CN103293318B - Method for detecting miRNAs (micro ribonucleic acids) by DIG labeling EDC cross-linking bridging method - Google Patents

Abstract

The invention discloses a method for detecting miRNAs by a DIG labeling EDC (carbodiimide) cross-linking bridging method. Once a DIG labeling system is utilized, the sensitivity of the DIG labeling system is improved by at least 50 times in contrast with that of a traditional DIG labeling Northern blots detection system; and the sensitivity of the DIG labeling system is equivalent with that of a radioisotope labeling Northern blots detection system; the method cannot cause any pollution on environment, and is very safe and free of a hybridization step; all operations can be completed in 9-10 hours; and therefore, the method is convenient, quick, and low in detection cost. As a result, the problems of low safety, complex procedure, high price, low sensitivity and the like of a current detection method are solved.

Description

Utilize digoxigenin labeled EDC cross-bridge connection to detect miRNAs method

Technical field

The invention provides one and utilize digoxigenin labeled EDC cross-bridge connection to detect miRNAs method, can on-radiation, fast and high-sensitivity detection microRNAs.

Background technology

In recent years, in biomedical research, one of most important discovery was exactly to find the non-coding microRNAs(miRNAs of size for about 22nt).These microRNAs are not translated into albumen, but can be matched and be acted on mRNA by base complementrity, make target mRNA degraded or translate suppressed.According to estimates, mammalian cell only has about 1% genome encoding miRNAs molecule, and these miRNAs regulate and control the encoding gene of mammal 1/3, it serves to show its importance to cell activities.

Research miRNA spatial-temporal expression spectrum in vivo can make researcher understand better the biological function of miRNAs.In order to reach this purpose, existing many technology are applied to the detection of miRNAs and other microRNAs s, comprise high-flux detection method based on micro-array chip and Microspheres Technique, rolling circle amplification method, primer invasion method, ELISA detection method, bead detection method, detection method based on fluorescence labeling technology and molecule detection.These technology or instrument that need to be special and expensive, or complex steps.Detecting at present the simple and the most most widely used method of microRNA is the Northern blots detection of application of radiation isotope labeling system.But, not only inconvenience but also dangerous of radioactive isotope, and many places do not use isotopic condition, and therefore the isotopic Northern blots of application of radiation detection method is very limited.On the contrary, compared with isotope labeling system, digoxin (DIG) Mk system has many advantages: the time shutter is short, and the storage time is long, and economy is especially safe.But the sensitivity of DIG Mk system, far away from labelled with radioisotope system, even if increase RNA amount, also cannot detect the microRNAs that expression is low sometimes.Although the rna probe of Kajigaya and colleague's application 3 '-DIG mark thereof can be brought up to the same with isotope the sensitivity that detects miRNA, but the miRNAs that some low abundance are expressed still cannot detect, and not only costliness but also be difficult for storage-stable of rna probe.The human hairs such as Bino John understand that a kind of LED method detects miRNAs, the method combines LNA and modifies probe, EDC is cross-linked and DIG Mk system, make not only safety but also have very high sensitivity of the method, but because LNA modification probe is synthetic all very expensive from being designed into, therefore these methods are not all used widely.

In view of traditional Northern blots detection method test period long, complex steps, has scholar to apply a kind of bridging technology for detection Microrna in recent years.One of this Technology Need design detects the special oligonucleotide fragment (bridge-clip) of miRNA, this fragment 3 ' end and the miRNA reverse complemental detecting, and 5 ' end is one and 5 ' to hold ³²the sequence of the random fragment reverse complemental of P mark.When renaturation, this bridge-clip can with miRNA and ³²the Probe Hybridization of P mark, thus both are pulled in together, centre only has a breach, and this breach can be repaired by T4 DNA ligase, thereby has made on miRNA mark ³²p.Bridging method is more convenient than traditional Northern blots, and sensitivity simultaneously, also than traditional at least 50 times of left and right of Northern blots method height, detects RNA concentration range also high 40 times than classic method.

By certain methods, RNA is linked to the sensitivity that can improve Northern blots method on film.But, some traditional methods such as, UV-crosslinked method but can not be cross-linked Microrna well.

Summary of the invention

The object of the invention is open one and utilize digoxigenin labeled EDC cross-bridge connection to detect miRNAs method, solved the problems such as current detection method security is not high, program is complicated, expensive, sensitivity is low.

The digoxigenin labeled EDC cross-bridge connection that utilizes provided by the invention detects miRNAs method (hereinafter to be referred as DSLE method), comprises the following steps:

1) extraction of sample cell total rna

With Trizol extraction sample cell total rna.

2) the catching and be connected of miRNA

First carry out catching of miRNA, system is: 7 μ l RNA+ddH ₂o, 1 μ l 1pmol/ μ l Bridge, 1 μ l 10 × Capture buffer, 1 μ l 1pmol/ μ l 3 ' DIG-probe, mixes, and carries out following program in PCR instrument: 94 DEG C, 1min; 65 DEG C, 2min; 37 DEG C, 10min.Then connect to adding T4 DNA ligase potpourri in above-mentioned reaction, system is: 1 μ l T4 DNA ligase, 1.5 μ l 10 × T4 buffer, 2.5 μ l ddH ₂o; At 30 DEG C, react 1h, this reaction product is final application of sample sample;

3) containing 15% polyacrylamide gel electrophoresis of 8M urea

(1) prepare according to following ratio the 15%PAGE glue that contains 8M urea: 3.75ml 40% acrylamidel, 1ml 5 × TBE, 4.2g Urea, 45 μ l 10%APS, 10 μ l TEMED, finally add water and mend to 10ml, mix rear encapsulating;

(2) preparation of samples: the sample equivalent in previous step and RNA loading buffer are mixed;

(3) sex change: evenly biased sample, 95 DEG C of sex change, after 3 minutes, are placed immediately on ice;

(4) prerunning: electrophoretic apparatus is correctly installed, is poured into 0.5 × TBE electrophoretic buffer, with the cull in syringe piping and druming electrophoresis hole, 120V, prerunning 20 minutes;

(5) point sample: again blow and beat electrophoresis hole, point sample with syringe after prerunning;

(6) constant voltage electrophoresis: 120V electrophoresis approximately 2.5 hours, bromjophenol blue is gone to glue bottom;

4) transferring film

According to the area of glue, the positively charged nylon membrane that cutting is onesize and WATMAN filter paper, make sandwich-like, by half-dried transfer instrument transferring film: transferring film 30 minutes;

5) EDC is crosslinked: after transferring film, film is taken out, be placed on the WATMAN filter paper that is soaked with EDC cross-linking reagent, 60 DEG C of effect 1-2h;

6) sealing

Film is put into 1% Blocking buffer, room temperature sealing 0.5 hour;

7) add antibody

Add the anti digoxin antibody (1:15000) in 1% Blocking of alkali phosphatase enzyme mark, incubated at room 0.5 hour;

8) washing

Washing buffer (Maleic Acid Buffer (pH7.5), 0.3% Tween 20 (v/v)) washes film 2 times, each 15 minutes;

Detecting buffer(Detecting buffer:6.055gTris and 2.9gNaCl add in 480ml water, are adjusted to PH 9.5 with concentrated hydrochloric acid, and constant volume is to 500ml) wash film 1 time, each 5 minutes;

9) develop

(1) the addition of C DP-Star ready-to-use reagent is put in film front, guarantees to be all paved with on film, and incubated at room squeezed out reagent after 5 minutes, blots raffinate with filter paper, puts into magazine X-ray and exposes;

(2) X-ray is taken out from magazine, put into D76 developer solution 1-2 minute, put into tap water and stop shadow, then put into stop bath photographic fixing 15 minutes, finally washing is dried.

good effect of the present invention is:

Overcome the shortcoming of traditional detection Microrna method, application digoxigenin labeled system, remolding sensitivity tradition digoxigenin labeled Northern blots detection system is at least high 50 times, suitable with labelled with radioisotope Northern blots detection system; Can not pollute environment, very safe, simultaneously without hybridization step, can within 9-10h, complete, fast and easy, testing cost is lower.

brief description of the drawings:

Fig. 1. the present invention detects HeLa cell hsa-miR-21 result;

Fig. 2. the present invention detects Giardia miR2 result;

Fig. 3. the present invention detects the absolute sensitivity analysis result of hsa-miR-21;

Fig. 4. the present invention and the detection comparative result of traditional digoxigenin labeled Northern blots detection system to hsa-miR-21 in HeLa cell total rna;

Fig. 5. the detection limit analysis of the present invention to hsa-miR-21 RNA in total HaLa cell RNA.

Embodiment

The following example is intended to further illustrate, instead of restriction the present invention.It will be appreciated by those skilled in the art that, do not deviating under the prerequisite of the spirit and principles in the present invention, all will fall into of the present invention awaiting the reply within the scope of claim to any parallel change of the present invention and change.

embodiment 1

dSLE method detects HeLa cell hsa-miR-21

In order to verify the feasibility of DSLE method, the hsa-miR-21 that author is chosen in high expressed in Hela cell is research object, and its sequence is 5'-phos-uagcuuaucagacugauguuga-3', and corresponding hsa-miR-21 Bridge sequence is

5'-GAATGTCATAAGCGTCAACATCAGTCTGATAAGCTA-3'；3’DIG-probe

For 5'-phos-CGCTTATGACATTC-DIG-3', synthetic by Shanghai bioengineering company limited.

1) extraction of HeLa cell total rna

With Trizol extraction HeLa cell total rna, carry out to specifications, recording concentration is 1 μ g/ μ l.

2) the catching and be connected of hsa-miR-21

Get respectively 2 μ g and the total RNA of 1 μ g for the experiment of catching of miRNAs, set up the negative control group that does not add RNA or Bridge simultaneously, the system of catching is as follows: 2 μ l HeLa RNA+5 μ l ddH ₂o (Rnase free) or 1 μ l HeLa RNA+6 μ l ddH ₂o (Rnase free), 1 μ l 1pmol/ μ l hsa-miR-21 Bridge, 1 μ l 10 × Capture buffer, 1 μ l 1pmol/ μ l 3 ' DIG-probe(negative control group RNA or Bridge water replace), mix, in PCR instrument, carry out following program: 94 DEG C, 1min; 65 DEG C, 2min; 37 DEG C, 10min.Then connect to adding T4 DNA ligase potpourri in above-mentioned reaction, system is: 1 μ l T4 DNA ligase, 1.5 μ l 10 × T4 buffer, 2.5 μ l ddH ₂o (Rnase free) reacts 1h after mixing at 30 DEG C.This reaction product is final loading sample.

3) containing 15% polyacrylamide gel electrophoresis of 8M urea

(1) prepare according to following ratio the 15%PAGE glue that contains 8M urea: 3.75ml 40% acrylamidel(Arc/ Bis=19/1), 1ml 5 × TBE, 4.2g Urea, 45 μ l 10%APS, 10 μ l TEMED, finally add water and mend to 10ml, mix rear encapsulating.

(2) preparation of samples: the 15 μ l of the sample equivalent in previous step and 15 μ l RNA loading buffer are mixed;

(3) sex change: evenly biased sample, 95 DEG C of sex change, after 3 minutes, are placed immediately on ice;

(4) prerunning: electrophoretic apparatus is correctly installed, is poured into 0.5 × TBE electrophoretic buffer, with the cull in syringe piping and druming electrophoresis hole, 120V, prerunning 20 minutes;

(5) point sample: again blow and beat electrophoresis hole, point sample with syringe after prerunning;

(6) constant voltage electrophoresis: 120V electrophoresis approximately 2.5 hours, bromjophenol blue is gone to glue bottom;

4) transferring film

According to the area of glue 30 cm ², the positively charged nylon membrane that cutting is onesize and WATMAN filter paper, made sandwich-like, by half-dried transfer instrument transferring film constant current 90mA transferring film 30 minutes;

5) EDC is crosslinked: after transferring film, film is taken out, be placed on the WATMAN filter paper that is soaked with EDC cross-linking reagent, 60 DEG C of effect 1-2h.

6) sealing

Film is put into 1% Blocking buffer, room temperature sealing 0.5 hour;

7) add antibody

Film is placed on to the anti digoxin antibody (1:15000) in 1% Blocking that adds alkali phosphatase enzyme mark, incubated at room 0.5 hour;

8) washing

Washing buffer washes film 2 times, each 15 minutes;

Detecting buffer washes film 1 time, each 5 minutes.

9) develop

(1) the addition of C DP-Star ready-to-use reagent is put in film front, guarantees to be all paved with on film, and incubated at room squeezed out reagent after 10 minutes, blots raffinate with filter paper, puts into magazine X-ray exposure 10 minutes;

(2) X-ray is taken out from magazine, put into D76 developer solution (the about 20 DEG C of left and right of temperature) 1-2 minute, put into tap water and stop shadow, then put into stop bath photographic fixing 15 minutes, finally washing is dried.

Result shows that DSLE method can detect hsa-miR-21 from HeLa cell total rna, and specific band (is shown in accompanying drawing 1: application DSLE method detects hsa-miR-21 from HeLa cell total rna nothing but, No RNA and the negative control group of No Bridge), negative control group, without band, illustrates that DSLE method has specificity.

embodiment 2

dSLE method detects low abundancegiardia miRNA

Giardia miR2 is by giardia lamblia snoRNA17(GlsR17) produce, research shows that its expression in On The Trophozoite of Giardia Lamblia is very low, traditional radioactivity Northern blots method cannot detect this miRNA.In order to verify that can DSLE method detect the miRNA that low abundance is expressed, this experiment, taking Giardia miR2 as research object, is carried out the detection of DSLE method.Giardia miR2 sequence is 5'-CAGCCUAAUCACCGCCCCUAUAGUCC-3', and corresponding Giardia miR2 Bridge sequence is

5'-GAATGTCATAAGCGGGACTATAGGGGCGGTGATTAGGCTG-3';?3’DIG-probe

For 5'-phos-CGCTTATGACATTC-DIG-3', synthetic by Shanghai bioengineering company limited.

1) extraction of Giardia cell total rna

With the total RNA of Trizol extraction Giardia lamblia trophozoite, carry out to specifications, recording concentration is 1 μ g/ μ l.

2) the catching and be connected of Giardia miR21

Get 5 μ g Giardia lamblia RNA for catching experiment, the system of catching is as follows: 5 μ l Giardia lamblia RNA, 2 μ l ddH ₂o (Rnase free), 1 μ l 1pmol/ μ l Giardia miR2 Bridge, 1 μ l 10 × Capture buffer, 1 μ l 1pmol/ μ l 3 ' DIG-probe, mixes, and carries out following program in PCR instrument: 94 DEG C, 1min; 65 DEG C, 2min; 37 DEG C, 10min.Then connect to adding T4 DNA ligase potpourri in above-mentioned reaction, system is: 1 μ l T4 DNA ligase, 1.5 μ l 10 × T4 buffer, 2.5 μ l ddH ₂o (Rnase free) reacts 1h after mixing at 30 DEG C.This reaction product is final loading sample.

3) containing 15% polyacrylamide gel electrophoresis of 8M urea

(1) prepare according to following ratio the 15%PAGE glue that contains 8M urea: 3.75ml 40% acrylamidel(Arc/ Bis=19/1), 1ml 5 × TBE, 4.2g Urea, 4513578985885 Xu Meng l 10%APS, 1013578985885 Xu Meng l TEMED, finally add water and mend to 10ml, mix rear encapsulating.

(2) preparation of samples: the 15 μ l of the sample equivalent in previous step and 15 μ l RNA loading buffer are mixed;

(3) sex change: evenly biased sample, 95 DEG C of sex change, after 3 minutes, are placed immediately on ice;

(4) prerunning: electrophoretic apparatus is correctly installed, is poured into 0.5 × TBE electrophoretic buffer, with the cull in syringe piping and druming electrophoresis hole, 120V, prerunning 20 minutes;

(5) point sample: again blow and beat electrophoresis hole, point sample with syringe after prerunning;

(6) constant voltage electrophoresis: 120V electrophoresis approximately 2.5 hours, bromjophenol blue is gone to glue bottom;

4) transferring film

According to the area of glue 10 cm ², the positively charged nylon membrane that cutting is onesize and WATMAN filter paper, made sandwich-like, by half-dried transfer instrument transferring film constant current 30mA transferring film 30 minutes;

5) EDC is crosslinked: after transferring film, film is taken out, be placed on the WATMAN filter paper that is soaked with EDC cross-linking reagent, 60 DEG C of effect 1-2h.

6) sealing

Film is put into 1% Blocking buffer, room temperature sealing 0.5 hour;

7) add antibody

Film is placed on to the anti digoxin antibody (1:15000) in 1% Blocking that adds alkali phosphatase enzyme mark, incubated at room 0.5 hour;

8) washing

Washing buffer washes film 2 times, each 15 minutes;

Detecting buffer washes film 1 time, each 5 minutes.

9) develop

(1) the addition of C DP-Star ready-to-use reagent is put in film front, guarantees to be all paved with on film, and incubated at room squeezed out reagent after 10 minutes, blots raffinate with filter paper, puts into magazine X-ray exposure 10 minutes;

(2) X-ray is taken out from magazine, put into D76 developer solution (the about 20 DEG C of left and right of temperature) 1-2 minute, put into tap water and stop shadow, then put into stop bath photographic fixing 15 minutes, finally washing is dried.

Result shows that DSLE method can detect Giardia miR2 from the total RNA of Giardia lamblia trophozoite, and can find out at the place of nt more than 100 have an other band, and the precursor GLsR17(that is miR2 is shown in the DNA probe of accompanying drawing 2:M:14nt; 1: application DSLE method detects g) middle miR2 result of Giardia lamblia trophozoite RNA(5 μ, the precursor that GlsR17 is miR2), illustrate that DSLE method can not only detect Microrna, can also detect the larger RNA with 5 ' terminal phosphate feature.

below test shows the good effect place of the inventive method:

test example 1

dSLE method absolute sensitivity is analyzed

Synthetic hsa-miR-21 RNA(5'-phos-uagcuuaucagacugauguuga-3' for this test) be research object, in order to determine the absolute sensitivity of DSLE method, according to synthetic instructions, hsa-miR-21 RNA is carried out to doubling dilution (200fmol, 40fmol, 20foml, 2fmol), result shows that DSLE method at least can detect that the hsa-miR-21 RNA(of 2fmol is shown in accompanying drawing 3:1-4: synthetic hsa-miR-21RNA concentration is: 200fmols, 40fmols, 20fmols, 2fmols).It is much higher that result shows that the sensitivity of DSLE method may detect miRNAs method than traditional DIG method, even can compare with radioactive label Northern blots method.

test example 2

the remolding sensitivity that DSLE method and traditional digoxigenin labeled Northern blots detection method detect miRNAs and

it detects the analysis of miRNA concentration limit

For the relatively sensitivity of DSLE method and Gaoxin method detection traditionally, this test, respectively from 7 μ g, detects hsa-miR-21 RNA by traditional digoxigenin labeled Northern blots method and DSLE method in 4 μ g and 2 μ g HeLa cell total rnas.

Tradition digoxigenin labeled Northern blots method detecting step is as follows: first, extract to specifications HeLa cell total rna with Trizol reagent, recording concentration is 1 μ g/ μ l.Remove respectively 7 μ l, 4 μ l, 2 μ l and isopyknic RNA Loading buffer mix, and 95 DEG C of sex change 3min, place 2min immediately on ice.Then contain 15% polyacrylamide gel electrophoresis of 8M urea, treat that bromophenol blue race is to stopping electrophoresis apart from glass plate 2cm place, carry out transferring film experiment: according to the area of glue 25 cm2, positively charged nylon membrane (Ambion) and WATMAN filter paper that cutting is onesize, make sandwich-like, by half-dried transfer instrument transferring film constant current 75mA transferring film 30 minutes.After transferring film, film is taken out, ultraviolet light cross-linking is fixed 1 minute, 80 DEG C of roasting films 1 hour.Then film is put into the hybrid pipe that contains appropriate high efficiency liquid phase hybridization solution (BioDev), hybrid pipe is put into 42 DEG C of hybrid heater (HL-2000 Hybrilinker, UVP), in, prehybridization adds the special digoxin labelled probe of 95 DEG C of sex change hsa-miR-21 of 1 minute (hsa-miR-21 DIG probe after 1 hour

5'-DIG-TCAACATCAGTCTGATAAGCTA-3'), 42 DEG C of hybridization 16h.After hybridization, with 2 × SSC/0.1%SDS, wash film twice for 42 DEG C, each 5 minutes; Washing Buffer room temperature is washed film once, 5 minutes; Then 1% Blocking room temperature sealing 0.5 hour; Add again the anti digoxin antibody (1:15000) in 1% Blocking of alkali phosphatase enzyme mark, incubated at room 0.5 hour; Washing Buffer washes film 2 times, each 15 minutes; Detecting Buffer washes film 1 time, each 5 minutes.Finally the addition of C DP-Star ready-to-use(Roche is put in film front) reagent, to guarantee to be all paved with on film, incubated at room squeezed out reagent after 10 minutes, blotted raffinate with filter paper, put into the magazine X-ray 10min that exposes; X-ray is taken out from magazine, put into D76 developer solution (the about 20 DEG C of left and right of temperature) 12 minutes, put into tap water and stop shadow, then put into stop bath photographic fixing 15 minutes.Use U6 RNA as internal reference, U6 digoxin labelled probe is simultaneously

U6?DIG?probe：5'-DIG-ATATGGAACGCTTCACGAATT-3'。

The detecting step of U6 RNA is same as above.

Separately, DSLE method detecting step is with described in embodiment.

Result was presented under the same time shutter, this method can obtain very strong signal, and Gaoxin method only obtains compared with weak signal that (U6 RNA Northern blots signal is fine traditionally, illustrate that Northern blots method itself is no problem), show by gray analysis software analysis, this method (is shown in that accompanying drawing 4:1-4 is that digoxigenin labeled bridging method detects hsa-miR-21 RNA than the sensitivity of the miRNAs of Gaoxin method detection is traditionally at least high 50 times; 5-6 is that traditional digoxigenin labeled Northern blots detects hsa-miR-21 RNA; (in two, the method time shutter is 10 minutes) 1-6:Hela cell total rna concentration is: 7 μ g, 4 μ g, 2 μ g, 7 μ g, 4 μ g, 2 μ are g).Simultaneously, author has also verified the detection limit of DSLE method to hsa-miR-21 RNA in total HeLa cell total rna, and result shows that DSLE method at least can detect that hsa-miR-21(is shown in that accompanying drawing 5:1-4:Hela cell total rna concentration is: 1 μ g, 0.5 μ g, 0.25 μ g, 0.13 μ g from 0.13 μ g HeLa RNA.*: non-specific assorted band (while extracting total RNA due to contaminating dna)), can find out the method and traditional radioactivity with reference to forefathers' result of study ³²it is suitable that P mark Northern blots detects the sensitivity of miRNAs.

Claims (1)

1. utilize digoxigenin labeled EDC cross-bridge connection to detect a miRNAs method, comprise the following steps:

1) extraction of sample cell total rna

With Trizol extraction sample cell total rna;

2) the catching and be connected of miRNA

First carry out catching of miRNA, system is: 7 μ l RNA+ddH ₂o, 1 μ l 1pmol/ μ l Bridge, 1 μ l 10 × Capture buffer, 1 μ l 1pmol/ μ l 3 ' DIG-probe, mixes, and carries out following program in PCR instrument: 94 DEG C, 1min; 65 DEG C, 2min; 37 DEG C, 10min;

Then connect to adding T4 DNA ligase potpourri in above-mentioned reaction, system is: 1 μ l T4 DNA ligase, 1.5 μ l 10 × T4 buffer, 2.5 μ l ddH ₂o; At 30 DEG C, react 1h, this reaction product is final application of sample sample;

3) containing 15% polyacrylamide gel electrophoresis of 8M urea

(1) prepare according to following ratio the 15%PAGE glue that contains 8M urea: 3.75ml 40% acrylamidel, 1ml 5 × TBE, 4.2g Urea, 45 μ l 10%APS, 10 μ l TEMED, finally add water and mend to 10ml, mix rear encapsulating;

(2) preparation of samples: the sample equivalent in previous step and RNA loading buffer are mixed;

(3) sex change: evenly biased sample, 95 DEG C of sex change, after 3 minutes, are placed immediately on ice;

(4) prerunning: electrophoretic apparatus is correctly installed, is poured into 0.5 × TBE electrophoretic buffer, with the cull in syringe piping and druming electrophoresis hole, 120V, prerunning 20 minutes;

(5) point sample: again blow and beat electrophoresis hole, point sample with syringe after prerunning;

(6) constant voltage electrophoresis: 120V electrophoresis approximately 2.5 hours, bromjophenol blue is gone to glue bottom;

4) transferring film

According to the area of glue, the positively charged nylon membrane that cutting is onesize and WATMAN filter paper, make sandwich-like, by half-dried transfer instrument transferring film, transferring film 30 minutes;

5) EDC is crosslinked: after transferring film, film is taken out, be placed on the WATMAN filter paper that is soaked with EDC cross-linking reagent, 60 DEG C of effect 1-2h;

6) sealing

Film is put into 1% Blocking buffer, room temperature sealing 0.5 hour;

7) add antibody

Add the anti digoxin antibody of alkali phosphatase enzyme mark in 1% Blocking, making its ultimate density is 1:15000, incubated at room 0.5 hour;

8) washing

Utilize pH7.5 to contain 0.3% Tween 20 Maleic Acid Buffer and wash film 2 times as Washing buffer, each 15 minutes; Utilize Detecting buffer to wash film 1 time, each 5 minutes; Detecting buffer collocation method is: 6.055gTris and 2.9gNaCl add in 480ml water, is adjusted to PH 9.5 with concentrated hydrochloric acid, and constant volume is to 500ml;

9) develop

(1) the addition of C DP-Star ready-to-use reagent is put in film front, guarantees to be all paved with on film, and incubated at room squeezed out reagent after 5 minutes, blots raffinate with filter paper, puts into magazine X-ray and exposes;

(2) X-ray is taken out from magazine, put into D76 developer solution 1-2 minute, put into tap water and stop shadow, then put into stop bath photographic fixing 15 minutes, finally washing is dried.