CN103235116B - Method for amplifying antibody marking signal or nucleic acid probe marking signal - Google Patents
Method for amplifying antibody marking signal or nucleic acid probe marking signal Download PDFInfo
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Abstract
The invention discloses a method for amplifying antibody marking signal or nucleic acid probe marking signal, and the main scheme is that: lipopolysaccharide of gram negative bacteria and an antibody are crosslinked, or fungi (1,3)-beta-D-glucan of fungi cell wall and the antibody or a nucleic acid probe are crosslinked, and then tachypleus amebocyte lysate and the gram negative bacteria lipopolysaccharide or fungi cell wall (1,3)-beta-D-glucan which is crosslinked with the antibody are specifically reacted, thereby improving the sensitivity of the antibody or nucleic acid probe detection. The system for amplifying antibody marking signal or nucleic acid probe marking signal can detect object substrates lower than pg level content.
Description
Technical field
The present invention relates to a kind of method of antibody marking signal or the amplification of labeled nucleic acid probe signal, be about to and the lipopolysaccharides (LPS) of the gramnegative bacterium of tachypleus amebocyte lysate generation specific reaction or the fungi (1 of fungal cell wall, 3)-callose labelled antibody or nucleic acid, signal for immunoassay process amplifies, and belongs to technical field of immunoassay.
Background technology
Antibody amplification system to have high sensitivity, the detectable mass signatures that specific reaction can occur on antibody or antigen molecule, by the reaction of label, the content of display detection material, simultaneously by the enhancing of signal, the signal of amplification detection, improves the detection sensitivity of material to be detected.
Current label comprises fluorescein, enzyme (colour developing of color emissivity substrate or chemiluminescence etc.) and radioactive nuclide etc., is called as 3 large immunolabelling techniques with the immunoassay technology that these 3 kinds of labels carry out marking.In addition, the immune marker of use also has chemiluminescent substance, ferritin and collaurum etc.The detection sensitivity of current employing radioisotope labeling is the highest, can reach pg level, low 1 to 3 orders of magnitude of additive method remolding sensitivity radioisotope labeling.The present invention adopts a kind of novel label, can with the molecule of tachypleus amebocyte lysate specific reaction, detection sensitivity is brought up to the level of radioisotope labeling.Tachypleus amebocyte lysate is the sterile cryo dry product be made up of the blood amebocyte lysate of sea life king crab, containing the proclotting enzyme that can be activated by trace of bacteria endotoxin and fungi glucosan, coagulagen, can accurately and rapidly in qualitative or quantitative detection sample whether containing bacterial endotoxin (hereinafter referred to as LPS) and (1,3)-callose (hereinafter referred to as glucosan).The method of bacterial detection endotoxin main at present and (1,3)-callose has gel method, nephelometry and colourimetry.
Summary of the invention
Technical matters to be solved by this invention is: provide a kind of detection sensitivity to reach the immunologic detection method of radioisotope labeling level, the invention provides the lipopolysaccharides of a kind of gramnegative bacterium or fungi (1, the 3)-callose labelled antibody method for amplifying signal of fungal cell wall or nucleic acid probe method for amplifying signal.
The present invention is achieved through the following technical solutions above-mentioned purpose:
1, the present invention provide firstly a kind of method that antibody marking signal amplifies, wherein for the label of labelled antibody be can with fungi (1, the 3)-callose of the lipopolysaccharides of the gramnegative bacterium of tachypleus amebocyte lysate generation specific reaction or fungal cell wall.
The detection that described signal amplifies adopts bacteria cell wall lipopolysaccharides to carry out connecting the detection amplifying signal being used as antibody test process by chemical bond and antibody.
The detection method that described signal amplifies adopts fungi (1, the 3)-callose of fungal cell wall to carry out connecting the detection amplifying signal being used as antibody test process by chemical bond and antibody.
Described detection amplifying signal, refers to the specificity Hirschfeld-Klinger reaction that bacteria cell wall lipopolysaccharides and tachypleus amebocyte lysate occur or specificity color reaction.
Described detection amplifying signal, refers to the specificity Hirschfeld-Klinger reaction that fungi (1, the 3)-callose of fungal cell wall and tachypleus amebocyte lysate occur or specificity color reaction.
2, the present invention still further provides a kind of method that labeled nucleic acid probe signal amplifies, wherein for the label of nucleic acid marking be can with fungi (1, the 3)-callose of the lipopolysaccharides of the gramnegative bacterium of tachypleus amebocyte lysate generation specific reaction or fungal cell wall.
The detection method that described signal amplifies adopts bacteria cell wall lipopolysaccharides to carry out connecting the detection amplifying signal being used as complementary nucleic acid hybridization check process by chemical bond and oligonucleotides.
The detection method that described signal amplifies adopts fungi (1, the 3)-callose of fungal cell wall to be connected with oligonucleotides by chemical bond, as the detection amplifying signal of complementary nucleic acid hybridization check process.
Described detection amplifying signal refers to the specificity Hirschfeld-Klinger reaction that bacteria cell wall lipopolysaccharides and tachypleus amebocyte lysate occur or specificity color reaction.
Described detection amplifying signal refers to the specificity Hirschfeld-Klinger reaction that fungi (1, the 3)-callose of fungal cell wall and tachypleus amebocyte lysate occur or specificity color reaction.
The collocation method that in the present invention, each solution is concrete is as follows:
The preparation of LPS solution: take lipopolysaccharides (LPS) 0.01g, is dissolved in the NaCO of 1 mL 0.1mol/L
3dissolve in solution, then add the NaIO of the 0.1mol/L that 1 mL newly configures
4solution, 55 DEG C after water-bath 60-120 minute, add 40 microliters isopropanol, put into 4 DEG C of refrigerators 30 minutes, centrifugal 10 minutes of 12000rpm/min, abandons supernatant, and precipitation washs 2 times with 1 mL pure water, each 5 seconds, after add the NaCO of 1 mL 0.1mol/L
3solubilize, preparation LPS solution.
The preparation of dextran solution: take glucosan 0.01g, adds the NaCO of 1 mL 0.1mol/L
3solution, adds the NaIO of the 0.1mol/L that 1 mL newly configures fully
4solution, 55 DEG C after water-bath 60-120 minute, add 40 microliters isopropanol, put into 4 DEG C of refrigerators 30 minutes, centrifugal 10 minutes of 12000rpm/min, abandons supernatant, and precipitation washs 2 times with 1 mL pure water, each 5 seconds, after add the NaCO of 1 mL 0.1mol/L
3solution, fully dissolves, and can obtain dextran solution.
The preparation of antibody-solutions: get antibody 0.01g to be marked, with the NaCO of 0.5 mL 0.1mol/L
3solution fully dissolves, and can obtain antibody-solutions.
The preparation of nucleic acid oligomer solution: get nucleic acid oligomer 1 OD to be marked, with the NaCO of 0.1mL 0.1mol/L
3solubilize, obtains nucleic acid oligomer solution.
The preparation of the antibody linked thing of LPS-: LPS solution is mixed by 1 to 1 volume ratio with antibody-solutions, on 37 DEG C of shaking tables, lucifuge reaction 30-60 minute under 100-200rpm/min, the antibody linked thing of preparation LPS-.After having reacted, add 0.1 g glycocoll, capping 15-30 minute.It is in the bag filter of 10,000 that all solution is loaded molecular cut off, by the phosphate buffer lucifuge dialysed overnight of 1 L 1 mmol/L pH 7.0.Take out solution in bag filter, put into reagent bottle, 4 DEG C of Refrigerator stores.
The preparation of LPS-nucleic acid oligomer cross-linking agent: LPS solution is mixed with nucleic acid oligomer solution 1 to 0.2 volume ratio, on 37 DEG C of shaking tables, lucifuge reaction 30-60 minute, preparation LPS-nucleic acid oligomer cross-linking agent under 100-200rpm/min.After having reacted, add 0.1 g glycocoll, capping 15-30 minute.It is in the bag filter of 10,000 that all solution is loaded molecular cut off, by the phosphate buffer lucifuge dialysed overnight of 1 L 1 mmol/L pH 7.0.Take out solution in bag filter, put into reagent bottle, 4 DEG C of Refrigerator stores.
The preparation of glucosan-antibody linked thing: mixed by 1 to 1 volume ratio with antibody-solutions by dextran solution, on 37 DEG C of shaking tables, under 100-200rpm/min, lucifuge reaction 30-60 minute, prepares glucosan-antibody linked thing.After having reacted, add 0.1 g glycocoll, capping 15-30 minute.It is in the bag filter of 10,000 that all solution is loaded molecular cut off, by the phosphate buffer lucifuge dialysed overnight of 1 L 1 mmol/L pH7.0.Take out solution in bag filter, put into reagent bottle, 4 DEG C of Refrigerator stores.
The preparation of glucosan-nucleic acid oligomer cross-linking agent: mixed by 1 to 0.2 volume ratio with nucleic acid oligomer solution by dextran solution, on 37 ° of C shaking tables, under 100-200rpm/min, lucifuge reaction 30-60 minute, prepares glucosan-nucleic acid oligomer cross-linking agent.After having reacted, add 0.1 g glycocoll, capping 15-30 minute.It is in the bag filter of 10,000 that all solution is loaded molecular cut off, by the phosphate buffer lucifuge dialysed overnight of 1 L 1 mmol/L pH 7.0.Take out solution in bag filter, put into reagent bottle, 4 DEG C of Refrigerator stores.
Remarkable advantage of the present invention:
This invention can realize the ultramicro-analysis technology detecting target product, has highly sensitive and outstanding advantages that is high specificity.The species analysis of pg rank can be reached, reach now the sensitiveest radiolabeled detection level.But compared with radioactive labeling techniques, the advantage such as label good stability, no radioactivity pollute that this technology generates, has the advantage that other labeling methods are incomparable.
The cross-linking method that the present invention relates to is method antibody and polysaccharide covalent combined, and passes through NaIO
4hydroxyl oxygen on glycan molecule is changed into aldehyde radical, then utilizes the amino of the hydroxyl of aldehyde radical and antibody or nucleic acid in the basic conditions cross-linking reaction to occur.LPS on cross-linking agent or glucosan, by reacting with tachypleus amebocyte lysate, play the effect that signal amplifies.The method, under prepared product dilutes the condition of 100 times, can detect the target product of pg rank.Antibody of the present invention and the crosslinked of polysaccharide are not merely limited in cross-linking method described above, and other adopt antibody link polysaccharide or other the method for attachment of Small molecular mediation, can prepare the product of suitable effect.
Accompanying drawing explanation
Fig. 1 is the sample distribution figure in enforcement 1 and embodiment 2, wherein A1, A2, A3 hole is 0.01pg/mL positive criteria sample, A4, A5, A6 hole is 0.1pg/mL positive criteria sample, A7, A8, A9 hole is 1pg/mL positive criteria sample, and A10, A11, A12 hole is 5pg/mL positive criteria sample; B1, B2, B3, B4, B5, B6 hole is the bovine serum albumin(BSA) negative sample of 1 μ g/mL; C1, C2, C3 hole is testing sample 1; C4, C5, C6 hole is testing sample 2; C7, C8, C9 hole is testing sample 3; C10, C11, C12 hole is testing sample 4; D1, D2, D3 hole is testing sample 5; D4, D5, D6 hole is testing sample 6; D7, D8, D9 hole is testing sample 7; D10, D11, D12 hole is testing sample 8; E1, E2, E3 hole is testing sample 9; E4, E5, E6 hole is testing sample 10.
Embodiment
Below by concrete exemplifying embodiment, technical scheme of the present invention is described further, but can not limits the scope of the invention with this.The application of sample distribution plan of following embodiment 1 and embodiment 2 is shown in Fig. 1.
embodiment 1
the mensuration (getting the 10 person-portion blood sample at certain Hospital Physical Examination center) of carcinomebryonic antigen in human serum
The preparation of LPS solution: take LPS 0.01g, is dissolved in the NaCO of 1 mL 0.1mol/L
3dissolve in solution, then add the NaIO of the 0.1mol/L that 1 mL newly configures
4solution, 55 DEG C of water-baths, after 120 minutes, add 40 microliters isopropanol, put into 4 DEG C of refrigerators 30 minutes, centrifugal 10 minutes of 12000rpm/min, abandon supernatant postprecipitation 1 mL pure water and wash 2 times, each 5 seconds, then use the NaCO of 1 mL 0.1mol/L
3dissolve in solution, preparation LPS solution.
The preparation of antibody-solutions: get rabbit anti-mouse antibody 0.01g to be marked, with the NaCO of 0.5 mL0.1mol/L
3solubilize, Dispersal risk solution.
The preparation of the antibody linked thing of LPS-: mixed by 1 to 1 volume ratio with antibody-solutions by LPS solution, on 37 DEG C of shaking tables, under 100rpm/min, lucifuge reacts 60 minutes, the antibody linked thing of preparation LPS-.After having reacted, add 0.1 g glycocoll, capping 30 minutes.It is in the bag filter of 10,000 that all solution is loaded molecular cut off, by the phosphate buffer lucifuge dialysed overnight of 1 L 1 mM pH 7.0.Take out solution in bag filter, put into reagent bottle, sterilized water is settled to 10 mL, 4 DEG C of Refrigerator stores stand-by (LPS labelled antibody used is below the cross-linking agent dilution prepared herein).
Sample detection methods:
1) bag quilt:
Positive bag quilt: be buffered liquid (0.05mol/L sodium carbonate liquor with 0.05mol/L pH9.0 sodium carbonate bag, pH to 9.0 is regulated with hydrochloric acid and NaOH) dissolve alpha-fetoprotein powder, configure the CEA protein solution of 0.01pg/mL, 0.1pg/mL, 1pg/mL, 5pg/mL respectively.By the solution configured, every hole adds 100 microlitres, and often kind of concentration repeats three holes (be placed in 3 holes respectively, finally average), and 4 DEG C are spent the night.Next day, discard solution in hole, wash 3 times with lavation buffer solution (phosphate buffer of the 0.01mol/L of pH7.0), each 3 minutes.(being called for short washing, lower same).
Sample well bag quilt: being buffered liquid (0.05mol/L sodium carbonate liquor regulates pH to 9.0 with hydrochloric acid and NaOH) dilution blood sample with 0.05mol/L pH9.0 sodium carbonate bag, is 10 μ g/ml by sampling hemodilution to total protein content.By the blood sample diluted, every hole adds 100 microlitres, and every person-portion repeats three holes (be placed in 3 holes respectively, finally average), and 4 DEG C are spent the night.Next day, discard solution in hole, wash with lavation buffer solution.
Negative hole bag quilt: be buffered liquid (0.05mol/L sodium carbonate liquor with 0.05mol/L pH9.0 sodium carbonate bag, pH to 9.0 is regulated with hydrochloric acid and NaOH) dissolve bovine serum albumin(BSA) powder, configuration concentration is the bovine serum albumin solution of 1 μ g/mL, by the solution configured, every hole adds 100 microlitres, repeat 6 holes (finally averaging), 4 DEG C are spent the night.Next day, discard solution in hole, wash with lavation buffer solution.
2) add first antibody: in each reacting hole, first antibody (diluting 100 times) the 0.1ml(Fuzhou Maixin biotechnology Development Co., Ltd adding the anti-carcinoembryonic antigen in the mouse source of diluted fresh buys, anti-CEA concentrated type antibody).Hatch 1 hour for 37 DEG C, washing.3) add LPS labelled antibody: in each reacting hole, add dilution 100 times of LPS labelled antibody 0.1ml of the above-mentioned preparation of diluted fresh.Hatch 1 hour for 37 DEG C, washing.4) result decision method 1: tachypleus amebocyte lysate (kit, purchased from company limited of tachypleus amebocyte lysate trial (demonstration) plant of Xiamen City) 0.1mL is added in each reacting hole, reacts 60 minutes.Polystyrene board is turned over turnback, if for negative under having liquid stream, if in freezing shape for positive.Result shows, 0.1 pg/mL and 1pg/mL positive hole presents positive reaction, shows that this detection method can be as accurate as 0.1pg/mL.In 10 parts of blood samples, gelation shape in 2 increment sample wells, this shows that the carcinomebryonic antigen concentration in this holes is positive, and least concentration is 0.1pg/mL.
Result decision method 2: tachypleus amebocyte lysate 0.1mL and chromogenic reagent (kit, purchased from company limited of tachypleus amebocyte lysate trial (demonstration) plant of Xiamen City) are added in hand-hole, reacts 60 minutes.Dynamic color method is adopted to detect (spectrophotometer detection).Detect with the concentration in positive hole as the typical curve with reference to preparation is for standard (y=0.1103 × light absorption value-0.0266, in formula, y value is sample concentration value, unit is pg/mL), positive blood tests result (being greater than 0.01pg/mL) has 4 holes, and the content of carcinomebryonic antigen is respectively 0.07 pg/mL, 0.92 pg/mL and 10.3 pg/mL.
embodiment 2
the mensuration (getting the 10 person-portion blood sample at certain Hospital Physical Examination center) of carcinomebryonic antigen in human serum
(1,3) (the present embodiment is abbreviated as-callose; Glucosan) preparation of solution: take glucosan 0.01g, in the NaCO of 1 mL 0.1mol/L
3dissolve in solution, then add the NaIO of the 0.1mol/L that 1 mL newly configures
4solution, 55 DEG C of water-baths, after 60 minutes, add 40 microliters isopropanol, put into 4 DEG C of refrigerators 30 minutes, then use 12000rcf centrifugal 10 minutes, abandon supernatant postprecipitation 1 mL pure water and wash 2 times, in each 5 seconds, then use the NaCO of 1 mL0.1mol/L
3dissolve in solution, prepare dextran solution.
The preparation of antibody-solutions: get rabbit against murine antiantibody 0.01g to be marked, with the NaCO of 0.5 mL0.1mol/L
3dissolve in solution, Dispersal risk solution.
The preparation of glucosan-antibody linked thing: mixed by 1 to 1 volume ratio with antibody-solutions by dextran solution, on 37 DEG C of shaking tables, under 200rpm/min, lucifuge reacts 30 minutes, prepares glucosan-antibody linked thing.After having reacted, add 0.1 g glycocoll, capping 15 minutes.It is in the bag filter of 10,000 that all solution is loaded molecular cut off, by the phosphate buffer lucifuge dialysed overnight of 1 L 1 mM pH 7.0.Take out solution in bag filter, put into reagent bottle, sterilized water is settled to 10 mL, 4 DEG C of Refrigerator stores stand-by (dextran markers antibody used is below the cross-linking agent dilution prepared herein).
Sample detection methods is:
Positive bag quilt: be buffered liquid (0.05mol/L sodium carbonate liquor with 0.05mol/L pH9.0 sodium carbonate bag, pH to 9.0 is regulated with hydrochloric acid and NaOH) dissolve alpha-fetoprotein powder, configure the CEA protein solution of 0.01pg/mL, 0.1pg/mL, 1pg/mL, 5pg/mL respectively.By the solution configured, every hole adds 100 microlitres, and often kind of concentration repeats three holes (be placed in 3 holes respectively, finally average), and 4 DEG C are spent the night.Next day, discard solution in hole, wash 3 times with lavation buffer solution (phosphate buffer of the 0.01mol/L of pH7.0), each 3 minutes.(being called for short washing, lower same).
Sample well bag quilt: being buffered liquid (0.05mol/L sodium carbonate liquor regulates pH to 9.0 with hydrochloric acid and NaOH) dilution blood sample with 0.05mol/L pH9.0 sodium carbonate bag, is 10 μ g/ml by sampling hemodilution to total protein content.By the blood sample diluted, every hole adds 100 microlitres, and every person-portion repeats three holes (be placed in 3 holes respectively, finally average), and 4 DEG C are spent the night.Next day, discard solution in hole, wash with lavation buffer solution.
Negative hole bag quilt: be buffered liquid (0.05mol/L sodium carbonate liquor with 0.05mol/L pH9.0 sodium carbonate bag, pH to 9.0 is regulated with hydrochloric acid and NaOH) dissolve bovine serum albumin(BSA) powder, configuration concentration is the bovine serum albumin solution of 1 μ g/mL, by the solution configured, every hole adds 100 microlitres, repeat 6 holes (finally averaging), 4 DEG C are spent the night.Next day, discard solution in hole, wash with lavation buffer solution.
2) add first antibody: in each reacting hole, first antibody (diluting 100 times) the 0.1ml(Fuzhou Maixin biotechnology Development Co., Ltd adding the anti-carcinoembryonic antigen in the mouse source of diluted fresh buys, anti-CEA concentrated type antibody).Hatch 0.5 hour for 37 DEG C, washing.3) add dextran markers antibody: in each reacting hole, add dilution 100 times of dextran markers antibody 0.1ml of the above-mentioned preparation of diluted fresh.Hatch 0.5 hour for 37 DEG C, washing.4) result decision method 1: tachypleus amebocyte lysate (kit, purchased from company limited of tachypleus amebocyte lysate trial (demonstration) plant of Xiamen City) 0.1mL is added in hand-hole, reacts 30 minutes.Polystyrene board is turned over turnback, if for negative under having liquid stream, if in freezing shape for positive.Result shows, 0.1 pg/mL and 1pg/mL positive hole presents positive reaction, shows that this detection method can be as accurate as 0.1pg/mL.In 10 parts of blood samples, gelation shape in 2 increment sample wells, this shows that the carcinomebryonic antigen concentration in this holes is positive, and least concentration is 0.1pg/mL.
Result decision method 2: tachypleus amebocyte lysate 0.1mL and chromogenic reagent (kit, purchased from company limited of tachypleus amebocyte lysate trial (demonstration) plant of Xiamen City) are added in hand-hole, reacts 30 minutes.Dynamic color method is adopted to detect (spectrophotometer detection).Detect with the concentration in positive hole as the typical curve with reference to preparation is for standard (y=0.2111 × light absorption value-0.2023, in formula, y value is sample concentration value, unit is pg/mL), blood testing 0.1 more than pg/mL concentration presents positive findings, in sample similarly to Example 1, (being greater than 0.1pg/mL) has holes, and the content of carcinomebryonic antigen is respectively 1.12pg/mL and 11.2 pg/mL.
embodiment 3
the detection (getting certain hospital's healthy pregnant women blood sample, totally 3 person-portions) of foetal DNA in maternal peripheral blood:
Microwell plate is Denmark Nunc Products, and probe is synthesized by American AB I company, probe sequence: 5 ' A TTC TT C GGC AGC ATC TCT GC 3 ' (3 ' hydroxylation).
The preparation of LPS solution: take LPS 0.01g, is dissolved in the NaCO of 1 mL 0.1mol/L
3dissolve in solution, then add the NaIO of the 0.1mol/L that 1 mL newly configures
4solution, 55 DEG C of water-baths are after 60 minutes, add 40 microliters isopropanol, put into 4 DEG C of refrigerators 30 minutes, use 12000rpm/min more centrifugal 10 minutes, abandon supernatant postprecipitation 1 mL pure water and wash 2 times, each 5 seconds, then dissolve with in the NaCO3 solution of 1 mL0.1mol/L, preparation LPS solution.
The preparation of probe solution: get DNA probe one to be marked and manage (1 OD), with the NaCO of 0.1 mL 0.1mol/L
3dissolve in solution, prepare probe solution.
The preparation of LPS-probe cross-linking agent: mixed by 1 to 0.2 volume ratio with probe solution by LPS solution, on 37 DEG C of shaking tables, under 150rpm/min, lucifuge reacts 45 minutes, preparation LPS-probe cross-linking agent.After having reacted, add 0.1 g glycocoll, capping 20 minutes.It is in the bag filter of 10,000 that all solution is loaded molecular cut off, by the phosphate buffer lucifuge dialysed overnight of 1 L 1 mM pH7.0.Take out solution in bag filter, put into reagent bottle, 4 DEG C of Refrigerator stores are stand-by.
The preparation of maternal peripheral blood plasma dna is undertaken (TIANGEN Biotech (Beijing) Co., Ltd., poba gene group extracts kit (DP332)) by extraction kit instructions.
Probe card bag quilt: by probe 95 DEG C of sex change 10 min, and join wrapper sheet liquid (Thermo Scientific company, Pierce DNA wraps by solution); Add microwell plate (100 microlitre) after being mixed with wrapper sheet liquid by LPS probe, hatch 5h for 50 DEG C; Wash plate 3 times, after leaving standstill 5 min, wash 3 times again; Natural drying 3 h, for subsequent use.
Needle pinhole plate hybridization procedures: (following positive hole is respectively 10copy, 30copy, 60copy, 90copy, 120copy, 2000copy, each sample 3 hole, totally 18 holes, negative hole 6 hole.）
It is 0.1%Tween-20 that the citric acid three sodium solution of 1 × SSCT(0.015mol/L adds final concentration) wash plate 3 times, positive hole respectively every hole adds the DNA(1 × SSCT reacted with probe specificity and dissolves) (concentration is 1 μ g/ml, total amount 100 microlitre); Negative hole is ten thousand/ calf thymus DNA (1 × SSCT dissolving) (concentration is 1 μ g/ml, total amount 100 microlitre); Sample treats that the every hole of gaging hole adds 1 × SSCT 90 microlitre and STb gene concentration to be measured is 1 μ g/ml extract 10 microlitre, hatches the every increment product of 1 h(for 48 DEG C and is placed in 3 holes respectively, finally average); Wash plate 3 times, add tachypleus amebocyte lysate and nitrite ion 100 microlitre (kit, purchased from company limited of tachypleus amebocyte lysate trial (demonstration) plant of Xiamen City), develop the color 60 minutes, by microplate reader in 450 nm wavelength place measurement results.Detect OD value and be greater than negative hole more than 10 times for positive hole.Pass through and the positive hole (typical curve y=1.137 × light absorption value-8.166 prepared for reference with the concentration in positive hole, in formula, y value is sample concentration value, unit is copy/mL)) contrast, the foetal DNA in the three person-portion blood of pregnant womens got is respectively the foetal DNA of every mL containing 92copy, 132copy and 87copy.
embodiment 4
the detection (getting certain hospital's healthy pregnant women blood sample, totally 3 person-portions) of foetal DNA in maternal peripheral blood:
Microwell plate is Denmark Nunc Products.Probe is synthesized by American AB I company.Probe sequence: 5 ' A TTC TT C GGC AGC ATC TCT GC 3 ' (3 ' hydroxylation).
(1,3) (the present embodiment is abbreviated as-callose; Glucosan) preparation of solution: take glucosan 0.01g, be dissolved in the NaCO of 1 mL 0.1mol/L
3dissolve in solution, then add the NaIO of the 0.1mol/L that 1 mL newly configures
4solution, 55 DEG C after water-bath 60-120 minute, add 40 microliters isopropanol, put into 4 DEG C of refrigerators 30 minutes, then use 12000rpm/min centrifugal 10 minutes, wash 2 times, in each 5 seconds, then use the NaCO of 1 mL0.1mol/L after taking out supernatant with 1 mL pure water
3dissolve in solution, prepare dextran solution.
The preparation of probe solution: get DNA probe one to be marked and manage (1 OD), with the NaCO of 0.1 mL0.1mol/L
3dissolve in solution, prepare probe solution.
The preparation of glucosan-probe cross-linking agent: mixed by 1 to 0.2 volume ratio with probe solution by dextran solution, on 37 DEG C of shaking tables, under 150rpm/min, lucifuge reacts 45 minutes, prepares glucosan-probe cross-linking agent.After having reacted, add 0.1 g glycocoll, capping 25 minutes.It is in the bag filter of 10,000 that all solution is loaded molecular cut off, by the phosphate buffer lucifuge dialysed overnight of 1 L 1 mM pH7.0.Take out solution in bag filter, put into reagent bottle, 4 DEG C of Refrigerator stores are stand-by.
The preparation of maternal peripheral blood plasma dna is undertaken by extraction kit instructions.
Probe card bag quilt: by probe 95 DEG C of sex change 10 min, and (Thermo Scientific company, Pierce DNA wraps by solution to join wrapper sheet liquid.); Add microwell plate (100 microlitre) after being mixed with wrapper sheet liquid by glucosan probe, hatch 5h for 50 DEG C; Wash plate 3 times, after leaving standstill 5 min, wash 3 times again; Natural drying 3 h, for subsequent use.
Needle pinhole plate hybridization procedures: (following positive hole is respectively 10copy, 30copy, 60copy, 90copy, 120copy, 2000copy, each sample 3 hole, totally 18 holes, negative hole 6 hole.）
It is 0.1%Tween-20 that the citric acid three sodium solution of 1 × SSCT(0.015mol/L adds final concentration) wash plate 3 times, positive hole DNA(1 × SSCT that every hole adds accordingly and probe specificity is reacted respectively dissolves) (concentration is 1 μ g/ml, total amount 100 microlitre); Negative hole is ten thousand/ calf thymus DNA (1 × SSCT dissolving) (concentration is 1 μ g/ml, total amount 100 microlitre); Sample treats that the every hole of gaging hole adds 1 × SSCT 90 microlitre and DNA extract 100 microlitre 48 DEG C to be measured hatches 1 h; Wash plate 3 times, add tachypleus amebocyte lysate and nitrite ion 100 microlitre (kit, purchased from company limited of tachypleus amebocyte lysate trial (demonstration) plant of Xiamen City), develop the color 20 minutes, by microplate reader in 450 nm wavelength place measurement results.Detect OD value and be greater than negative hole more than 10 times for positive hole.Pass through and the positive hole (typical curve y=3.156 × light absorption value-13.778 prepared for reference with the concentration in positive hole, in formula, y value is sample concentration value, unit is copy/mL)) contrast, the foetal DNA in the three person-portion blood of pregnant womens got is respectively the foetal DNA of every mL containing 97copy, 128copy and 82copy.
Claims (2)
1. the method for an antibody marking signal amplification, it is characterized in that: described method for amplifying antibody marking signal be adopt can with the fungi (1 of the lipopolysaccharides of the gramnegative bacterium of tachypleus amebocyte lysate generation specific reaction or fungal cell wall, 3)-callose is connected with antibody by chemical bond, as the detection amplifying signal of antibody test process, concrete steps are as follows:
(1) preparation of lipopolysaccharides or (1,3)-callose solution: take lipopolysaccharides or glucosan 0.01g, is dissolved in the Na of 1 mL 0.1mol/L
2cO
3dissolve in solution, then add the NaIO of the 0.1mol/L that 1 mL newly configures
4solution, 55 DEG C after water-bath 60-120 minute, add 40 microliters isopropanol, put into 4 DEG C of refrigerators 30 minutes, centrifugal 10 minutes of 12000rpm/min, abandons supernatant, precipitation washs 2 times with 1 mL pure water, each 5 seconds, after add the Na of 1 mL 0.1mol/L
2cO
3solubilize, prepares lipopolysaccharides or dextran solution;
(2) preparation of antibody-solutions: get antibody 0.01g to be marked, with the Na of 0.5 mL 0.1mol/L
2cO
3solution fully dissolves, and can obtain antibody-solutions;
(3) lipopolysaccharides or (1,3) preparation of-callose-antibody linked thing: lipopolysaccharides or dextran solution are mixed by 1 to 1 volume ratio with antibody-solutions, on 37 DEG C of shaking tables, under 100-200rpm/min, lucifuge reaction 30-60 minute, prepares lipopolysaccharides or glucosan-antibody linked thing; After having reacted, add 0.1 g glycocoll, capping 15-30 minute; It is in the bag filter of 10,000 that all solution is loaded molecular cut off, by the phosphate buffer lucifuge dialysed overnight of 1 L 1 mmol/L pH 7.0; Take out solution in bag filter, put into reagent bottle, 4 DEG C of Refrigerator stores.
2. the method for a labeled nucleic acid probe signal amplification, it is characterized in that: the detection method that described labeled nucleic acid probe signal amplifies be adopt can with the fungi (1 of the lipopolysaccharides of the gramnegative bacterium of tachypleus amebocyte lysate generation specific reaction or fungal cell wall, 3)-callose is connected with oligonucleotides by chemical bond, as the detection amplifying signal of complementary nucleic acid hybridization check process, concrete steps are as follows:
(1) preparation of lipopolysaccharides or (1,3)-callose solution: take lipopolysaccharides or glucosan 0.01g, is dissolved in the Na of 1 mL 0.1mol/L
2cO
3dissolve in solution, then add the NaIO of the 0.1mol/L that 1 mL newly configures
4solution, 55 DEG C after water-bath 60-120 minute, add 40 microliters isopropanol, put into 4 DEG C of refrigerators 30 minutes, centrifugal 10 minutes of 12000rpm/min, abandons supernatant, precipitation washs 2 times with 1 mL pure water, each 5 seconds, after add the Na of 1 mL 0.1mol/L
2cO
3solubilize, prepares lipopolysaccharides or dextran solution;
(2) preparation of nucleic acid oligomer solution: get nucleic acid oligomer 1 OD to be marked, with the Na of 0.1mL 0.1mol/L
2cO3 solubilize, obtains nucleic acid oligomer solution;
(3) lipopolysaccharides or (1,3) preparation of-callose-nucleic acid oligomer cross-linking agent: lipopolysaccharides or dextran solution are mixed with nucleic acid oligomer solution 1 to 0.2 volume ratio, on 37 DEG C of shaking tables, under 100-200rpm/min, lucifuge reaction 30-60 minute, prepares lipopolysaccharides or glucosan-nucleic acid oligomer cross-linking agent; After having reacted, add 0.1 g glycocoll, capping 15-30 minute; It is in the bag filter of 10,000 that all solution is loaded molecular cut off, by the phosphate buffer lucifuge dialysed overnight of 1 L 1 mmol/L pH 7.0, takes out solution in bag filter, puts into reagent bottle, 4 DEG C of Refrigerator stores.
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| CN101292045A (en) * | 2005-10-20 | 2008-10-22 | 株式会社日立制作所 | Method of analyzing nucleic acid |
| CN101400802A (en) * | 2006-01-05 | 2009-04-01 | 凯奇拜基因有限公司 | A quantitative method for HBV-DNA, a primer and probe for detecting HBV-DNA, and a detecting kit comprising the same |
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| CN101400802A (en) * | 2006-01-05 | 2009-04-01 | 凯奇拜基因有限公司 | A quantitative method for HBV-DNA, a primer and probe for detecting HBV-DNA, and a detecting kit comprising the same |
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| Amplification Immunoassay for the Detection of Hepatitis B Surface Antigen.;Atsushi Seki, et al.;《Applied Biochemistry and Biotechnology》;19910331;第27卷(第3期);摘要，第260页第18行-第262页第27行，图1 * |
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