CN103235116A - 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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- CN103235116A CN103235116A CN2013101269044A CN201310126904A CN103235116A CN 103235116 A CN103235116 A CN 103235116A CN 2013101269044 A CN2013101269044 A CN 2013101269044A CN 201310126904 A CN201310126904 A CN 201310126904A CN 103235116 A CN103235116 A CN 103235116A
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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 the method that a kind of antibody marking signal or labeled nucleic acid probe signal amplify, 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, the signal that is used for the immunoassay process amplifies, and belongs to the immuno analytical method field.
Background technology
The antibody amplification system is to have high sensitivity, the detectable material mark of specific reaction that can take place on antibody or antigen molecule, reaction by label, the content that shows detection material, enhancing by signal simultaneously, the signal of amplification detection improves the detection sensitivity of material to be detected.
Present label comprises fluorescein, enzyme (colour developing of color emissivity substrate or chemiluminescence etc.) 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.In addition, the immune marker of use also has chemiluminescent substance, ferritin and collaurum etc.Adopt the detection sensitivity of radioisotope labeling the highest at present, can reach the pg level, additive method remolding sensitivity radioisotope labeling hangs down 1 to 3 order of magnitude.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 aseptic freeze drying product of being made by the blood amebocyte lysate of sea life king crab, containing can be by the proclotting enzyme of micro-bacterial endotoxin and the activation of fungi glucosan, coagulagen, whether contain bacterial endotoxin (hereinafter to be referred as LPS) and (1,3)-callose (hereinafter to be referred as glucosan) accurately and rapidly in the qualitative or quantitative test sample.At present main bacterial detection endotoxin and the method for (1,3)-callose have 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 fungi (1,3)-callose labelled antibody method for amplifying signal or the nucleic acid probe method for amplifying signal of lipopolysaccharides or the fungal cell wall of a kind of gramnegative bacterium.
The present invention is achieved through the following technical solutions above-mentioned purpose:
1, the present invention at first provides the method that a kind of antibody marking signal amplifies, the label that wherein is used for labelled antibody be can with the lipopolysaccharides of the gramnegative bacterium of tachypleus amebocyte lysate generation specific reaction or fungi (1, the 3)-callose of fungal cell wall.
The detection that described signal amplifies is to adopt the bacteria cell wall lipopolysaccharides to connect the detection amplifying signal that is used as the antibody test process with antibody by chemical bond.
The detection method that described signal amplifies is to adopt fungi (1, the 3)-callose of fungal cell wall to connect the detection amplifying signal that is used as the antibody test process with antibody by chemical bond.
Described detection amplifying signal refers to specificity Hirschfeld-Klinger reaction or specificity color reaction that bacteria cell wall lipopolysaccharides and tachypleus amebocyte lysate take place.
Described detection amplifying signal refers to fungi (1, the 3)-callose of fungal cell wall and specificity Hirschfeld-Klinger reaction or the specificity color reaction that tachypleus amebocyte lysate takes place.
2, the present invention also provides the method that a kind of labeled nucleic acid probe signal amplifies in addition, the label that wherein is used for nucleic acid marking be can with the lipopolysaccharides of the gramnegative bacterium of tachypleus amebocyte lysate generation specific reaction or fungi (1, the 3)-callose of fungal cell wall.
The detection method that described signal amplifies is to adopt the bacteria cell wall lipopolysaccharides to connect the detection amplifying signal that is used as the complementary hybridization of nucleic acid testing process with oligonucleotides by chemical bond.
The detection method that described signal amplifies is to adopt fungi (1, the 3)-callose of fungal cell wall to connect with oligonucleotides by chemical bond, as the detection amplifying signal of the complementary hybridization of nucleic acid testing process.
Described detection amplifying signal refers to specificity Hirschfeld-Klinger reaction or the specificity color reaction that bacteria cell wall lipopolysaccharides and tachypleus amebocyte lysate take place.
Described detection amplifying signal refers to fungi (1, the 3)-callose of fungal cell wall and specificity Hirschfeld-Klinger reaction or the specificity color reaction that tachypleus amebocyte lysate takes place.
The concrete collocation method of each solution is as follows among the present invention:
The preparation of LPS solution: take by weighing lipopolysaccharides (LPS) 0.01g, be dissolved in the NaCO of 1 mL 0.1mol/L
3Dissolve in the solution, add the NaIO of the 0.1mol/L of the new configuration of 1 mL then
4Solution, 55 ℃ after water-bath 60-120 minute, add 40 microlitre isopropyl alcohols, put into 4 ℃ of refrigerators 30 minutes, centrifugal 10 minutes of 12000rpm/min abandons supernatant, and precipitation is with 1 mL pure water washing 2 times, each 5 seconds, the NaCO of back adding 1 mL 0.1mol/L
3The solution dissolving, preparation LPS solution.
The preparation of dextran solution: take by weighing glucosan 0.01g, add the NaCO of 1 mL 0.1mol/L
3Solution, fully the back adds the NaIO of the 0.1mol/L of the new configuration of 1 mL
4Solution, 55 ℃ after water-bath 60-120 minute, add 40 microlitre isopropyl alcohols, put into 4 ℃ of refrigerators 30 minutes, centrifugal 10 minutes of 12000rpm/min abandons supernatant, and precipitation is with 1 mL pure water washing 2 times, each 5 seconds, the NaCO of back adding 1 mL 0.1mol/L
3Solution, fully dissolving can make 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 make 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
3The solution dissolving namely gets 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, and on 37 ℃ of shaking tables, the lucifuge reaction is 30-60 minute under the 100-200rpm/min, the antibody linked thing of preparation LPS-.After reaction is finished, add 0.1 g glycocoll, capping 15-30 minute.Be in 10,000 the bag filter, with the phosphate buffer lucifuge dialysed overnight of 1 L, 1 mmol/L pH 7.0 with all solution molecular cut off of packing into.Take out solution in the bag filter, put into reagent bottle, 4 ℃ of refrigerators are preserved.
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, and on 37 ℃ of shaking tables, the lucifuge reaction is 30-60 minute under the 100-200rpm/min, preparation LPS-nucleic acid oligomer cross-linking agent.After reaction is finished, add 0.1 g glycocoll, capping 15-30 minute.Be in 10,000 the bag filter, with the phosphate buffer lucifuge dialysed overnight of 1 L, 1 mmol/L pH 7.0 with all solution molecular cut off of packing into.Take out solution in the bag filter, put into reagent bottle, 4 ℃ of refrigerators are preserved.
The preparation of glucosan-antibody linked thing: dextran solution is mixed by 1 to 1 volume ratio with antibody-solutions, and on 37 ℃ of shaking tables, the lucifuge reaction is 30-60 minute under the 100-200rpm/min, preparation glucosan-antibody linked thing.After reaction is finished, add 0.1 g glycocoll, capping 15-30 minute.Be in 10,000 the bag filter, with the phosphate buffer lucifuge dialysed overnight of 1 L, 1 mmol/L pH7.0 with all solution molecular cut off of packing into.Take out solution in the bag filter, put into reagent bottle, 4 ℃ of refrigerators are preserved.
The preparation of glucosan-nucleic acid oligomer cross-linking agent: dextran solution is mixed by 1 to 0.2 volume ratio with nucleic acid oligomer solution, and on 37 ° of C shaking tables, the lucifuge reaction is 30-60 minute under the 100-200rpm/min, preparation glucosan-nucleic acid oligomer cross-linking agent.After reaction is finished, add 0.1 g glycocoll, capping 15-30 minute.Be in 10,000 the bag filter, with the phosphate buffer lucifuge dialysed overnight of 1 L, 1 mmol/L pH 7.0 with all solution molecular cut off of packing into.Take out solution in the bag filter, put into reagent bottle, 4 ℃ of refrigerators are preserved.
Remarkable advantage of the present invention:
This invention can realize detecting the ultramicro-analysis technology of target product, has highly sensitive and outstanding advantage high specificity.Can reach other species analysis of pg level, reach the sensitiveest radiolabeled detection level now.But compare with the radioactive label technology, advantages such as the label good stability that this technology generates, "dead" pollution have the incomparable advantage of other labeling methods.
The cross-linking method that the present invention relates to is the method that antibody and polysaccharide covalent are combined, and passes through NaIO
4Hydroxyl oxygen on the glycan molecule is changed into aldehyde radical, utilize the amino of the hydroxyl of aldehyde radical and antibody or nucleic acid under alkali condition, cross-linking reaction to take place then.LPS on the cross-linking agent or glucosan by reacting with tachypleus amebocyte lysate, play the effect that signal amplifies.This method can detect other target product of pg level under the condition of 100 times of prepared product dilutions.Antibody of the present invention and polysaccharide crosslinked not merely is limited in cross-linking method described above, and other adopt little numerator mediated antibody link polysaccharide or other method of attachment, can both prepare the product of suitable effect.
Description of drawings
Fig. 1 is the sample distribution figure among enforcement 1 and the embodiment 2, wherein A1, A2, A3 hole are 0.01pg/mL positive criteria sample, A4, A5, A6 hole are 0.1pg/mL positive criteria sample, A7, A8, A9 hole are 1pg/mL positive criteria sample, and A10, A11, A12 hole are 5pg/mL positive criteria sample; B1, B2, B3, B4, B5, B6 hole are the bovine serum albumin(BSA) negative sample of 1 μ g/mL; C1, C2, C3 hole are testing sample 1; C4, C5, C6 hole are testing sample 2; C7, C8, C9 hole are testing sample 3; C10, C11, C12 hole are testing sample 4; D1, D2, D3 hole are testing sample 5; D4, D5, D6 hole are testing sample 6; D7, D8, D9 hole are testing sample 7; D10, D11, D12 hole are testing sample 8; E1, E2, E3 hole are testing sample 9; E4, E5, E6 hole are 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 seen Fig. 1.
The mensuration of carcinomebryonic antigen in the human serum (getting 10 person-portion blood sample of certain hospital's MEC)
The preparation of LPS solution: take by weighing LPS 0.01g, be dissolved in the NaCO of 1 mL 0.1mol/L
3Dissolve in the solution, add the NaIO of the 0.1mol/L of the new configuration of 1 mL then
4Solution, 55 ℃ of water-baths added 40 microlitre isopropyl alcohols after 120 minutes, put into 4 ℃ of refrigerators 30 minutes, and centrifugal 10 minutes of 12000rpm/min abandons the supernatant postprecipitation with 1 mL pure water washing 2 times, in each 5 seconds, used the NaCO of 1 mL 0.1mol/L then
3Dissolve preparation LPS solution in the 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
3The solution dissolving, the preparation antibody-solutions.
The preparation of the antibody linked thing of LPS-: LPS solution is mixed by 1 to 1 volume ratio with antibody-solutions, and on 37 ℃ of shaking tables, the lucifuge reaction is 60 minutes under the 100rpm/min, the antibody linked thing of preparation LPS-.After reaction is finished, add 0.1 g glycocoll, capping 30 minutes.Be in 10,000 the bag filter, with the phosphate buffer lucifuge dialysed overnight of 1 L, 1 mM pH 7.0 with all solution molecular cut off of packing into.Take out solution in the bag filter, put into reagent bottle, sterilized water is settled to 10 mL, and 4 ℃ of refrigerators are preserved stand-by (following used LPS labelled antibody is the cross-linking agent dilution of preparation herein).
The sample detection method:
1) bag quilt:
Positive bag quilt: be cushioned liquid (0.05mol/L sodium carbonate liquor with 0.05mol/L pH9.0 sodium carbonate bag, regulate pH to 9.0 with hydrochloric acid and NaOH) dissolve the alpha-fetoprotein powder, dispose the carcinomebryonic antigen protein solution of 0.01pg/mL, 0.1pg/mL, 1pg/mL, 5pg/mL respectively.With the solution that configures, every hole adds 100 microlitres, and every kind of concentration repeats three holes (place 3 holes respectively, average at last), and 4 ℃ are spent the night.Discarded solution in the hole next day, with lavation buffer solution (phosphate buffer of the 0.01mol/L of pH7.0) washing 3 times, each 3 minutes.(being called for short washing, down together).
Sample well bag quilt: be cushioned liquid (the 0.05mol/L sodium carbonate liquor is regulated pH to 9.0 with hydrochloric acid and NaOH) dilution blood sample with 0.05mol/L pH9.0 sodium carbonate bag, hemodilution to the total protein content of will taking a sample is 10 μ g/ml.The blood sample that dilution is good, every hole adds 100 microlitres, and everyone part repeats three holes (place 3 holes respectively, average at last), and 4 ℃ are spent the night.Discard solution in the hole next day, washs with lavation buffer solution.
Negative hole bag quilt: be cushioned liquid (0.05mol/L sodium carbonate liquor with 0.05mol/L pH9.0 sodium carbonate bag, regulate pH to 9.0 with hydrochloric acid and NaOH) dissolving bovine serum albumin(BSA) powder, configuration concentration is the bovine serum albumin solution of 1 μ g/mL, with the solution that configures, every hole adds 100 microlitres, repeat 6 holes (averaging at last), 4 ℃ are spent the night.Discard solution in the hole next day, washs 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 of anticancer embryonal antigen that adds the mouse source of fresh dilution buys anti-CEA concentrated type antibody).Hatched 1 hour washing for 37 ℃.3) add the LPS labelled antibody: in each reacting hole, add 100 times of LPS labelled antibodies of dilution 0.1ml of the above-mentioned preparation of fresh dilution.Hatched 1 hour washing for 37 ℃.4) decision method 1 as a result: 0.1mL adds in each reacting hole with tachypleus amebocyte lysate (kit is available from Xiamen City's tachypleus amebocyte lysate trial (demonstration) plant company limited), reacts 60 minutes.Polystyrene board is turned over turnback, if to have liquid to flow down negative, to freeze shape positive if be.The result shows that 0.1 pg/mL and 1pg/mL positive hole present positive reaction, shows that this detection method can be as accurate as 0.1pg/mL.In 10 parts of blood samples, gelation shape in the 2 duplicate samples holes, this shows that the carcinomebryonic antigen concentration in this two hole is positive, least concentration is 0.1pg/mL.
The mensuration of carcinomebryonic antigen in the human serum (getting 10 person-portion blood sample of certain hospital's MEC)
(present embodiment is abbreviated as (1,3)-callose; Glucosan) preparation of solution: take by weighing glucosan 0.01g, in the NaCO of 1 mL 0.1mol/L
3Dissolve in the solution, add the NaIO of the 0.1mol/L of the new configuration of 1 mL then
4Solution, 55 ℃ of water-baths added 40 microlitre isopropyl alcohols after 60 minutes, put into 4 ℃ of refrigerators 30 minutes, used 12000rcf more centrifugal 10 minutes, abandoned the supernatant postprecipitation with 1 mL pure water washing 2 times, in each 5 seconds, used the NaCO of 1 mL0.1mol/L then
3Dissolve the preparation dextran solution in the solution.
The preparation of antibody-solutions: get the anti-mouse-anti antibody of rabbit to be marked 0.01g, with the NaCO of 0.5 mL0.1mol/L
3Dissolve the preparation antibody-solutions in the solution.
The preparation of glucosan-antibody linked thing: dextran solution is mixed by 1 to 1 volume ratio with antibody-solutions, and on 37 ℃ of shaking tables, the lucifuge reaction is 30 minutes under the 200rpm/min, preparation glucosan-antibody linked thing.After reaction is finished, add 0.1 g glycocoll, capping 15 minutes.Be in 10,000 the bag filter, with the phosphate buffer lucifuge dialysed overnight of 1 L, 1 mM pH 7.0 with all solution molecular cut off of packing into.Take out solution in the bag filter, put into reagent bottle, sterilized water is settled to 10 mL, and 4 ℃ of refrigerators are preserved stand-by (following used glucosan labelled antibody is the cross-linking agent dilution of preparation herein).
The sample detection method is:
Positive bag quilt: be cushioned liquid (0.05mol/L sodium carbonate liquor with 0.05mol/L pH9.0 sodium carbonate bag, regulate pH to 9.0 with hydrochloric acid and NaOH) dissolve the alpha-fetoprotein powder, dispose the carcinomebryonic antigen protein solution of 0.01pg/mL, 0.1pg/mL, 1pg/mL, 5pg/mL respectively.With the solution that configures, every hole adds 100 microlitres, and every kind of concentration repeats three holes (place 3 holes respectively, average at last), and 4 ℃ are spent the night.Discarded solution in the hole next day, with lavation buffer solution (phosphate buffer of the 0.01mol/L of pH7.0) washing 3 times, each 3 minutes.(being called for short washing, down together).
Sample well bag quilt: be cushioned liquid (the 0.05mol/L sodium carbonate liquor is regulated pH to 9.0 with hydrochloric acid and NaOH) dilution blood sample with 0.05mol/L pH9.0 sodium carbonate bag, hemodilution to the total protein content of will taking a sample is 10 μ g/ml.The blood sample that dilution is good, every hole adds 100 microlitres, and everyone part repeats three holes (place 3 holes respectively, average at last), and 4 ℃ are spent the night.Discard solution in the hole next day, washs with lavation buffer solution.
Negative hole bag quilt: with being cushioned liquid (0.05mol/L sodium carbonate liquor with 0.05mol/L pH9.0 sodium carbonate bag, regulate pH to 9.0 with hydrochloric acid and NaOH) dissolving bovine serum albumin(BSA) powder, configuration concentration is the bovine serum albumin solution of 1 μ g/mL, with the solution that configures, every hole adds 100 microlitres, repeat 6 holes (averaging at last), 4 ℃ are spent the night.Discard solution in the hole next day, washs 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 of anticancer embryonal antigen that adds the mouse source of fresh dilution buys anti-CEA concentrated type antibody).Hatched 0.5 hour washing for 37 ℃.3) add the glucosan labelled antibody: in each reacting hole, add 100 times of glucosan labelled antibodies of dilution 0.1ml of the above-mentioned preparation of fresh dilution.Hatched 0.5 hour washing for 37 ℃.4) decision method 1 as a result: 0.1mL adds in the hand-hole with tachypleus amebocyte lysate (kit is available from Xiamen City's tachypleus amebocyte lysate trial (demonstration) plant company limited), reacts 30 minutes.Polystyrene board is turned over turnback, if to have liquid to flow down negative, to freeze shape positive if be.The result shows that 0.1 pg/mL and 1pg/mL positive hole present positive reaction, shows that this detection method can be as accurate as 0.1pg/mL.In 10 parts of blood samples, gelation shape in the 2 duplicate samples holes, this shows that the carcinomebryonic antigen concentration in this two hole is positive, least concentration is 0.1pg/mL.
The detection of foetal DNA in the maternal peripheral blood (getting certain hospital's healthy pregnant women blood sample, totally 3 person-portions):
Microwell plate is Denmark Nunc company product, and probe is synthetic 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 by weighing LPS 0.01g, be dissolved in the NaCO of 1 mL 0.1mol/L
3Dissolve in the solution, add the NaIO of the 0.1mol/L of the new configuration of 1 mL then
4Solution, 55 ℃ of water-baths are after 60 minutes, add 40 microlitre isopropyl alcohols, put into 4 ℃ of refrigerators 30 minutes, used 12000rpm/min more centrifugal 10 minutes, and abandoned the supernatant postprecipitation and wash 2 times each 5 seconds with 1 mL pure water, dissolve preparation LPS solution then in the NaCO3 solution with 1 mL0.1mol/L.
The preparation of probe solution: get dna probe one pipe (1 OD) to be marked, with the NaCO of 0.1 mL 0.1mol/L
3Dissolve the preparation probe solution in the solution.
The preparation of LPS-probe cross-linking agent: LPS solution is mixed by 1 to 0.2 volume ratio with probe solution, and on 37 ℃ of shaking tables, the lucifuge reaction is 45 minutes under the 150rpm/min, preparation LPS-probe cross-linking agent.After reaction is finished, add 0.1 g glycocoll, capping 20 minutes.Be in 10,000 the bag filter, with the phosphate buffer lucifuge dialysed overnight of 1 L, 1 mM pH7.0 with all solution molecular cut off of packing into.Take out solution in the bag filter, put into reagent bottle, 4 ℃ of refrigerators are preserved stand-by.
The preparation of maternal peripheral blood plasma dna is carried out (TIANGEN Biotech (Beijing) Co., Ltd., the poba gene group is extracted kit (DP332)) by extracting the kit instructions.
Probe card bag quilt: with 95 ℃ of sex change 10 min of probe, and join wrapper sheet liquid (Thermo Scientific company, Pierce DNA wraps by solution); The LPS probe is mixed the back add microwell plate (100 microlitre) with wrapper sheet liquid, hatch 5h for 50 ℃; Wash plate 3 times, wash again 3 times after leaving standstill 5 min; Air dry 3 h, standby.
Probe micropore plate hybridization program: (following positive hole is respectively 10copy, 30copy, 60copy, 90copy, 120copy, 2000copy, each sample 3 hole, totally 18 holes, negative hole 6 holes.）
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, respectively every hole, positive hole adds the DNA(1 * SSCT dissolving with the probe specificity reaction) (concentration is 1 μ g/ml, total amount 100 microlitres); Negative hole is ten thousand/ calf thymus DNA (1 * SSCT dissolving) (concentration is 1 μ g/ml, total amount 100 microlitres); Sample treats that the every hole of gaging hole adds 1 * SSCT, 90 microlitres and total DNA concentration to be measured is 1 μ g/ml extract, 10 microlitres, hatches the every duplicate samples of 1 h(for 48 ℃ and places 3 holes respectively, averages at last); Wash plate 3 times, add tachypleus amebocyte lysate and colour developing liquid 100 microlitres (kit is available from Xiamen City's tachypleus amebocyte lysate trial (demonstration) plant company limited), developed the color 60 minutes, use microplate reader in 450 nm wavelength place measurement results.Detect the OD value greater than negative hole positive hole more than 10 times.Through (concentration with positive hole serves as with reference to the typical curve y=1.137 * light absorption value-8.166 for preparing with positive hole, the y value is the sample concentration value in the formula, unit is copy/mL)) contrast, the foetal DNA in the three person-portion pregnant woman blood of getting is respectively the foetal DNA that every mL contains 92copy, 132copy and 87copy.
The detection of foetal DNA in the maternal peripheral blood (getting certain hospital's healthy pregnant women blood sample, totally 3 person-portions):
Microwell plate is Denmark Nunc company product.Probe is synthetic by American AB I company.Probe sequence: 5 ' A TTC TT C GGC AGC ATC TCT GC 3 ' (3 ' hydroxylation).
(present embodiment is abbreviated as (1,3)-callose; Glucosan) preparation of solution: take by weighing glucosan 0.01g, be dissolved in the NaCO of 1 mL 0.1mol/L
3Dissolve in the solution, add the NaIO of the 0.1mol/L of the new configuration of 1 mL then
4Solution, 55 ℃ after water-bath 60-120 minute, add 40 microlitre isopropyl alcohols, put into 4 ℃ of refrigerators 30 minutes, used 12000rpm/min more centrifugal 10 minutes, wash 2 times with 1 mL pure water after taking out supernatant, in each 5 seconds, use the NaCO of 1 mL0.1mol/L then
3Dissolve the preparation dextran solution in the solution.
The preparation of probe solution: get dna probe one pipe (1 OD) to be marked, with the NaCO of 0.1 mL0.1mol/L
3Dissolve the preparation probe solution in the solution.
The preparation of glucosan-probe cross-linking agent: dextran solution is mixed by 1 to 0.2 volume ratio with probe solution, and on 37 ℃ of shaking tables, the lucifuge reaction is 45 minutes under the 150rpm/min, preparation glucosan-probe cross-linking agent.After reaction is finished, add 0.1 g glycocoll, capping 25 minutes.Be in 10,000 the bag filter, with the phosphate buffer lucifuge dialysed overnight of 1 L, 1 mM pH7.0 with all solution molecular cut off of packing into.Take out solution in the bag filter, put into reagent bottle, 4 ℃ of refrigerators are preserved stand-by.
The preparation of maternal peripheral blood plasma dna is undertaken by extracting the kit instructions.
Probe card bag quilt: with 95 ℃ of sex change 10 min of probe, and (Thermo Scientific company, Pierce DNA wraps by solution to join wrapper sheet liquid.); The glucosan probe is mixed the back add microwell plate (100 microlitre) with wrapper sheet liquid, hatch 5h for 50 ℃; Wash plate 3 times, wash again 3 times after leaving standstill 5 min; Air dry 3 h, standby.
Probe micropore plate hybridization program: (following positive hole is respectively 10copy, 30copy, 60copy, 90copy, 120copy, 2000copy, each sample 3 hole, totally 18 holes, negative hole 6 holes.）
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 every hole respectively adds DNA(1 * SSCT dissolving corresponding and that probe specificity is reacted) (concentration is 1 μ g/ml, total amount 100 microlitres); Negative hole is ten thousand/ calf thymus DNA (1 * SSCT dissolving) (concentration is 1 μ g/ml, total amount 100 microlitres); Sample treats that the every hole of gaging hole adds 1 * SSCT, 90 microlitres and DNA extract to be measured 100 microlitres are hatched 1 h for 48 ℃; Wash plate 3 times, add tachypleus amebocyte lysate and colour developing liquid 100 microlitres (kit is available from Xiamen City's tachypleus amebocyte lysate trial (demonstration) plant company limited), developed the color 20 minutes, use microplate reader in 450 nm wavelength place measurement results.Detect the OD value greater than negative hole positive hole more than 10 times.Through (concentration with positive hole serves as with reference to the typical curve y=3.156 * light absorption value-13.778 for preparing with positive hole, the y value is the sample concentration value in the formula, unit is copy/mL)) contrast, the foetal DNA in the three person-portion pregnant woman blood of getting is respectively the foetal DNA that every mL contains 97copy, 128copy and 82copy.
Claims (10)
1. the antibody marking signal method of amplifying is characterized in that: the label that is used for labelled antibody for can with the lipopolysaccharides of the gramnegative bacterium of tachypleus amebocyte lysate generation specific reaction or fungi (1, the 3)-callose of fungal cell wall.
2. the labeled nucleic acid probe signal method of amplifying is characterized in that: the label that is used for nucleic acid marking for can with the lipopolysaccharides of the gramnegative bacterium of tachypleus amebocyte lysate generation specific reaction or fungi (1, the 3)-callose of fungal cell wall.
3. a kind of antibody marking signal according to claim 1 method of amplifying, it is characterized in that: described detection signal amplification method is to adopt the bacteria cell wall lipopolysaccharides to connect the detection amplifying signal that is used as the antibody test process with antibody by chemical bond.
4. a kind of antibody marking signal according to claim 1 method of amplifying, it is characterized in that: the detection method that described signal amplifies is to adopt fungi (1, the 3)-callose of fungal cell wall to connect the detection amplifying signal that is used as the antibody test process with antibody by chemical bond.
5. a kind of antibody marking signal according to claim 3 method of amplifying is characterized in that: described detection amplifying signal refers to specificity Hirschfeld-Klinger reaction or the specificity color reaction of the generation of bacteria cell wall lipopolysaccharides and tachypleus amebocyte lysate.
6. a kind of antibody marking signal according to claim 4 method of amplifying, it is characterized in that: described detection amplifying signal, refer to fungi (1, the 3)-callose of fungal cell wall and specificity Hirschfeld-Klinger reaction or the specificity color reaction that tachypleus amebocyte lysate takes place.
7. a kind of labeled nucleic acid probe signal according to claim 2 method of amplifying, it is characterized in that: the detection method that described signal amplifies is to adopt the bacteria cell wall lipopolysaccharides to connect with oligonucleotides by chemical bond to be used as the complementary detection amplifying signal of hybridizing testing process of nucleic acid.
8. a kind of labeled nucleic acid probe signal according to claim 2 method of amplifying, it is characterized in that: the detection method that described signal amplifies is the fungi (1 of adopting fungal cell wall, 3)-callose connects with oligonucleotides by chemical bond, as the detection amplifying signal of the complementary hybridization of nucleic acid testing process.
9. a kind of labeled nucleic acid probe signal according to claim 7 method of amplifying is characterized in that: described detection amplifying signal refers to specificity Hirschfeld-Klinger reaction or the specificity color reaction of bacteria cell wall lipopolysaccharides and tachypleus amebocyte lysate generation.
10. a kind of labeled nucleic acid probe signal according to claim 8 method of amplifying, it is characterized in that: described detection amplifying signal refers to fungi (1, the 3)-callose of fungal cell wall and specificity Hirschfeld-Klinger reaction or the specificity color reaction that tachypleus amebocyte lysate takes place.
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