CN1055122C - Structural method of anti-body albumen A-DNA probe - Google Patents
Structural method of anti-body albumen A-DNA probe Download PDFInfo
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- CN1055122C CN1055122C CN95111578A CN95111578A CN1055122C CN 1055122 C CN1055122 C CN 1055122C CN 95111578 A CN95111578 A CN 95111578A CN 95111578 A CN95111578 A CN 95111578A CN 1055122 C CN1055122 C CN 1055122C
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Abstract
The present invention relates to a method for constituting antibody / protein A-DNA probes, which belongs to the technical field of biology engineering, wherein antibodies are primary antibodies and secondary antibodies. The method comprises three essential procedures: reacting with anitbodies or protein and p-benzoquinone (PBQ), adding polyethylene imine (PEI) and adding crosslinking agents of glutaraldehyde. The constituted probes have the advantages of good stability, no trouble of adoption of molecule connection and other middle procedures, high reaction sensitivity and accuracy of immune-PCR, little nonspecific amplification, high universality with general chemical agents, etc. The present invention is suitable for constituting various antibody / protein A-DNA probes in different types.
Description
The present invention relates to the construction process of a kind of antibody/albumin A-dna probe, antibody refers to that one is anti-and two anti-, belongs to technical field of bioengineering.
Background technology.In the basic and applied research in fields such as clinical medicine, medical jurisprudence, veterinary science, agronomy and molecular biology, all relate to the detection technique of trace or ultramicron antigen-antibody.On October 2nd, 1992, Tadeshi Sano etc. are at ' science (Science) ' magazine, the 258th volume is delivered the paper that is entitled as ' immunity-polymerase chain reaction: utilize the chimeric highly sensitive Detection of antigen technology of specific antibody-DNA (Immuno-PCR:VerySensilive Anligen Detection by Means of SpecificAntibody-DNA Conjugates) ' on (VOL.258), propose sensitivity high, detect antigenic immunity-polymerase chain reaction (Immuno-PCR).This technology is by using one to DNA and the active link molecule of antibody tool dual combination, be attached to the dna molecular of thing as a token of on the antibody complex specifically, this DNA mark can carry out pcr amplification with special primer, according to the product of specific PCR, whether proves antigenic existence.The advantage of this technology is that the high degree of specificity of antigen antibody reaction in the DNA cloning ability of PCR and the immunology is combined, and has improved the sensitivity that antigen-antibody detects widely, no radiocontamination.The shortcoming of this technology is that the link molecule that is used to make up antibody-dna probe is streptavidin-albumin A fusion rotein, and not commercialization as yet at present is difficult to obtain.
The objective of the invention is to release a kind of easy and direct, have powerful connections technological merit and do not have the construction process of the antibody/albumin A-dna probe of background technology shortcoming.
Now describe technology contents of the present invention in detail.The present invention relates to the construction process of a kind of antibody/albumin A-dna probe, antibody refers to that one is anti-and two anti-, it is characterized in that, this method comprises three essential steps: with antibody or albumin A and para benzoquinone (PBQ) reaction, purport makes antibody or albumin A activation, add polymine (PEI), purport further activates antibody or albumin A, add the linking agent glutaraldehyde, purport makes activatory antibody or albumin A and single strand dna covalent attachment, be built into antibody/albumin A-dna probe, the Overall Steps of this method is enumerated as follows:
The 1st step was got antibody or the albumin A of 0.01～10mg, be dissolved in 440 μ l's, concentration and pH value are respectively in the phosphoric acid buffer of 90m mol/l and 6.0, with antibody is raw material, the probe that finally is built into is antibody-dna probe, with the albumin A is raw material, and the probe that finally is built into is universal albumin A-dna probe;
The 2nd step added the PBQ solution of 120 μ l in the solution of the 1st step preparation, again mixed solution being placed temperature is 37 ℃ dark surrounds, and the reaction times is 1 hour;
The 3rd step is Sepladex G100 post on the reaction solution in the 2nd step, and is that the NaCl solution of 0.1～0.2mol/l is washed post with concentration;
The 4th step was collected the washings 4ml that the 3rd step formed;
The 5th step added 360 μ l's in the solution of the 4th step collection, concentration is the NaHCO of 1mol/l
3Solution;
The 6th step added the PEI of 266 μ g in the solution of the 5th step preparation after, placing temperature is 37 ℃ dark surrounds, and the reaction times is 2 hours;
The 7th step packed the reaction solution in the 6th step in the dialysis tubing into, and the phosphoric acid buffer that concentration and pH value is respectively 5m mol/l and 6.8 is dialysed, and dialysis time is 12 hours;
The 8th step was collected the reaction solution through dialysis in the 7th step, was deposited in temperature and is 4 ℃ environment, and is standby;
The 9th step learnt from else's experience and extracts the plasmid PNC DNA 1 μ g of purifying, was dissolved in 20 μ l, and concentration and pH value are respectively in the phosphoric acid buffer of 5m mol/l and 6.8;
It is 100 ℃ boiling water that the 10th step placed temperature with the solution of the 9th step preparation, and the time is 3 minutes, again solution is placed ice, and the time is 3 minutes, repeats aforesaid operations three times;
The 11st step added the refrigeration solution in the 8th step of 20 μ l in the solution of the 10th step formation;
The 12nd step added 6 μ l's in the mixed solution in the 11st step, concentration is 5% glutaraldehyde solution, hatched 10 minutes under temperature is 37 ℃ environment again;
The reaction solution of the 13rd step with the 12nd step replaces the 2nd reaction solution that goes on foot, and repeats for the 3rd, 4 steps, promptly gets antibody-dna probe or albumin A-dna probe.
Compare with background technology, the present invention has following advantage:
1. antibody/albumin A-the dna probe that makes up with the method that the present invention relates to is a covalent attachment between its antibody/albumin A and the DNA, the good stability of probe.
2. the present invention adopts antibody/albumin A directly to be attached to construction process on the dna molecular, has saved and has adopted the trouble that links molecule and other intermediate steps, has increased the sensitivity and the accuracy of immunity-PCR reaction, has reduced non-specific amplification.
3. the reagent of the present invention's employing is general chemistry reagent, and convenient sources, economy are easy to apply.
4. the method for the structure probe that the present invention relates to has versatility, can be used for the structure of each sharp dissimilar antibody-dna probe.
Therefore, the method that the present invention relates to is particularly suitable for being used for easy and the trace that often relate in the basic and applied research directly fields such as structure detection clinical medicine, medical jurisprudence, veterinary science, agronomy and molecular biology or the antibody/albumin A-dna probe of ultramicron antigen-antibody.
Claims (1)
1. the construction process of antibody/albumin A-dna probe, antibody refers to that one is anti-and two anti-, it is characterized in that, this method comprises three essential steps: with antibody or albumin A and para benzoquinone (PBQ) reaction, add polymine (PEI), add the linking agent glutaraldehyde, the Overall Steps of this method is enumerated as follows:
The 1st step was got antibody or the albumin A of 0.01～10mg, be dissolved in 440 μ l's, concentration and pH value are respectively in the phosphoric acid buffer of 90m mol/l and 6.0, with antibody is raw material, the probe that finally is built into is antibody-dna probe, with the albumin A is raw material, and the probe that finally is built into is universal albumin A-dna probe;
The 2nd step added the PBQ solution of 120 μ l in the solution of the 1st step preparation, again mixed solution being placed temperature is 37 ℃ dark surrounds, and the reaction times is 1 hour;
The 3rd step is Sephadex G 100 posts on the reaction solution in the 2nd step, and is that the NaCl solution of 0.1～0.2mol/l is washed post with concentration;
The 4th step was collected the washings 4ml that the 3rd step formed;
The 5th step added 360 μ l's in the solution of the 4th step collection, concentration is the NaHCO of 1mol/l
3Solution;
The 6th step added the PEI of 266 μ g in the solution of the 5th step preparation after, placing temperature is 37 ℃ dark surrounds, and the reaction times is 2 hours;
The 7th step packed the reaction solution in the 6th step in the dialysis tubing into, and the phosphoric acid buffer that concentration and pH value is respectively 5m mol/l and 6.8 is dialysed, and dialysis time is 12 hours;
The 8th step was collected the reaction solution through dialysis in the 7th step, was deposited in temperature and is 4 ℃ environment, and is standby;
The 9th step learnt from else's experience and extracts the plasmid PNC DNA 1 μ g of purifying, was dissolved in 20 μ l, and concentration and pH value are respectively in the phosphoric acid buffer of 5m mol/l and 6.8;
It is 100 ℃ boiling water that the 10th step placed temperature with the solution of the 9th step preparation, and the time is 3 minutes, again solution is placed ice, and the time is 3 minutes, repeats aforesaid operations three times;
The 11st step added the refrigeration solution in the 8th step of 20 μ l in the solution of the 10th step formation;
The 12nd step added 6 μ l's in the mixed solution in the 11st step, concentration is 5% glutaraldehyde solution, hatched 10 minutes under temperature is 37 ℃ environment again;
The reaction solution of the 13rd step with the 12nd step replaces the 2nd reaction solution that goes on foot, and repeats for the 3rd, 4 steps, promptly gets antibody-dna probe or albumin A-dna probe.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN95111578A CN1055122C (en) | 1995-03-29 | 1995-03-29 | Structural method of anti-body albumen A-DNA probe |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN95111578A CN1055122C (en) | 1995-03-29 | 1995-03-29 | Structural method of anti-body albumen A-DNA probe |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1132250A CN1132250A (en) | 1996-10-02 |
| CN1055122C true CN1055122C (en) | 2000-08-02 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN95111578A Expired - Fee Related CN1055122C (en) | 1995-03-29 | 1995-03-29 | Structural method of anti-body albumen A-DNA probe |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105358984B (en) * | 2013-03-15 | 2020-02-18 | 普罗格诺西斯生物科学公司 | Methods for detecting peptide/MHC/TCR binding |
| CN109569524A (en) * | 2018-11-30 | 2019-04-05 | 广西科技大学 | Fibroin immobilized DNA sorbent preparation method based on glutaraldehyde cross-linking effect and its application in aflatoxin elimination |
-
1995
- 1995-03-29 CN CN95111578A patent/CN1055122C/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| NUCLEIC ACIDS RESEARCH VOL21 NO25 1993.1.1 HONG ZHOU ET AL * |
| NUCLEIC ACIDS RESEARCH VOL21 NO25 1993.1.1 HONG ZHOU ET AL;SCIENCE VOL.260 1993.4.30 TAKESHI SANO ET AL;SCIENCL VOL.258 1992.10.2 TAKESHI SANO ET AL * |
| SCIENCE VOL.260 1993.4.30 TAKESHI SANO ET AL * |
| SCIENCL VOL.258 1992.10.2 TAKESHI SANO ET AL * |
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| CN1132250A (en) | 1996-10-02 |
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