CN101441221A - Method for amplifying antibody marking signal - Google Patents
Method for amplifying antibody marking signal Download PDFInfo
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- CN101441221A CN101441221A CNA2008102434739A CN200810243473A CN101441221A CN 101441221 A CN101441221 A CN 101441221A CN A2008102434739 A CNA2008102434739 A CN A2008102434739A CN 200810243473 A CN200810243473 A CN 200810243473A CN 101441221 A CN101441221 A CN 101441221A
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Abstract
The invention provides a marked-antibody signal amplification method, comprising (1) weighing 0.1g of low-melting-point agarose, dissolving with purified water by water bath, adding 0.1M of NaIO4, reacting for 45-120 mins in water bath, adding 9-15ml of anhydrous alcohol for the generation of precipitate, then carrying out centrifugal separation for 5-20 mins, removing the supernatant liquid, adding the purified water for cleaning the precipitate and repeating twice, after the cleaning is completed, adding purified water for dissolving the precipitate by water bath to produce solution A; (2) taking the antibody to be marked, adjusting the pH value to 8.6-9.6, adding 100-500mul of solution A, stirring uniformly, reacting for 30-60 mins at 37 DEG C to produce solution B; (3) weighing Horseradish Peroxidase (HRP), dissolving it in distilled water, adding 0.1M of NaIO4 solution, reacting for 10-30 mins under the condition of no light, adding ethylene glycol for reacting for 8-15 mins under the condition of no light to produce solution C; (4) mixing the solution B and the solution C in a volume ratio of 1:1-1:3, reacting for 25-45 mins under the condition of no light, adding isovolumetric aldehyde-based confining liquid and reacting for 15-30 mins, then putting all the solutions into a bag filter, dialyzing for 12-24 hours with 1mM of phosphate buffer with the pH of 8.0 under the condition of no light, after the dialysis is finished, taking out the marked antibody from the bag filter. The minimum detection limit may be improved to below 10<-18> mol/l.
Description
Technical field
The present invention relates to a kind of method for amplifying antibody marking signal, belong to the immuno analytical method field.
Background technology
Immunolabelling technique is that not only easily mensuration but also material mark with high susceptibility were on specific antigen or antibody molecule with some, and the enhancing enlarge-effect by these labels shows the character and the content of antigen in the reactive system or antibody.Label commonly used comprises fluorescein, enzyme and radioactive nuclide etc., and the immunoassay technology that carries out mark with these 3 kinds of labels is called as 3 big immunolabelling techniques.All avirulences can have the enzyme of colour chemistry reaction again, and all can be used as mark usefulness in principle, but the enzyme that the antibody that serves as a mark is used should satisfy following requirement: (1) convenient sources is easy to purifying; (2) specific activity height, stable in properties; (3) enzymatic activity and amount can be measured with straightforward procedure.Because (stable, molecular weight is little for Horseradish Peroxidase, HRP) specific activity height, and pure enzyme prepares easily, so the most frequently used for horseradish peroxidase.The detection method of using HRP labelled antibody technology at present detects lower limit and can reach 10
-16Mol/L, but still can't satisfy the detection requirement.
Summary of the invention
Technical matters to be solved by this invention is: provide a kind of realization can make the detection lower limit reach 10
-18The horseradish peroxidase-labeled antibody method for amplifying signal that mol/L is following.
Technical scheme of the present invention is:
(1), take by weighing low melting-point agarose 0.1g, be dissolved in the 10ml purified water, after 45～50 ℃ of water-baths dissolving, add and newly join 0.1MNaIO
45～10ml, 50～60 ℃ of water-baths are after 45～120 minutes, add 9～15ml absolute ethyl alcohol, after precipitation was separated out, centrifugal 5～20 minutes of 8000～12000rpm added 10ml purified water washing and precipitating behind the removal supernatant solution, repetitive operation 2 times, after cleaning end, add 45～50 ℃ of water-bath dissolution precipitations of 50ml purified water, make solution A;
(2), measure antibody 5mg to be marked, transfer pH to 8.6～9.6 with NaOH, add 100～500ul solution A, after stirring, solution B is made in 37 ℃ of reactions 30～60 minutes;
(3), take by weighing the 5mg horseradish peroxidase and be dissolved in the 1ml distilled water, after the dissolving, add the 0.1M NaIO that 100～200ul newly joins fully
4Solution, 30～37 ℃ of lucifuge reactions added 10% ethylene glycol, the 10～20ul that now joins after 10～30 minutes, and 30～37 ℃ of lucifuge reactions were made solution C after 8～15 minutes;
(4), with solution B and solution C by volume 1:1～1:3 mix, 30～37 ℃ of lucifuge reactions are after 25～45 minutes, add 30～37 ℃ of reactions of isopyknic aldehyde radical confining liquid 15-30 minute, to mix back solution then packs in the bag filter, phosphate buffer lucifuge with 1mM PH8.0 was dialysed 12～24 hours, will mix back solution after dialysis finishes and take out from bag filter.
Beneficial effect: method for amplifying antibody marking signal of the present invention, earlier by using NaIO
4The hydroxyl oxygen of agarose is changed into aldehyde radical, utilize the characteristics that contain multidigit point aldehyde radical after the agarose oxidation, under alkali condition with to be marked antibody linked, and then cross-linking antibody carried out the HRP mark, played the signal amplification, under same antigen concentration condition, obtain 5-10 signal doubly and amplify.Method for amplifying signal provided by the invention can bring up to 10 with detecting lower limit by reaching the signal amplification effect with low melting-point agarose crosslinked labeling antibody after oxidation
-18Below the mol/L.
Embodiment
Further specify the specific embodiment of the present invention below in conjunction with embodiment.
Embodiment 1
(1), take by weighing low melting-point agarose 0.1g, be dissolved in the 10ml purified water, after 45 ℃ of water-baths dissolving, add and newly join 0.1M NaIO
45ml, 50 ℃ of water-baths added the 9ml absolute ethyl alcohol after 45 minutes, after precipitation is separated out, centrifugal 5 minutes of 8000rpm adds 10ml purified water washing and precipitating, repetitive operation 2 times behind the removal supernatant solution, after cleaning end, add 45 ℃ of water-bath dissolution precipitations of 50ml purified water, make solution A;
(2), measure antibody 5mg to be marked, transfer pH to 8.6 with NaOH, add the 100ul solution A, after stirring, solution B is made in 37 ℃ of reactions 30 minutes;
(3), take by weighing the 5mg horseradish peroxidase and be dissolved in the 1ml distilled water, after the dissolving, add the 0.1M NaIO that 100ul now joins fully
4Solution, 30 ℃ of lucifuge reactions added and newly join 10% ethylene glycol 10ul after 10 minutes, and 30 ℃ of lucifuge reactions were made solution C after 8 minutes;
(4), with solution B and solution C by volume 1:1 mix, 30 ℃ of lucifuge reactions are after 25 minutes, add 30 ℃ of reactions of isopyknic aldehyde radical confining liquid 15 minutes, to mix back solution then packs in the bag filter, phosphate buffer lucifuge with 1mM PH8.0 was dialysed 12 hours, will mix back solution after dialysis finishes and take out from bag filter.
Embodiment 2
(1), take by weighing low melting-point agarose 0.1g, be dissolved in the 10ml purified water, after 45 ℃ of water-baths dissolving, add and newly join 0.1M NaIO
46ml, 50 ℃ of water-baths added the 10ml absolute ethyl alcohol after 65 minutes, after precipitation is separated out, centrifugal 8 minutes of 9000rpm adds 10ml purified water washing and precipitating, repetitive operation 2 times behind the removal supernatant solution, after cleaning end, add 45 ℃ of water-bath dissolution precipitations of 50ml purified water, make solution A;
(2), measure antibody 5mg to be marked, transfer pH to 8.8 with NaOH, add the 180ul solution A, after stirring, solution B is made in 37 ℃ of reactions 34 minutes;
(3), take by weighing the 5mg horseradish peroxidase and be dissolved in the 1ml distilled water, after the dissolving, add the 0.1M NaIO that 128ul now joins fully
4Solution, 32 ℃ of lucifuge reactions added and newly join 10% ethylene glycol 12ul after 14 minutes, and 32 ℃ of lucifuge reactions were made solution C after 10 minutes;
(4), with solution B and solution C by volume 1:1 mix, 32 ℃ of lucifuge reactions are after 31 minutes, add 32 ℃ of reactions of isopyknic aldehyde radical confining liquid 18 minutes, to mix back solution then packs in the bag filter, phosphate buffer lucifuge with 1mM PH8.0 was dialysed 15 hours, will mix back solution after dialysis finishes and take out from bag filter.
Embodiment 3
(1), take by weighing low melting-point agarose 0.1g, be dissolved in the 10ml purified water, after 48 ℃ of water-baths dissolving, add and newly join 0.1M NaIO
47ml, 54 ℃ of water-baths added the 11ml absolute ethyl alcohol after 82 minutes, after precipitation is separated out, centrifugal 12 minutes of 9800rpm adds 10ml purified water washing and precipitating, repetitive operation 2 times behind the removal supernatant solution, after cleaning end, add 48 ℃ of water-bath dissolution precipitations of 50ml purified water, make solution A;
(2), measure antibody 5mg to be marked, transfer pH to 9.0 with NaOH, add the 280ul solution A, after stirring, solution B is made in 37 ℃ of reactions 40 minutes;
(3), take by weighing the 5mg horseradish peroxidase and be dissolved in the 1ml distilled water, after the dissolving, add the 0.1M NaIO that 144ul now joins fully
4Solution, 34 ℃ of lucifuge reactions added and newly join 10% ethylene glycol 14ul after 18 minutes, and 34 ℃ of lucifuge reactions were made solution C after 10 minutes;
(4), with solution B and solution C by volume 1:2 mix, 34 ℃ of lucifuge reactions are after 35 minutes, add 34 ℃ of reactions of isopyknic aldehyde radical confining liquid 20 minutes, to mix back solution then packs in the bag filter, phosphate buffer lucifuge with 1mM PH8.0 was dialysed 18 hours, will mix back solution after dialysis finishes and take out from bag filter.
Embodiment 4
(1), take by weighing low melting-point agarose 0.1g, be dissolved in the 10ml purified water, after 48 ℃ of water-baths dissolving, add and newly join 0.1M NaIO
48ml, 54 ℃ of water-baths added the 12ml absolute ethyl alcohol after 90 minutes, after precipitation is separated out, centrifugal 16 minutes of 10800rpm adds 10ml purified water washing and precipitating, repetitive operation 2 times behind the removal supernatant solution, after cleaning end, add 48 ℃ of water-bath dissolution precipitations of 50ml purified water, make solution A;
(2), measure antibody 5mg to be marked, transfer pH to 9.2 with NaOH, add the 320ul solution A, after stirring, solution B is made in 37 ℃ of reactions 44 minutes;
(3), take by weighing the 5mg horseradish peroxidase and be dissolved in the 1ml distilled water, after the dissolving, add the 0.1M NaIO that 168ul newly joins fully
4Solution, 37 ℃ of lucifuge reactions added and newly join 10% ethylene glycol 16ul after 22 minutes, and 37 ℃ of lucifuge reactions were made solution C after 12 minutes;
(4), with solution B and solution C by volume 1:2 mix, 37 ℃ of lucifuge reactions are after 38 minutes, add 37 ℃ of reactions of isopyknic aldehyde radical confining liquid 23 minutes, to mix back solution then packs in the bag filter, phosphate buffer lucifuge with 1mM PH8.0 was dialysed 20 hours, will mix back solution after dialysis finishes and take out from bag filter.
Embodiment 5
(1), take by weighing low melting-point agarose 0.1g, be dissolved in the 10ml purified water, after 50 ℃ of water-baths dissolving, add and newly join 0.1M NaIO
49ml60 ℃ of water-bath added the 14ml absolute ethyl alcohol after 106 minutes, after precipitation is separated out, centrifugal 18 minutes of 11200rpm adds 10ml purified water washing and precipitating, repetitive operation 2 times behind the removal supernatant solution, after cleaning end, add 50 ℃ of water-bath dissolution precipitations of 50ml purified water, make solution A;
(2), measure antibody 5mg to be marked, transfer pH to 9.4 with NaOH, add the 440ul solution A, after stirring, solution B is made in 37 ℃ of reactions 52 minutes;
(3), take by weighing the 5mg horseradish peroxidase and be dissolved in the 1ml distilled water, after the dissolving, add the 0.1M NaIO that 188ul now joins fully
4Solution, 37 ℃ of lucifuge reactions added and newly join 10% ethylene glycol 1gul after 26 minutes, and 37 ℃ of lucifuge reactions were made solution C after 15 minutes;
(4), with solution B and solution C by volume 1:3 mix, 37 ℃ of lucifuge reactions are after 42 minutes, add 37 ℃ of reactions of isopyknic aldehyde radical confining liquid 26 minutes, to mix back solution then packs in the bag filter, phosphate buffer lucifuge with 1mM PH8.0 was dialysed 22 hours, will mix back solution after dialysis finishes and take out from bag filter.
Embodiment 6
(1), take by weighing low melting-point agarose 0.1g, be dissolved in the 10ml purified water, after 50 ℃ of water-baths dissolving, add and newly join 0.1M NaIO
460 ℃ of water-baths of 10ml added the 15ml absolute ethyl alcohol after 120 minutes, after precipitation is separated out, centrifugal 20 minutes of 12000rpm adds 10ml purified water washing and precipitating, repetitive operation 2 times behind the removal supernatant solution, after cleaning end, add 50 ℃ of water-bath dissolution precipitations of 50ml purified water, make solution A;
(2), measure antibody 5mg to be marked, transfer pH to 9.6 with NaOH, add the 500ul solution A, after stirring, solution B is made in 37 ℃ of reactions 60 minutes;
(3), take by weighing the 5mg horseradish peroxidase and be dissolved in the 1ml distilled water, after the dissolving, add the 0.1M NaIO that 200ul now joins fully
4Solution, 37 ℃ of lucifuge reactions added and newly join 10% ethylene glycol 20ul after 30 minutes, and 37 ℃ of lucifuge reactions were made solution C after 15 minutes;
(4), with solution B and solution C by volume 1:3 mix, 37 ℃ of lucifuge reactions are after 45 minutes, add 37 ℃ of reactions of isopyknic aldehyde radical confining liquid 30 minutes, to mix back solution then packs in the bag filter, phosphate buffer lucifuge with 1mM PH8.0 was dialysed 24 hours, will mix back solution after dialysis finishes and take out from bag filter.
Table 1 is the part of detecting result of embodiment of the invention 1-6 and conventional labeling method
The raw material that is used in the table 1 test is respectively:
Ankyrin: AFP (Alpha-Fetoprotein) antibody (the rich match in Henan biological production);
Labelled antibody: AFP second antibody (the rich match in Henan biological production);
Horseradish Peroxidase (HRP): pierce company produces;
Used general chemical reagent is analyzes pure rank;
Reaction washing lotion: 0.1mol/l Tris-Hcl (biological production of U.S. season of Shanghai);
Luminous substrate: SuperSignal ELISA Femto Maximum Sensitivity Substrate (U.S. pierce production);
Centrifugal dryer: produce for my company designs voluntarily voluntarily.
Specking spotting robot with the U.S. biodot company during test is put ankyrin after surface of glass slide, reads signal with the chip reaction respectively with the antibody of mark among the embodiment and conventional labeling method, analyzes.
| Embodiment | 1 | 2 | 3 | 4 | 5 | 6 | Conventional method |
| Signal value | 1860 | 1920 | 2060 | 2260 | 2180 | 1960 | 220 |
Claims (1)
1, a kind of method for amplifying antibody marking signal is characterized in that, comprises the steps:
(1), take by weighing low melting-point agarose 0.1g, be dissolved in the 10ml purified water, after 45～50 ℃ of water-baths dissolving, add and newly join 0.1M NaIO
45～10ml, 50～60 ℃ of water-baths are after 45～120 minutes, add 9～15ml absolute ethyl alcohol, after precipitation was separated out, centrifugal 5～20 minutes of 8000～12000rpm added 10ml purified water washing and precipitating behind the removal supernatant solution, repetitive operation 2 times, after cleaning end, add 45～50 ℃ of water-bath dissolution precipitations of 50ml purified water, make solution A;
(2), measure antibody 5mg to be marked, transfer pH to 8.6～9.6 with NaOH, add 100～500ul solution A, after stirring, solution B is made in 37 ℃ of reactions 30～60 minutes;
(3), take by weighing the 5mg horseradish peroxidase and be dissolved in the 1ml distilled water, after the dissolving, add the 0.1M NaIO that 100～200ul newly joins fully
4Solution, 30～37 ℃ of lucifuge reactions added and newly join 10% ethylene glycol, 10～20ul after 10～30 minutes, and 30～37 ℃ of lucifuge reactions were made solution C after 8～15 minutes;
(4), with solution B and solution C by volume 1:1～1:3 mix, 30～37 ℃ of lucifuge reactions are after 25～45 minutes, add 30～37 ℃ of reactions of isopyknic aldehyde radical confining liquid 15-30 minute, to mix back solution then packs in the bag filter, phosphate buffer lucifuge with 1mM PH8.0 was dialysed 12～24 hours, will mix back solution after dialysis finishes and take out from bag filter.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101825628A (en) * | 2010-05-04 | 2010-09-08 | 武汉伊艾博科技有限公司 | Competitive immunological detection kit produced by antibody univalent polymerized marking method, use method thereof and application thereof |
| CN102809648A (en) * | 2011-05-31 | 2012-12-05 | 深圳出入境检验检疫局食品检验检疫技术中心 | High-sensitivity immunoassay method for enzyme signal cyclic amplification |
| CN114397464A (en) * | 2022-03-24 | 2022-04-26 | 天津德祥生物技术有限公司 | Coupling compound of erythrocyte membrane fragments and carrier, coupling method and application thereof |
-
2008
- 2008-12-19 CN CNB2008102434739A patent/CN100559183C/en active Active
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101825628A (en) * | 2010-05-04 | 2010-09-08 | 武汉伊艾博科技有限公司 | Competitive immunological detection kit produced by antibody univalent polymerized marking method, use method thereof and application thereof |
| CN102809648A (en) * | 2011-05-31 | 2012-12-05 | 深圳出入境检验检疫局食品检验检疫技术中心 | High-sensitivity immunoassay method for enzyme signal cyclic amplification |
| CN102809648B (en) * | 2011-05-31 | 2015-11-25 | 深圳出入境检验检疫局食品检验检疫技术中心 | A kind of enzyme signal cycle amplifies High-sensitivity immunoassay method |
| CN114397464A (en) * | 2022-03-24 | 2022-04-26 | 天津德祥生物技术有限公司 | Coupling compound of erythrocyte membrane fragments and carrier, coupling method and application thereof |
| CN114397464B (en) * | 2022-03-24 | 2022-06-10 | 天津德祥生物技术有限公司 | Coupling compound of erythrocyte membrane fragments and carrier, coupling method and application thereof |
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Denomination of invention: Method for amplifying antibody marking signal Effective date of registration: 20190726 Granted publication date: 20091111 Pledgee: Agricultural Bank of China Limited by Share Ltd Wuxi science and Technology Branch Pledgor: Jiangsu Sanlian Bioengineering Co., Ltd. Registration number: 2019990000792 |
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Date of cancellation: 20200810 Granted publication date: 20091111 Pledgee: Agricultural Bank of China Limited by Share Ltd. Wuxi science and Technology Branch Pledgor: SUNLANT BIOLOGICAL ENGINEERING Co.,Ltd. Registration number: 2019990000792 |
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Address after: No. 35-305, Changjiang South Road, Xinwu District, Wuxi, Jiangsu 214111 Patentee after: Jiangsu Sanlian Bioengineering Co.,Ltd. Address before: A science and Technology Park Comprehensive incubator building Xin'an District Road 214101 Jiangsu Xingyuan University City of Wuxi Province, first floor Patentee before: SUNLANT BIOLOGICAL ENGINEERING CO.,LTD. |
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