CN105886500A - Universal antibody-oligonucleotide probe and kit - Google Patents
Universal antibody-oligonucleotide probe and kit Download PDFInfo
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Abstract
The invention provides a universal antibody-oligonucleotide probe and a kit. The universal antibody-oligonucleotide probe comprises an oligonucleotide probe part and an antibody part, wherein the oligonucleotide probe part is obtained by carrying out aldehyde group modification on the 5' terminals of oligonucleotides, and the antibody part is obtained by carrying out hydrazine group modification on antibodies; and the oligonucleotide probe part and the antibody part are coupled to obtain the universal antibody-oligonucleotide probe. In the universal antibody-oligonucleotide probe, the different oligonucleotides can be coupled with the different antibodies, thus QPCR can be carried out, the universal parallel testing on a plurality of corresponding antigens in cells is realized, and therefore, the practicability is strong.
Description
Technical field
The present invention relates to immunity field, polymerase chain reaction, be specifically related to a kind of universal antibody
-oligonucleotide probe and test kit.
Background technology
Immunity polymerase chain reaction (Polymerase Chain Reaction, PCR), is a kind of profit
The one set up with the specificity of antigen antibody reaction and the susceptiveness of pcr amplification reaction is micro-
Amount Antigen Detection Techniques.In order to measure certain specific antigen, it is necessary to prepare corresponding antibody-core
Thuja acid probe.And specifically antibody is extremely complex with the cross-linking process of nucleotide, and do not have
Versatility.It is typically according to specific antibody structure, designs specific crosslinking method.
Such as the Chinese patent of Application No. 201110315281.6, its patent name is " detection
The targeting molecule of pathogenic cells and application thereof ", disclose a kind of for targeting detection source of disease
The test kit of cell, wherein disclosed targeting molecule is by even to folic acid or folic acid-cysteine
Connection thing and oligonucleotide probe are obtained by C-S key coupling.But the method only can get leaf
Acid or the discernible pathogenic cells of folic acid-cysteine conjugate, versatility is the strongest, it is impossible to real
The purpose of existing Multiple detection.And general cell detection is often required for detecting multiple resisting simultaneously
Former, a sample is carried out operation repeatedly, is not only added significantly to workload, extends inspection
In the survey cycle, add probability of failure, the problem that simultaneously may bring sample contamination, testing result
Reliability the highest.
Further, coupling oligonucleotide can the affinity of antagonist have an impact, and makes to combine few core
The affinity that the antibody specificity of thuja acid combines is the highest, thus affects the accuracy of detection.
Summary of the invention
It is an object of the invention to provide a kind of universal antibody-oligonucleotide acid probe, can be to sample
Multiple surface antigens carry out parallel detection by quantitative, and improve the sensitivity and accurately of detection
Property.
It is a further object to provide containing above-mentioned universal antibody-oligonucleotide acid probe
Test kit.
The present invention is achieved through the following technical solutions:
A kind of universal antibody-oligonucleotide acid probe, it is characterised in that: described universal antibody-
Oligonucleotide probe includes oligonucleotide probe part and antibody moiety；Described oligonucleotide probe
Part be the 5' end of oligonucleotide is carried out aldehyde group modified after obtain, described antibody moiety be by
Antibody obtains after carrying out diazanyl modification；Described oligonucleotide probe part and described antibody moiety
Described universal antibody-oligonucleotide acid probe is obtained through coupling.
Further, a length of 16-22nt of described oligonucleotide, 5 ' ends are 6-14nt length
Primer recognition sequence, 3 ' end extension primers carry out extend extension.
Further, a length of 50-80nt of described extension primer, it 3 ' end ends with
3 ' end complementary pairings of oligonucleotide, 5 ' ends can form hairpin structure.Extend the sequence of primer
It is preferably SEQ ID NO:14.
Described oligonucleotide probe part is with amido modified oligonucleotide by 5 ' ends, with rubbing
Your equivalent be the SFB of 5-20 times carry out aldehyde group modified after obtain；Described antibody moiety be by
Antibody molar equivalent is to obtain after the SANH of 10-50 times carries out diazanyl modification.
Wherein, above-mentioned SFB refers to 4-carbamoyl benzoate N-succinimide ester；Above-mentioned
SANH refers to 4-(N-maleimidomethyl) hexamethylene-1-carboxylic acid succinimide ester.
Described antibody-oligonucleotide acid probe is to be (7-10) by mol ratio: the oligonucleotide probe portion of 1
Dividing and antibody moiety, reaction obtained after 4-24 hour at ambient temperature.
Preferably, the sequence of described oligonucleotide is arbitrary shown in SEQ ID NO:1-13
Individual.
In another aspect of this invention, it is provided that a kind of containing above-mentioned universal antibody-oligonucleotide
The test kit of acid probe, described test kit includes:
Universal antibody-oligonucleotide acid probe；
For universal antibody-oligonucleotide acid probe being carried out the amplimer of PCR amplification and delaying
Rush liquid；
Also include fluorescent probe.
Described amplimer includes a pair for being carried out by universal antibody-oligonucleotide acid probe
The forward of PCR amplification, reverse primer；5 ' ends of described forward primer are complementary with oligonucleotide.
Further, described test kit also includes extend primer, the length of described extension primer
For 50-80nt, its 3 ' end ends and 3 ' end complementary pairings of oligonucleotide, 5 ' ends can be with shape
Become hairpin structure.
Universal antibody-oligonucleotide acid probe in described test kit is that CK19-oligonucleotide is visited
Pin.
There is advantages that
1. oligonucleotide and antibody are first oriented modification and obtain oligonucleotide by the present invention respectively
Probe portion and antibody moiety, such that it is able to avoid crosslinking inside antibody, make oligonucleotide
The coupling of probe portion and antibody moiety has high degree of specificity, and the hydrazone key conjugate obtained is stable
Property is good.And the reaction condition of directed modification and coupling is gentle, is not required to additionally use reducing agent,
Antibody not changeableness and inactivation.
2. due to the affinity of antibody moiety meeting in a certain degree in antibody-oligonucleotide acid probe
Increase along with oligonucleotide chain length and decline, and inventor finds to control the length of oligonucleotide
During for 16-22nt, the affinity of antibody moiety is barely affected.
3. with extending the primer 3 ' the end amplifications to oligonucleotide, it is ensured that oligonucleotide chain is too short
Time PCR expand requirement, and extend the hairpin structure that primer 5 ' holds and can increase the heap of base
Block power, thus strengthen probe acute, make universal antibody-oligonucleotide acid probe detect more
Sensitive.
4. hold complementary pairing with extending primer 3 ' due to oligonucleotide, therefore extend extension
Process can react voluntarily in PCR amplification procedure, anti-with after extending again after extending extension
Body-oligonucleotide is that template expands, single step reaction, and reaction efficiency is high.
Oligonucleotide different in universal antibody-oligonucleotide acid probe the most of the present invention is permissible
With different types of antibody coupling, such that it is able to carry out multiplex PCR, general Parallel testing is thin
Multiple corresponding antigen in born of the same parents, practical.
Detailed description of the invention
By the following specific examples further illustrate the invention:
The present invention to obtain universal antibody-oligonucleotide acid probe, and it is by first by oligonucleoside
Acid and antibody carry out the directed modification of aldehyde radical and diazanyl respectively and obtain oligonucleotide part and antibody
Part, then carry out hydrazone key coupling and obtain.Preferably, described oligonucleotide probe part is by 5 '
End, with amido modified oligonucleotide, carries out aldehyde radical with the SFB that molar equivalent is 5-20 times
Obtaining after modification, preferably molar equivalent is 10 times；Described antibody moiety is with rubbing by antibody
Your equivalent is to obtain after the SANH of 10-50 times carries out diazanyl modification, and preferably molar equivalent is
25 times.Wherein, SFB is 4-carbamoyl benzoate N-succinimide ester, and SANH is 4-(N-
Maleimidomethyl) hexamethylene-1-carboxylic acid succinimide ester.Oligonucleoside is modified with SFB
Amino on 5 ' ends of acid can obtain the oligonucleotide of aldehyde radical activation, uses SANH modified antibodies
The antibody protein of diazanyl activation can be obtained.The two reaction is available for public technology,
Such as China's document " immuno-chip antibody based on making nucleic acid molecular hybridization fixes new method ", " analyze
Chemistry ", the 2nd phase in 2013, Sha Sha etc. is disclosed.The condition such as response time, reaction temperature
Can make according to literature content and well known to a person skilled in the art adjustment.Currently preferred technology
Scheme is: by 5 ' ends with the amido modified PBS that oligonucleotide pH value is 7.4 buffering
Liquid dissolves, and is then that the SFB solution of oligonucleotide 10 times mixes with molar equivalent, and room temperature is anti-
Answering 2.5 hours, repurity obtains aldehyde group modified oligonucleotide probe part.By antibody pH
After value is the PBS dilution of 7.4, with the SANH solution that molar equivalent is antibody 25 times
Mixing, room temperature reaction 2.5 hours, repurity obtains the antibody moiety that diazanyl is modified.Finally will
Mol ratio is (7-10): the at room temperature coupling of the oligonucleotide probe part of 1 and antibody moiety is anti-
Described universal antibody-oligonucleotide acid probe should be obtained.
1 be further described by the following examples, remaining unaccounted actual conditions and
Experimental technique, conventionally and condition, or selects according to catalogue.
" room temperature " described in the present embodiment refers to the indoor temperature of routine, generally 15-30
℃。
Sequence is SEQ ID NO:(1-13 by the present embodiment) Oligo be abbreviated as Oligo (1-13),
Oligo is oligonucleotide.
PBS in the present embodiment is phosphate buffer, and MES buffer is 2-(N-
Quinoline generation) ethanesulfonic acid buffer.
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 by the present embodiment is: the PBS of differently configured pH value, at this
In the detailed description of the invention of invention, specifically include the PBS that pH value is 6.0, and
PH value is the PBS of 7.4, and configures the MES buffer that pH value is 5.0,
Filtration sterilization processes, and deposits for 4 DEG C.
Embodiment 1CK19-oligonucleotide probe
(1) oligonucleotide probe is aldehyde group modified
By the Oligo1 (oligonucleotide) of the 50nmol phosphoric acid buffer of the pH 7.4 of 0.1M
Liquid is configured to solution.Weigh the SFB (4-carbamoyl benzoate N-succinimide ester) of 500nmol,
After dissolving by dry DMF (DMF), at room temperature reaction 2.5h, cross post
Purification obtains Oligo-FB (aldehyde group modified oligonucleotide).
The concentration of detection Oligo-FB: detect A with Nanodrop spectrophotometer260Value,
The concentration calculating Oligo-FB is 0.68nmol/ μ L.
Detect aldehyde group modified rate: repair with quantitative 2-hydrazine pyridine-2-HCI solution detection aldehyde radical
Decorations rate.Take above-mentioned Oligo-FB and join in 2-hydrazine pyridine-2-HCI solution, after vibration mixing
React 1h in 37 DEG C, be 1.41 with the light absorption value at Nanodrop detection 360nm, calculate
Its modification rate, A360Under modification rate be 0.85.
(2) diazanyl of antibody CK19 is modified
Antagonist CK19 after carrying out desalting and purifying by Nanodrop detection antibody protein concentration is
7.9mg/mL。
Diluting antibody CK19 with the phosphate buffer that pH value is 7.4 is 2mg/mL to concentration.
Weigh the SANH (antibody is 1:25 with the mol ratio of SANH) of 25 times amount, be dissolved in anhydrous
In DMF, it is then added in antibody, at room temperature reaction 2.5h, crosses column purification and obtain CK19-SANH
(CK19 that diazanyl is modified).
The concentration of detection CK19-SANH: calculate the CK19-SANH after modifying by BCA method
Concentration be 1.80mg/mL.
Detection diazanyl modification rate: modify with quantitative 2-formyl benzene sulfonyl sodium salt solution detection diazanyl
Rate.Take in the 2-formyl benzene sulfonyl sodium salt solution that antibody-SANH after purification joins, vortex
After mixing, the light absorption value at 37 DEG C of reaction 1h, Nanodrop detection 348nm is 0.43.
The hydrazine of CK19-SANH is calculated by the densitometer of the light absorption value at 348nm and CK19-SANH
Base modification rate is 3.7.
(3) coupling of Oligo-FB Yu CK19-SANH
Being mixed according to mol ratio 7:1 mixing vortex by Oligo-FB with CK19-SANH, room temperature is anti-
Answering 4h, the product finally obtained i.e. can get described universal after crossing column purification
CK19-Oligo probe.
Take CK19-Oligo probe and carry out Nanodrop detection.Substantially absorption is had under 354nm
Peak occurs, the coupling success of Oligo-FB Yu CK19-SANH is described.
Take CK19-Oligo probe and carry out SDS-PAGE detection, analyze CK19 Yu oligo coupling
Degree, with pure CK19 compares, obtains a plurality of electrophoretic band, coupling is described on CK19 not
Oligonucleotide with quantity.
Take CK19-Oligo probe and carry out BCA method Concentration Testing.BCA method Concentration Testing calculates even
The concentration of connection thing antibody.By the concentration of Oligo1 in the quantitative CK19-Oligo of strand quantification kit.
The ratio of CK19 Yu Oligo1 can be calculated, it is possible to and modification rate results contrast.Explanation
The coupling ratio of Oligo1 and antibody in CK19-Oligo molecule, result is as shown in table 1.
Oligo and the coupling ratio of antibody in table 1 CK19-Oligo molecule
The QPCR augmentation detection of embodiment 2 Oligo molecule
Oligo (1-13) molecule is diluted to 10 respectively8Molecular number/μ l.Then according to shown in table 2
Configuration QPCR amplification reaction solution, obtains respective test kit.Wherein, extension primer is RT-P,
Its 3 ' end ends and 3 ' end complementary pairings of oligonucleotide, 5 ' ends can form hairpin structure,
And the complementary of intermediate sequence and MGB probe (MGty), the RT-P of the present embodiment
By sequence for as a example by shown in SEQ ID NO:14.Forward primer is FP, and reverse sequence is RP.
FP is by sequence for as a example by shown in SEQ ID NO:15, and RP is with sequence for SEQ ID NO:16 institute
Being shown as example, MGty is by sequence for as a example by shown in SEQ ID NO:17, and the fluorophor that 5 ' hold is used
FAM labelling, 3 ' ends are MGB.
The QPCR amplification kit of table 2 Oligo molecule
| Reagent | 1 part of consumption (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 according to the program of table 3 carries out QPCR amplification respectively to respective test kit:
Table 3 QPCR amplification program
Amplification is as shown in table 4:
Table 4 embodiment 2 amplification
From table 4, it can be seen that after adding RT-P, the Oligo of short chain can also be carried out effectively
PCR amplification.This is because Oligo can first carry out with RT-P extending extension, the most again to prolong
Stretching the chain after extension is that template carries out PCR amplification.
The QPCR specific amplification detection of embodiment 3 Oligo molecule
It is 25000 molecular number/μ l, 83333 molecular number by Oligo1-13 molecular dilution to concentration
/ μ l and 250000 molecular number/μ l tri-concentration.Then as a example by Oligo1-3, by Oligo1-3
Mix under same concentrations, obtain biased sample A, B, C, as shown in table 5.
Table 5 embodiment 3 sample formulations
Above-mentioned sample is configured QPCR amplification kit according to table 2, then according to shown in table 3
Program Oligo1-3 is carried out QPCR amplification, and in biased sample A, B, C
Oligo1-3 each carries out QPCR amplification, and the Ct value of amplification is as shown in table 6.Used
RT-P, FP, RP, MGty are the most same as in Example 2, and amplification is to extend the Oligo1-3 after extending
For template.
Table 6 amplification Ct value
| Oligo1 | Oligo2 | Oligo3 | |
| 25000 molecular number/μ l | 25.33 | 25.92 | 23.98 |
| 83333 molecular number/μ l | 23.50 | 24.17 | 22.43 |
| 250000 molecular number/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 biased sample A, B, C, in same concentrations
The Ct value of lower amplification is about 0.1 with the amplification Ct value of single Oligo.Meanwhile, according to every
Regression straight line drawn by the variable concentrations sample of Oligo, calculates to obtain regression beeline equation and correlation coefficient
R2 is 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 A-C sample that every corresponding for Oligo is calculated according to separate equation, calculates score
Subnumber is divided by theory of correspondences molecular number, and gained detection efficiency is 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, expanding Ct difference between parallel sample is about 0.1, and detection efficiency is
Between 98.5%-110.5%, illustrate that three Oligo do not have non-specific expansion with remaining two respectively
Increase, do not interfere with amplification efficiency each other.Remaining Oligo is identified can be tied equally
Really, illustrate between a plurality of Oligo of this experimental design without cross influence, it is ensured that multiplex PCR
Specificity, accuracy and sensitivity.Other Oligo can obtain same conclusion, due to
Length reason describes in detail the most one by one.
Embodiment 4 makes standard curve
According to the step of embodiment 1, with the SFB that molar equivalent is 5 times, Oligo2 is repaiied
Decorations obtain Oligo-FB, with the SANH that molar equivalent is 10 times, CK19 are carried out diazanyl modification
Obtain CK19-SANH, by Oligo-FB Yu CK19-SANH that mol ratio is 10:1 in room temperature
Under the conditions of react after 16 hours and obtain CK19-Oligo2.
According to the step of embodiment 1, with the SFB that molar equivalent is 20 times, Oligo3 is carried out
Modification obtains Oligo-FB, with the SANH that molar equivalent is 50 times, CK19 is carried out diazanyl and repaiies
Decorations obtain CK19-SANH, by Oligo-FB Yu CK19-SANH that mol ratio is 8:1 in room temperature
Under the conditions of react after 24 hours and obtain CK19-Oligo3.
By residue Oligo4-13 all according to the step of embodiment 1, obtain CK19-Oligo (4-13).
CK19-Oligo (1-13) is carried out QPCR amplification according to the test kit of table 2 and the program of table 3,
The amplification obtained is consistent with table 4, and the expansion of Oligo1-13 Yu CK19-Oligo (1-13) is described
Increasing Efficiency is consistent, and the antibody moiety of coupling expands not impact to the PCR of Oligo.
With standard dilutions, CK19-Oligo1 being diluted to concentration is: 833,2500,8333,
25000,83333 and 250000 Molecules/2.5 μ l, totally six concentration, blank group
For deionized water.
According to the recipe configuration PCR amplification kit shown in table 2, then according to shown in table 3
The CK19-Oligo1 of above each concentration is carried out for template by program to extend the Oligo1 after extending
QPCR expands, and by amplification according to the variable concentrations of CK19-Oligo1, draws standard curve,
Carry out linear regression calculating, obtain regression beeline equation and correlation coefficient is as follows:
Y=-3.3353x+39.193, R2=0.99609.
Antigen levels detection on embodiment 5 cell
In order to calculate antigen levels on each cell, the following experiment of design, with on MCF-7 cell
CK19 Detection of antigen as a example by:
(1) all 1.5mlEP pipes use reaction buffer to be coated 15min.
(2) take the MCF-7 cell of fresh cultured, cell counting after digestion, take 1*105Individual
Cell centrifugation removes supernatant.Removal supernatant, adds 100 μ l blocking after 400 μ l buffer are resuspended
DNA room temperature closes 20min；
(3) add CK19-Oligo1 probe, terminate reaction after incubated at room 40min and be centrifuged
Remove supernatant.
(4) 120 μ l eluent mix homogeneously, ice are added after using PBS washed cell 3 times
On hatch 2min, centrifuging and taking 100 μ l supernatant, eluting unreacted CK19-Oligo1 probe；
It is eventually adding 20 μ L reaction neutralizers to neutralize.
(5) with embodiment 4 similarity condition carry out QPCR amplification come detecting step (4) obtain
Sample Ct value, the standard curve Ct value in corresponding embodiment 4 can calculate in acquisition sample
The concentration (molecular number) of CK19-Oligo1.By total molecular number of recording divided by the cell number added
The molecular number combined on available each cell, result is total CK19-Oligo1 molecular number
157589726.3, on each cell, CK19-Oligo1 molecular number is 1575.9.
Experiment each cell of gained on combine CK19-oligo molecular number with pass through western
The data result that blot method measures is essentially identical.
The response rate that embodiment 6 circulating tumor cell (CTC) detects
The response rate=(averagely detecting cell quantity-negative controls cell quantity)/incorporation
Cell quantity * 100%
In order to detect the response rate that circulating tumor cell (CTC) detects, the following experiment of design, with
As a example by MCF-7 cell detection:
(1) every 3ml mixes from the blood of Healthy People withdraw 0 respectively, 1,10,
50,100 MCF-7 cells, with mix 0 MCF-7 cell blood as negative controls,
It is subsequently adding 12mL cell pyrolysis liquid, gently reverse fully mixing.Then sample is placed in
Centrifugal supernatant of abandoning, addition 10mLPBS washed cell after cracking 15min in 2-8 DEG C of refrigerator；Again
It is centrifuged and abandons supernatant, add 400 μ LPBS re-suspended cells.
(2) add 100 μ l blocking buffer room temperatures and close 20min；Add CK19-Oligo1
Probe, terminates after incubated at room 40min reacting and being centrifuged removing supernatant.
(3) 120 μ l eluent mix homogeneously, ice are added after using PBS washed cell 3 times
On hatch 2min, centrifuging and taking 100 μ l supernatant；Eluting unreacted CK19-Oligo1 probe；
It is eventually adding 20 μ L reaction neutralizers to neutralize.
Calculating MCF-7 number of cells by mark song, result is as shown in table 7:
Table 7 CTC detects the response rate
As can be seen from Table 7, the CK19-Oligo1 probe in detecting cell recoveries of the present invention is high
In the response rate of magnetic bead screening and highly sensitive.
For those skilled in the art, former without departing from the embodiment of the present invention
On the premise of reason, it is also possible to make some improvements and modifications, these improvements and modifications are also considered as this
The protection domain of inventive embodiments.
Claims (10)
1. a universal antibody-oligonucleotide acid probe, it is characterised in that: described universal antibody
-oligonucleotide probe includes oligonucleotide probe part and antibody moiety；Described oligonucleotide is visited
Pin part be the 5' end of oligonucleotide is carried out aldehyde group modified after obtain, described antibody moiety is
Obtain after antibody is carried out diazanyl modification；Described oligonucleotide probe part and described antibody portion
Lease making is crossed coupling and is obtained described universal antibody-oligonucleotide acid probe.
Universal antibody-oligonucleotide acid probe the most according to claim 1, its feature exists
In: a length of 16-22nt of described oligonucleotide, 5 ' ends are the primer identification of 6-14nt length
Sequence, 3 ' end extension primers carry out extending extension.
Universal antibody-oligonucleotide acid probe the most according to claim 2, its feature exists
In: a length of 50-80nt of described extension primer, its 3 ' end ends and the 3 ' of oligonucleotide
End complementary pairing, 5 ' ends can form hairpin structure.
Universal antibody-oligonucleotide acid probe the most according to claim 1, its feature exists
In: described oligonucleotide probe part is with amido modified oligonucleotide by 5 ' ends, with rubbing
Your equivalent be the SFB of 5-20 times carry out aldehyde group modified after obtain；Described antibody moiety be by
Antibody molar equivalent is to obtain after the SANH of 10-50 times carries out diazanyl modification.
Universal antibody-oligonucleotide acid probe the most according to claim 1, its feature exists
It it is to be (7-10) by mol ratio in: described antibody-oligonucleotide acid probe: the oligonucleotide probe portion of 1
Dividing and antibody moiety, reaction obtained after 4-24 hour at ambient temperature.
Universal antibody-oligonucleotide acid probe the most according to claim 1, its feature exists
In: the sequence of described oligonucleotide is any one shown in SEQ ID NO:1-13.
7. the test kit containing universal antibody-oligonucleotide acid probe described in claim 1,
It is characterized in that: described test kit includes:
Universal antibody-oligonucleotide acid probe；
For universal antibody-oligonucleotide acid probe being carried out the amplimer of PCR amplification and delaying
Rush liquid；
Also include fluorescent probe.
Test kit the most according to claim 7, it is characterised in that: in described amplimer
Including a pair for universal antibody-oligonucleotide acid probe being carried out the forward of PCR amplification, anti-
To primer；5 ' ends of described forward primer are complementary with oligonucleotide.
Test kit the most according to claim 7, it is characterised in that: in described test kit also
Including extending primer, a length of 50-80nt of described extension primer, its 3 ' end ends are with few
3 ' end complementary pairings of nucleotide, 5 ' ends can form hairpin structure.
Test kit the most according to claim 7, it is characterised in that: in described test kit
Universal antibody-oligonucleotide acid probe be CK19-oligonucleotide probe.
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| CN108753941A (en) * | 2018-06-22 | 2018-11-06 | 广东顺德工业设计研究院(广东顺德创新设计研究院) | Double labeling magnetic bead and its preparation method and application |
| CN113528611A (en) * | 2021-06-01 | 2021-10-22 | 上海交通大学 | Protein detection kit and detection method |
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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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| 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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| JIANG HE 等: "An improved method for covalently conjugating morpholino oligomers to antitumor antibodies", 《BIOCONJUGATE CHEM》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108753941A (en) * | 2018-06-22 | 2018-11-06 | 广东顺德工业设计研究院(广东顺德创新设计研究院) | Double labeling magnetic bead and its preparation method and application |
| CN113528611A (en) * | 2021-06-01 | 2021-10-22 | 上海交通大学 | Protein detection kit and detection method |
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| CN105886500B (en) | 2018-11-30 |
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Inventor after: Cai Hongdong Inventor after: Zhang Xianglin Inventor after: Chen Changyue Inventor after: Li Jing Inventor after: Deng Wenbin Inventor after: Gan Guangli Inventor before: Chen Changyue Inventor before: Li Jing Inventor before: Cai Hongdong Inventor before: Deng Wenbin Inventor before: Gan Guangli Inventor before: Zhang Xianglin |