CN105886500B - A kind of universal antibody-oligonucleotide acid probe and kit - Google Patents
A kind of universal antibody-oligonucleotide acid probe and kit Download PDFInfo
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Abstract
The present invention provides a kind of universal antibody-oligonucleotide acid probe and kit, the universal antibody-oligonucleotide acid probe includes oligonucleotide probe part and antibody moiety；The oligonucleotide probe part be the end 5' of oligonucleotides is carried out it is aldehyde group modified after obtain, the antibody moiety be will antibody carry out diazanyl modification after obtain；The oligonucleotide probe part and the antibody moiety pass through coupling and obtain the universal antibody-oligonucleotide acid probe.Different oligonucleotides can be with different types of antibody coupling in the universal antibody-oligonucleotide acid probe, and so as to carry out QPCR, a variety of corresponding antigens in general Parallel testing cell are practical.
Description
Technical field
The present invention relates to immune polymerase chain reaction fields, and in particular to a kind of universal antibody-oligonucleotide acid probe
And kit.
Background technique
Immune polymerase chain reaction (Polymerase Chain Reaction, PCR) is a kind of to utilize antigen-antibody
The specificity of reaction and the sensitivity of pcr amplification reaction and a kind of trace antigen detection technique for establishing.In order to measure certain spy
Determine antigen, it is necessary to prepare corresponding antibody-nucleotide probe.And the cross-linking process of specific antibody and nucleotide is extremely complex,
And do not have versatility.Usually according to specific antibody structure, specific crosslinking method is designed.
Such as application No. is 201110315281.6 Chinese patent, patent name is《Detect the targeting of pathogenic cells
Molecule and its application》, disclose it is a kind of for targeting detection pathogenic cells kit, disclosed in targeting molecule be
Folic acid or folic acid-cysteine conjugate and oligonucleotide probe are coupled by C-S key.But this method can only obtain
To folic acid or the identifiable pathogenic cells of folic acid-cysteine conjugate, versatility is not strong, cannot achieve the mesh of Multiple detection
's.And general cell detection often requires to detect multiple antigens simultaneously, carries out operation repeatedly to a sample, not only greatly
The problem of width increases workload, extends detection cycle, increases probability of failure, while may pollute with sample, detection
As a result reliability is not high.
Also, being coupled oligonucleotides can have an impact to the compatibility of antibody, keep the antibody for combining oligonucleotides special
Property combine affinity it is not high, thus influence detection accuracy.
Summary of the invention
The object of the present invention is to provide a kind of universal antibody-oligonucleotide acid probes, can be to multiple surface antigens of sample
Parallel quantitative detection is carried out, and improves the sensitivity and accuracy of detection.
It is a further object to provide the kits for containing above-mentioned universal antibody-oligonucleotide acid probe.
The present invention is achieved through the following technical solutions：
A kind of universal antibody-oligonucleotide acid probe, it is characterised in that：The universal antibody-oligonucleotide acid probe packet
Include oligonucleotide probe part and antibody moiety；The oligonucleotide probe part is that the end 5' of oligonucleotides is carried out aldehyde radical to repair
It is obtained after decorations, the antibody moiety is obtained after antibody is carried out diazanyl modification；The oligonucleotide probe part and institute
It states antibody moiety and obtains the universal antibody-oligonucleotide acid probe by coupling.
Further, the length of the oligonucleotides is 16-22nt, and 5 ' ends are the primer recognition sequence of 6-14nt length,
3 ' ends carry out extension extension with extension primer.
Further, the length of the extension primer is 50-80nt, its 3 ' end ends and the 3 ' of oligonucleotides are held mutually
It recruits pair, 5 ' ends can form hairpin structure.The sequence of extension primer is preferably SEQ ID NO：14.
The oligonucleotide probe part is by 5 ' ends with amido modified oligonucleotides, is 5-20 times with molar equivalent
SFB carry out it is aldehyde group modified after obtain；The antibody moiety is that antibody is carried out hydrazine with the SANH that molar equivalent is 10-50 times
It is obtained after base modification.
Wherein, above-mentioned SFB refers to 4- carbamoyl benzoate N- succinimide ester；Above-mentioned SANH refers to p- third hydrazone group
Pyridine carboxylic acid N-hydroxy-succinamide ester, No. CAS is 362522-50-7.
It is (7-10) that the antibody-oligonucleotide acid probe, which is by molar ratio,:1 oligonucleotide probe part and antibody portion
Point, it is obtained after reacting 4-24 hours at room temperature.
Preferably, the sequence of the oligonucleotides is SEQ ID NO：Shown in 1-13 any one.
In another aspect of this invention, a kind of kit containing above-mentioned universal antibody-oligonucleotide acid probe is provided,
The kit includes：
Universal antibody-oligonucleotide acid probe；
For universal antibody-oligonucleotide acid probe to be carried out to the amplimer and buffer of PCR amplification；
It further include fluorescence probe.
Include in the amplimer a pair of forward direction for universal antibody-oligonucleotide acid probe to be carried out to PCR amplification,
Reverse primer；5 ' ends of the forward primer are complementary with oligonucleotides.
It further, further include extension primer in the kit, the length of the extension primer is 50-80nt, it
3 ' end complementary pairings of 3 ' end ends and oligonucleotides, 5 ' ends can form hairpin structure.
Universal antibody-oligonucleotide acid probe in the kit is CK19- oligonucleotide probe.
The present invention has the advantages that：
1. oligonucleotides and antibody are first oriented modification respectively and obtain oligonucleotide probe part and antibody by the present invention
Part makes the coupling of oligonucleotide probe part and antibody moiety have height so as to avoid crosslinking inside antibody
Specificity, obtained hydrazone bond conjugate stability are good.And directed modification and the reaction condition of coupling are mild, are not required in addition use
Reducing agent, antibody not mutability and inactivation.
2. since the compatibility of antibody moiety in antibody-oligonucleotide acid probe to a certain extent can be with oligonucleotide chain
Length increases and decline, and when inventor has found the length of control oligonucleotides as 16-22nt, the compatibility of antibody moiety is almost
It is unaffected.
3. being expanded with 3 ' ends of the extension primer to oligonucleotides, it is ensured that PCR amplification when oligonucleotide chain is too short is wanted
Ask, and the hairpin structure held of extension primer 5 ' can increase base pile up power, to enhance probe acute, make universal
The detection of antibody-oligonucleotide acid probe is sensitiveer.
4. the process for extending extension expands in PCR since oligonucleotides and extension primer are to hold complementary pairing 3 '
It can voluntarily be reacted in increasing process, extend extension and expanded again using the antibody-oligonucleotide acid after extending as template later, a step is anti-
Should, reaction efficiency is high.
5. different oligonucleotides can resist with different types of in universal antibody-oligonucleotide acid probe of the present invention
Body coupling, so as to carry out multiplex PCR, a variety of corresponding antigens in general Parallel testing cell are practical.
Specific embodiment
By the following specific examples further illustrate the invention：
The present invention will obtain universal antibody-oligonucleotide acid probe, be by first by oligonucleotides and antibody respectively into
The directed modification of row aldehyde radical and diazanyl obtains oligonucleotides part and antibody moiety, then carries out hydrazone bond and be coupled to obtain.Preferably, institute
Stating oligonucleotide probe part is to carry out at 5 ' ends with the SFB that molar equivalent is 5-20 times with amido modified oligonucleotides
It is obtained after aldehyde group modified, preferably molar equivalent is 10 times；It with molar equivalent is 10-50 times that the antibody moiety, which is by antibody,
It is obtained after SANH progress diazanyl modification, preferably molar equivalent is 25 times.Wherein, SFB is that 4- carbamoyl benzoate N- succinyl is sub-
Amine ester, SANH are p- third hydrazone group pyridine carboxylic acid N-hydroxy-succinamide ester, and No. CAS is 362522-50-7.It is modified with SFB few
The oligonucleotides of the available aldehyde radical activation of amino on 5 ' ends of nucleotide, is activated with the available diazanyl of SANH modification antibody
Antibody protein.The two reactions are that public technology is available, such as Chinese document " the immune core based on making nucleic acid molecular hybridization
Piece antibody fixes new method ",《Analytical chemistry》, the 2nd phase in 2013, disclosed in Sha Sha etc..The conditions such as reaction time, reaction temperature
It can make that well known to a person skilled in the art adjustment according to literature content.Currently preferred technical solution is：5 ' ends are had
The PBS buffer solution that amido modified oligonucleotides pH value is 7.4 dissolves, and is then 10 times of oligonucleotides with molar equivalent
The mixing of SFB solution, reacts at room temperature 2.5 hours, repurity obtains aldehyde group modified oligonucleotide probe part.By antibody pH value
After 7.4 PBS buffer solution dilution, the SANH solution for being 25 times of antibody with molar equivalent is mixed, and is reacted at room temperature 2.5 hours, then
Purifying obtains the antibody moiety of diazanyl modification.It is finally (7-10) by molar ratio：1 oligonucleotide probe part and antibody moiety
Coupling reaction obtains the universal antibody-oligonucleotide acid probe at room temperature.
It 1 is further described by the following examples, remaining unaccounted actual conditions and experimental method, according to normal
Method and condition is advised, or is selected according to product manual.
" room temperature " described in the present embodiment refers to conventional room temperature, generally 15-30 DEG C.
Sequence is SEQ ID NO by the present embodiment：The Oligo of (1-13) is abbreviated as Oligo (1-13), and Oligo is few core
Thuja acid.
PBS buffer solution in the present embodiment is phosphate buffer, and MES buffer is 2- (N- morpholino) ethanesulfonic acid buffering
Liquid.
QPCR in the present embodiment is real-time fluorescence quantitative PCR.
Nanodrop in the present embodiment is spectrophotometer.
BCA method in the present embodiment refers to the quantitative approach of determination of protein concentration.
Buffer used in the present embodiment is：The PBS buffer solution for configuring different pH value, in a specific embodiment of the invention
In, it specifically includes PBS buffer solution that pH value is 6.0 and pH value is 7.4 PBS buffer solution, and configuring pH value is 5.0
MES buffer, filtration sterilization processing, 4 DEG C of storages.
Embodiment 1CK19- oligonucleotide probe
(1) oligonucleotide probe is aldehyde group modified
The Oligo1 (oligonucleotides) of 50nmol is configured to solution with the phosphate buffer of the pH 7.4 of 0.1M.It weighs
The SFB (4- carbamoyl benzoate N- succinimide ester) of 500nmol, after anhydrous DMF (n,N-Dimethylformamide) dissolution,
2.5h is reacted at room temperature, column purification is crossed and obtains Oligo-FB (aldehyde group modified oligonucleotides).
Detect the concentration of Oligo-FB：A is detected with Nanodrop spectrophotometer260Value, calculate the concentration of Oligo-FB
For 0.68nmol/ μ L.
Detect aldehyde group modified rate：Aldehyde group modified rate is detected with quantitative 2- hydrazine pyridine -2- HCI solution.It takes above-mentioned
Oligo-FB is added in 2- hydrazine pyridine -2- HCI solution, in 37 DEG C of reaction 1h after oscillation mixing, is detected with Nanodrop
Light absorption value at 360nm is 1.41, calculates its modification rate, A360Under modification rate be 0.85.
(2) the diazanyl modification of antibody CK19
It is 7.9mg/mL to antibody protein concentration is detected with Nanodrop after antibody CK19 progress desalting and purifying.
The phosphate buffer for being 7.4 with pH value dilute antibody CK19 to concentration be 2mg/mL.The SANH for weighing 25 times of amounts is (anti-
The molar ratio of body and SANH are 1:25) it, is dissolved in anhydrous DMF, is then added in antibody, in room temperature reaction 2.5h, it is pure to cross column
Change and obtains CK19-SANH (CK19 of diazanyl modification).
Detect the concentration of CK19-SANH：It is 1.80mg/mL by the concentration that BCA method calculates the CK19-SANH after modification.
Detect diazanyl modification rate：Diazanyl modification rate is detected with quantitative 2- formyl benzene sulfonyl sodium salt solution.It takes after purification
In the 2- formyl benzene sulfonyl sodium salt solution that antibody-SANH is added to, it is vortexed after mixing in 37 DEG C of reaction 1h, Nanodrop detections
Light absorption value at 348nm is 0.43.The hydrazine of CK19-SANH is calculated by the densimeter of light absorption value and CK19-SANH at 348nm
Base modification rate is 3.7.
(3) coupling of Oligo-FB and CK19-SANH
By Oligo-FB and CK19-SANH according to molar ratio 7:1 mixing, which is vortexed, to be mixed, and is reacted at room temperature 4h, is finally obtained
The universal CK19-Oligo probe can be obtained after crossing column purification in product.
CK19-Oligo probe is taken to carry out Nanodrop detection.There is obvious absorption peaks appearance at 354nm, illustrates Oligo-
The coupling success of FB and CK19-SANH.
CK19-Oligo probe is taken to carry out SDS-PAGE detection, analysis CK19 and oligo coupling degree, with pure CK19 ratio
Compared with obtaining a plurality of electrophoretic band, illustrate the oligonucleotides for being coupled different number on CK19.
CK19-Oligo probe is taken to carry out BCA method Concentration Testing.The concentration of BCA method Concentration Testing calculating conjugate antibody.
The concentration of Oligo1 in CK19-Oligo is quantified with single-stranded quantification kit.The ratio of CK19 and Oligo1 can be calculated, and
It can compare with modification rate result.The coupling ratio of Oligo1 and antibody in CK19-Oligo molecule are illustrated, as a result such as 1 institute of table
Show.
The coupling ratio of Oligo and antibody in 1 CK19-Oligo molecule of table
The QPCR augmentation detection of 2 Oligo molecule of embodiment
Oligo (1-13) molecule is diluted to 10 respectively8Molecular number/μ l.Then anti-according to the amplification of configuration QPCR shown in table 2
Liquid is answered, respective kit is obtained.Wherein, extension primer RT-P, its 3 ' end ends and 3 ' ends of oligonucleotides mutually recruit
Right, 5 ' ends can form hairpin structure, and intermediate sequence is complementary with the sequence of MGB probe (MGty), the RT- of the present embodiment
P is with sequence for SEQ ID NO：For shown in 14.Forward primer is FP, reverse sequence RP.FP is with sequence for SEQ ID NO：
For shown in 15, RP is with sequence for SEQ ID NO：For shown in 16, MGty is with sequence for SEQ ID NO：For shown in 17,5 '
The fluorophor at end is marked with FAM, and 3 ' ends are MGB.
The QPCR amplification kit of 2 Oligo molecule of table
| Reagent | 1 part of dosage (μ l) | Final concentration |
| Nuclear free water | 1.35 | |
| RT-P(1uM) | 0.25 | 25nM |
| FP(10uM) | 0.3 | 300nM |
| RP(10uM) | 0.3 | 300nM |
| MGty(10uM) | 0.1 | 100nM |
| Premix Ex Taq(2×) | 5 | |
| Oligo | 2.5 |
Then QPCR amplification is carried out respectively to respective kit according to the program of table 3：
3 QPCR amplification program of table
Amplification is as shown in table 4：
4 embodiment of table, 2 amplification
From table 4, it can be seen that the Oligo of short chain can also carry out effective PCR amplification after joined RT-P.This be because
Extension extension first can be carried out with RT-P for Oligo, then carry out PCR amplification as template to extend the chain after extending again.
The QPCR specific amplification of 3 Oligo molecule of embodiment detects
It is 25000 molecular numbers/μ l, 83333 molecular numbers/μ l and 250000 molecules by Oligo1-13 molecular dilution to concentration
Tri- concentration of number/μ l.Then by taking Oligo1-3 as an example, Oligo1-3 is mixed under same concentrations, obtain mixing sample A, B,
C, as shown in table 5.
5 embodiment of table, 3 sample formulations
By above-mentioned sample according to table 2 configure QPCR amplification kit, then according to program shown in table 3 to Oligo1-3 into
Row QPCR amplification, and QPCR amplification, the Ct value such as table of amplification are respectively carried out to the Oligo1-3 in mixing sample A, B, C
Shown in 6.RT-P, FP, RP, MGty used is same as Example 2, and amplification is to extend the Oligo1-3 after extending as mould
Plate.
6 amplification Ct value of table
| Oligo1 | Oligo2 | Oligo3 | |
| 25000 molecular numbers/μ l | 25.33 | 25.92 | 23.98 |
| 83333 molecular numbers/μ l | 23.50 | 24.17 | 22.43 |
| 250000 molecular numbers/μ l | 21.96 | 22.54 | 20.60 |
| A | 25.25 | 25.88 | 24.03 |
| B | 23.40 | 24.08 | 22.27 |
| C | 21.81 | 22.56 | 20.69 |
As can be seen from Table 6, the Oligo1-3 in sample A, B, C is mixed, the Ct value expanded under same concentrations and single
The amplification Ct value of Oligo is 0.1 or so.Meanwhile regression straight line is drawn according to the various concentration sample of every Oligo, it calculates to return
Return linear equation and coefficient R 2 as follows：
Oligo1：Y=-3.3664x+40.11, R2=0.99937；
Oligo2：Y=-3.384x+40.809, R2=0.99997；
Oligo3：Y=-3.3824x+38.928, R2=0.99463.
The corresponding A-C sample of every Oligo is calculated according to separate equation, calculate molecular number divided by theory of correspondences
Molecular number, gained detection efficiency are as follows：
| Oligo1 | Oligo2 | Oligo3 | |
| A | 104.0% | 103.4% | 101.6% |
| B | 110.5% | 105.6% | 101.0% |
| C | 109.0% | 99.0% | 98.5% |
Therefore, it is 0.1 or so that Ct difference is expanded between parallel sample, and detection efficiency is between 98.5%-110.5%, explanation
Three Oligo do not have non-specific amplification with remaining two respectively, will not influence amplification efficiency between each other.Remaining Oligo warp
It identifies available equifinality, illustrates between a plurality of Oligo of this experimental design without cross influence, it is ensured that multiplex PCR
Specificity, accuracy and sensitivity.The available same conclusion of other Oligo, due to length not one by one specifically
It is bright.
Embodiment 4 makes standard curve
The step of according to embodiment 1, modifies Oligo2 to obtain Oligo-FB with the SFB that molar equivalent is 5 times, uses
The SANH that molar equivalent is 10 times carries out diazanyl to CK19 and modifies to obtain CK19-SANH, is 10 by molar ratio:1 Oligo-FB
CK19-Oligo2 is obtained after reacting 16 hours at room temperature with CK19-SANH.
The step of according to embodiment 1, modifies Oligo3 to obtain Oligo-FB with the SFB that molar equivalent is 20 times,
Diazanyl is carried out to CK19 with the SANH that molar equivalent is 50 times to modify to obtain CK19-SANH, is 8 by molar ratio:1 Oligo-FB
CK19-Oligo3 is obtained after reacting 24 hours at room temperature with CK19-SANH.
The step of by remaining Oligo4-13 all in accordance with embodiment 1, obtains CK19-Oligo (4-13).By CK19-Oligo
(1-13) carries out QPCR amplification according to the kit of table 2 and the program of table 3, and obtained amplification and table 4 are consistent, explanation
Oligo1-13 is consistent with the amplification efficiency of CK19-Oligo (1-13), and the antibody moiety of coupling does not have the PCR amplification of Oligo
It influences.
CK19-Oligo1, which is diluted to concentration, with standard dilutions is：833,2500,8333,25000,83333 and
250000 Molecules/2.5 μ l, totally six concentration, blank control group are deionized water.
According to recipe configuration PCR amplification kit shown in table 2, then according to program shown in table 3 to above each concentration
CK19-Oligo1 using extend extension after Oligo1 be template progress QPCR amplification, by amplification according to CK19-Oligo1
Various concentration, draw standard curve, carry out linear regression calculating, obtain regression beeline equation and related coefficient is as follows：
Y=-3.3353x+39.193, R2=0.99609.
Antigen levels detect on 5 cell of embodiment
In order to calculate antigen levels on each cell, following experiment is designed, is detected as with the CK19 antigen on MCF-7 cell
Example：
(1) all 1.5mlEP pipes are coated with 15min using reaction buffer.
(2) the MCF-7 cell of fresh cultured is taken, cell count after digestion takes 1*105Supernatant is removed in a cell centrifugation.It removes
100 μ l blocking DNA room temperatures closing 20min is added in supernatant, 400 μ l buffers after being resuspended；
(3) CK19-Oligo1 probe is added, terminate reaction after being incubated at room temperature 40min and is centrifuged supernatant.
(4) 120 μ l eluents are added after washing cell 3 times using PBS to be uniformly mixed, are incubated for 2min, centrifuging and taking 100 on ice
μ l supernatant elutes unreacted CK19-Oligo1 probe；20 μ L reaction neutralizer is eventually adding to neutralize.
(5) QPCR amplification is carried out with 4 similarity condition of embodiment obtain sample Ct value, corresponding embodiment 4 come detecting step (4)
In standard curve Ct value can calculate obtain sample in CK19-Oligo1 concentration (molecular number).The total molecular number that will be measured
The molecular number combined on each cell can be obtained divided by the cell number of addition, result is total CK19-Oligo1 molecular number
157589726.3, CK19-Oligo1 molecular number is 1575.9 on each cell.
It tests the CK19-oligo molecular number combined on each cell of gained and passes through what western blot method measured
Data result is essentially identical.
The rate of recovery of 6 circulating tumor cell of embodiment (CTC) detection
The rate of recovery=(averagely detection cell quantity-negative controls cell quantity)/incorporation cell quantity * 100%
In order to detect the rate of recovery of circulating tumor cell (CTC) detection, following experiment is designed, is with MCF-7 cell detection
Example：
(1) 0,1,10,50,100 MCF-7 cell is mixed respectively from 3ml every in the blood of Healthy People withdraw, with
The blood for mixing 0 MCF-7 cell is negative controls, and 12mL cell pyrolysis liquid is then added, gently overturns and mixes well.So
Sample is placed in 2-8 DEG C of refrigerator to centrifugation after cracking 15min afterwards and abandons supernatant, 10mLPBS is added and washs cell；It is centrifuged in abandoning again
Clearly, 400 μ LPBS are added, cell is resuspended.
(2) 100 μ l blocking buffer room temperatures are added and close 20min；CK19-Oligo1 probe is added, room temperature is incubated
Reaction is terminated after educating 40min and is centrifuged supernatant.
(3) 120 μ l eluents are added after washing cell 3 times using PBS to be uniformly mixed, are incubated for 2min, centrifuging and taking 100 on ice
μ l supernatant；Elute unreacted CK19-Oligo1 probe；20 μ L reaction neutralizer is eventually adding to neutralize.
MCF-7 number of cells is calculated by mark is bent, the results are shown in Table 7：
7 CTC of table detects the rate of recovery
As can be seen from Table 7, CK19-Oligo1 probe in detecting cell recoveries of the invention are higher than the recycling of magnetic bead screening
Rate, and high sensitivity.
For those skilled in the art, without departing from the principles of the embodiments of the present invention, also
Several improvements and modifications can be made, these improvements and modifications are also considered as the protection scope of the embodiment of the present invention.
Claims (8)
1. a kind of universal antibody-oligonucleotide acid probe, it is characterised in that：The universal antibody-oligonucleotide acid probe includes
Oligonucleotide probe part and antibody moiety；The oligonucleotide probe part is that the end the 5' progress of oligonucleotides is aldehyde group modified
It obtains afterwards, the antibody moiety is obtained after antibody is carried out diazanyl modification；The oligonucleotide probe part and described
Antibody moiety obtains the universal antibody-oligonucleotide acid probe by coupling；The sequence of the oligonucleotides is SEQ ID
NO：Shown in 1-13 any one；The SEQ ID NO：1-13 sequence has following feature：The 6-14nt long of its 5 ' end starting
The sequence of degree can be identified that the sequence at 3 ' ends can carry out extension extension with extension primer by primer；The extension primer tool
There is following feature：3 ' end ends of extension primer sequence can be with above-mentioned SEQ ID NO：The sequence at 3 ' ends of sequence shown in 1-13
5 ' ends of complementary pairing, extension primer sequence can form hairpin structure.
2. universal antibody-oligonucleotide acid probe according to claim 1, it is characterised in that：The length of the oligonucleotides
Degree is 16-22nt.
3. universal antibody-oligonucleotide acid probe according to claim 1, it is characterised in that：The length of the extension primer
Degree is 50-80nt.
4. universal antibody-oligonucleotide acid probe according to claim 1, it is characterised in that：The oligonucleotide probe
Part is by 5 ' ends with amido modified oligonucleotides, the SFB for being 5-20 times with molar equivalent carry out it is aldehyde group modified after obtain
's；The antibody moiety is obtained after the SANH for being 10-50 times with molar equivalent by antibody carries out diazanyl modification.
5. universal antibody-oligonucleotide acid probe according to claim 1, it is characterised in that：The antibody-oligonucleotide acid
It is (7-10) that probe, which is by molar ratio,:1 oligonucleotide probe part and antibody moiety react 4-24 hours at room temperature
It obtains afterwards.
6. a kind of kit containing universal antibody-oligonucleotide acid probe described in claim 1, it is characterised in that：The examination
Agent box includes：Universal antibody-oligonucleotide acid probe；For universal antibody-oligonucleotide acid probe to be carried out to the expansion of PCR amplification
Increase primer and buffer；It further include fluorescence probe；
It include a pair of in the amplimer for universal antibody-oligonucleotide acid probe to be carried out the positive, reversed of PCR amplification
Primer；5 ' ends of the forward primer are complementary with oligonucleotides；
It further include extension primer in the kit, 3 ' end ends of the extension primer and 3 ' ends of oligonucleotides mutually recruit
Right, 5 ' ends can form hairpin structure.
7. kit according to claim 6, it is characterised in that：The length of the extension primer is 50-80nt.
8. kit according to claim 6, it is characterised in that：Universal antibody-oligonucleotide acid in the kit
Probe is CK19- oligonucleotide probe.
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| CN102901830A (en) * | 2012-10-15 | 2013-01-30 | 浙江大学 | Construction method for high-throughput micro-fluidic chip detecting system |
| CN103159854A (en) * | 2013-03-07 | 2013-06-19 | 浙江大学 | Connection method of antibody and microsphere |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102901830A (en) * | 2012-10-15 | 2013-01-30 | 浙江大学 | Construction method for high-throughput micro-fluidic chip detecting system |
| CN103159854A (en) * | 2013-03-07 | 2013-06-19 | 浙江大学 | Connection method of antibody and microsphere |
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| An improved method for covalently conjugating morpholino oligomers to antitumor antibodies;Jiang He 等;《Bioconjugate Chem》;20071231;第18卷(第3期);第983-988页 * |
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