Fc fragment of first antibody carry positive charge, wherein, fixing agent used can be the ammoniacal liquor (NH of 1% volumetric concentration
4oH) solution;
Next, utilize deionized water that the immune biochip of processing through immobilization is rinsed 5 times;
Then, immune biochip through rinsing is mixed with total length second antibody, so that making total length second antibody is combined by electrostatic interaction with the Fc fragment of carrying the first antibody of positive charge, thereby form the immune biochip that carries second antibody, particularly, the phosphate buffer (PBS) that the immune biochip through rinsing is placed in to total length second antibody soaks 15～20min.Wherein, the phosphate buffer of total length second antibody is the mixed solution of the phosphate buffer of total length second antibody and pH=7.0～7.5, and in the phosphate buffer of total length second antibody, the concentration of total length second antibody is 50～100mg/L, and phosphatic concentration is 0.03～0.5mol/L.
Then, the immune biochip that carries second antibody is placed in to the PBS solution of pH=7.4, and preserves in 4 ℃ of refrigerators, standby.
Thus, the immune biochip that carries second antibody of acquisition, the immune biochip of regenerating, can be combined with corresponding antigen effectively, thus can be effectively for the detection of corresponding antigens.
In addition, by before preserving with the immune biochip of second antibody, can further include and utilize sealer the immune biochip that carries second antibody to be sealed to the step of processing, particularly, the immune biochip that carries second antibody is placed in to sealer and soaks 5～10min, wherein sealer used is bovine serum albumin(BSA) phosphate buffer, and it is to obtain by the bovine serum albumin(BSA) of 1～5g/L (BSA) being dissolved in the PBS solution of pH=7.4.
Of the present invention immune biochip renovation process described in the present embodiment, can remove the antigen being combined on total length first antibody, decompose and be combined in the total length first antibody in chip basal body, make it eliminate immunobiologic activity, and do not affect the immune response after regeneration, and can effectively in chip basal body, fix new antibody, i.e. total length second antibody.In addition, it should be noted that, the immune biochip renovation process described in the present embodiment is suitable for immune biochip to regenerate for the first time.
Embodiment 2
With reference to Fig. 3, immune biochip renovation process of the present invention can also comprise following steps:
First, by comprising total length the 3rd antibody, the Fc fragment of first antibody and the chip of chip basal body that are combined with antigen, be placed in eluent solution and soak 5～10min, so that using eluant, eluent processes immune biochip, in connection with having, total length the 3rd antibody of antigen is separated the Fc fragment of first antibody, wherein, eluant, eluent used is 0.04～4g/L, the NaOH of pH=11.0～13.0 (NaOH) aqueous solution;
Secondly, utilize deionized water that the immune biochip of processing through eluant, eluent is rinsed 5 times, to remove total length the 3rd antibody that is combined with antigen;
Then, immune biochip through rinsing is placed in to fixative solution and soaks 5～10min, to use fixing agent to process immune being fixed of biochip, make the Fc fragment of first antibody carry positive charge, wherein, fixing agent used can be the ammoniacal liquor (NH of 1% volumetric concentration
4oH) solution;
Next, utilize deionized water that the immune biochip of processing through immobilization is rinsed 5 times;
Then, immune biochip through rinsing is mixed with total length second antibody, so that making total length second antibody is combined by electrostatic interaction with the Fc fragment of carrying the first antibody of positive charge, thereby form the immune biochip that carries second antibody, particularly, the phosphate buffer (PBS) that the immune biochip through rinsing is placed in to total length second antibody soaks 15～20min.Wherein, the phosphate buffer of total length second antibody is the mixed solution of the phosphate buffer of total length second antibody and pH=7.0～7.5, and in the phosphate buffer of total length second antibody, the concentration of total length second antibody is 50～100mg/L, and phosphatic concentration is 0.03～0.5mol/L.
Then, the immune biochip that carries second antibody is placed in to the PBS solution of pH=7.4, and preserves in 4 ℃ of refrigerators, standby.
Thus, the immune biochip that carries second antibody of acquisition, the immune biochip of regenerating, can be combined with corresponding antigen effectively, thus can be effectively for the detection of corresponding antigens.
In addition, before the immune biochip that carries second antibody is preserved, can further include and utilize sealer the immune biochip that carries second antibody to be sealed to the step of processing, particularly, the immune biochip that carries second antibody is placed in to sealer and soaks 5～10min, wherein sealer used is bovine serum albumin(BSA) phosphate buffer, and it is to obtain by the bovine serum albumin(BSA) of 1～5g/L (BSA) being dissolved in the PBS solution of pH=7.4.
Of the present invention immune biochip renovation process described in the present embodiment, can remove the total length that is combined with antigen the 3rd antibody being combined in chip basal body, and in chip basal body, retighten new antibody, be i.e. total length second antibody.In addition, it should be noted that, the immune biochip renovation process described in the present embodiment is suitable for the immune biochip of used regeneration of the present invention to regenerate.
In the description of this instructions, the description of reference term " embodiment ", " some embodiment ", " example ", " concrete example " or " some examples " etc. means to be contained at least one embodiment of the present invention or example in conjunction with specific features, structure, material or the feature of this embodiment or example description.In this manual, the schematic statement of above-mentioned term is not necessarily referred to identical embodiment or example.And the specific features of description, structure, material or feature can be with suitable mode combinations in any one or more embodiment or example.
Although illustrated and described embodiments of the invention, those having ordinary skill in the art will appreciate that: in the situation that not departing from principle of the present invention and aim, can carry out multiple variation, modification, replacement and modification to these embodiment, scope of the present invention is limited by claim and equivalent thereof.
Claims (27)
1. an immune biochip renovation process, described immune biochip comprises the Fc fragment of chip basal body and first antibody, the Fc fragment of described first antibody is formed in described chip basal body,
Wherein, described method comprises:
Use fixing agent to process described immune being fixed of biochip, to make the Fc fragment of described first antibody carry positive charge; And
The immune biochip of processing through immobilization is mixed with total length second antibody, so as to make described total length second antibody with described in carry the first antibody of positive charge Fc fragment by electrostatic interaction, be combined, thereby the immune biochip of second antibody is carried in formation,
Described fixing agent is ammoniacal liquor, metal chlorate, metal sulfate or metal cyanide salt,
Wherein,
The Fc fragment of described first antibody is by being used distintegrant that total length first antibody is digested and obtained, and described total length first antibody is formed in described chip basal body, and described total length first antibody is combined with antigen; Or
The Fc fragment of described first antibody is by being used eluant, eluent that the second immune biochip is processed and obtained, described the second immune biochip comprises Fc fragment and total length the 3rd antibody of chip basal body, first antibody, the Fc fragment of described first antibody is formed in described chip basal body, the Fc fragment of described total length the 3rd antibody and described first antibody is by electrostatical binding, and described total length the 3rd antibody is combined with antigen.
2. method according to claim 1, is characterized in that, described distintegrant is pepsin.
3. method according to claim 2, is characterized in that, described distintegrant is 1～10g/L, the pepsin aqueous solution of pH=0.5～2.0.
4. method according to claim 3, is characterized in that, the temperature of described pepsin aqueous solution is 25～37 ℃.
5. method according to claim 1, is characterized in that, uses distintegrant to digest 5～10 minutes total length first antibody.
6. method according to claim 1, is characterized in that, described eluant, eluent is to be selected from any one of strong acid or highly basic.
7. method according to claim 6, is characterized in that, described strong acid is to be selected from least one of hydrochloric acid, sulfuric acid and nitric acid.
8. method according to claim 6, is characterized in that, described highly basic is to be selected from least one of NaOH and potassium hydroxide.
9. method according to claim 8, is characterized in that, described eluant, eluent is 0.04～4g/L, the sodium hydrate aqueous solution of pH=11.0～13.0.
10. method according to claim 1, is characterized in that, uses eluant, eluent to process 5～10 minutes immune biochip.
11. methods according to claim 1, is characterized in that, utilize coupling agent that the Fc fragment of described first antibody is connected with described chip basal body.
12. methods according to claim 11, is characterized in that, described coupling agent is to be selected from least one of silane reagent, high molecular polymer, sulfydryl alkanol and mercaptan acid.
13. methods according to claim 1, is characterized in that, carry out described immobilization processing before and afterwards, further comprise described immune biochip and described step of cleaning through the immune biochip of immobilization processing.
14. methods according to claim 13, is characterized in that, utilize deionized water to carry out described cleaning 5 times.
15. methods according to claim 1, it is characterized in that, described metal chlorate is to be selected from least one of ferric trichloride, cupric chloride, aluminum chloride and manganese chloride, described metal sulfate is to be selected from least one of iron sulfate, ferrous sulphate, aluminium sulphate and manganese sulfate, and described metal cyanide salt is to be selected from least one of ferricyanide, copper cyanider and cyaniding manganese.
16. methods according to claim 15, is characterized in that, described fixing agent is the ammonia spirit of 1% volumetric concentration or the ferric chloride aqueous solutions of 10～30g/L.
17. methods according to claim 1, is characterized in that, carry out described immobilization and process 5～10 minutes.
18. methods according to claim 1, is characterized in that, described total length second antibody provides with the form of the damping fluid of total length second antibody.
19. methods according to claim 18, is characterized in that, described total length second antibody provides with the form of the phosphate buffer of total length second antibody.
20. methods according to claim 19, it is characterized in that, the phosphate buffer of described total length second antibody is the mixed solution of the phosphate buffer of described total length second antibody and pH=7.0～7.5, and in the phosphate buffer of described total length second antibody, the concentration of described total length second antibody is 50～100mg/L, and phosphatic concentration is 0.03～0.5mol/L.
21. methods according to claim 1, is characterized in that, the described immune biochip of processing through immobilization is mixed 15～20 minutes with described total length second antibody.
22. methods according to claim 1, is characterized in that, further comprise and utilize sealer the described immune biochip that carries second antibody to be sealed to the step of processing.
23. methods according to claim 22, is characterized in that, described sealer for be selected from bovine serum albumin(BSA), skimmed milk power, calf serum, sheep blood serum and caseic at least one.
24. methods according to claim 23, is characterized in that, described sealer is bovine serum albumin(BSA) phosphate buffer.
25. methods according to claim 24, is characterized in that, described bovine serum albumin(BSA) phosphate buffer is to obtain by the bovine serum albumin(BSA) of 1～5g/L being dissolved in the phosphate buffer of pH=7.4.
26. methods according to claim 22, is characterized in that, carry out described sealing and process 5～10 minutes.
27. 1 kinds of immune biochips of regeneration, is characterized in that, it is to be obtained by the method described in claim 1-26 any one.
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| Optimizing antibody immobilization strategies for the construction of protein microarrays;Paul Peluso;《Analytical Biochemistry》;20031231;第312卷;第113-124页 * |
| Paul Peluso.Optimizing antibody immobilization strategies for the construction of protein microarrays.《Analytical Biochemistry》.2003,第312卷第113-124页. |
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