CN108593615A - The method for screening PD1/PD-L1 blocking agents using HTRF one-step method - Google Patents
The method for screening PD1/PD-L1 blocking agents using HTRF one-step method Download PDFInfo
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- CN108593615A CN108593615A CN201810407194.5A CN201810407194A CN108593615A CN 108593615 A CN108593615 A CN 108593615A CN 201810407194 A CN201810407194 A CN 201810407194A CN 108593615 A CN108593615 A CN 108593615A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The invention discloses a kind of methods for screening PD1/PD L1 blocking agents using HTRF one-step method, belong to blocking agent screening technique field.A method of PD1/PD L1 blocking agents being screened using HTRF one-step method, are included the following steps：Step 1 configures PD1 the and PD L1 albumen with different labels built, sample to be screened；Step 2 sequentially adds sample, PD1, PD L1 and the tag antibody for being coupled Donor and Acceptor respectively to be screened in microwell plate；Step 3, incubation at room temperature read detection fluorescence with microplate reader, and the ratio of 665nm/620nm is initial data.Screening technique through the invention realizes screening PD1/PD L1 blocking agents on biochemistry level, more directly quick without building cell.
Description
Technical field
The invention belongs to blocking agent screening technique fields, and in particular to a kind of to screen PD1/PD-L1 using HTRF one-step method
The method of blocking agent.
Background technology
Procedural apoptotic proteins PD1 is the immunologic test point receptor expressed on T cell surface, as PD1 and its ligand PD-
After L1 is combined, T cell will be accordingly suppressed.Immunologic test, which orders blocking agent, can block the formation of PD1/PD-L1 complexs,
To make T cell restore immunocompetence again.So ordering blocking agent Field of Drug Discovery in immunologic test, PD1/PD-L1 is
As one of the most hot target spot of domestic and international medicament research and development.
Currently, common screening technique is FACS and ELISA.FACS is fluidic cell fluorescence sorting technology, will be constructed
The cell line of PD1 or PD-L1 memebrane proteins is dyed through special fluorochrome or is combined with the antibody with fluorescent marker, PD1/PDL1
The latter is generally used in experiment.The antibody of tape label is added after cell and sample to be tested (monoclonal antibody) are mixed, is put into mixture
Sample is squeezed into the flow chamber containing sheath fluid by sample cell, gas pressure, and under the pressure of sheath fluid, cell passes through inspection in the form of single-row
Survey channel, be detected channel in excitation, transmitting light can be detected by detector, if sample can be incorporated into PD1 or
On PD-L1, then it is positive signal, is not combined into negative antibody.
EL ISA, enzyme-linked immunosorbent assay, 96 holes that the PD1 or PDL1 albumen of recombination purifying is coated on to high absorption are examined
In drafting board, excess protein is washed off after overnight incubation, and sample to be tested is added, washes off extra unbonded sample after incubation again, then
Another albumen is added, is incubated and washs for the third time, is then added and is marked with the secondary antibody of HRP (horseradish peroxidase), the 4th
Secondary incubation washing, is eventually adding the colour reagent of HRP, is read under microplate reader after reacting half an hour.According to signal judgement sample
The positive it is negative.
FACS needs special FCM analysis instrument, and instrument is expensive, and individual cells cross column, and flux is very low, and only
It is suitble to do the screening of antibody medicine, is not suitable for small molecule, what is filtered out is mostly binding molecule, as long as can be in conjunction with PD1 or PD-L1
Can, the combination of the two might not can be blocked, other experiment evidences are usually also needed to.EL ISA are deposited as traditional method
The shortcomings that some are difficult to overcome, such as：1) experimental procedure is more, and time-consuming, needs by multiple board-washing, sample-adding, colour developing etc., at least
Need one day time；2) it is unable to reach high throughput, it is difficult to solve the problems, such as that drug screening sample is more；3) have can for coated albumen
Some protein locis can be shielded, cause to miss positive to get to false negative result；4) experiment is easily caused since step is more
Error leads to result poor repeatability, unstable.
Invention content
In order to overcome the deficiencies of the prior art, it is blocked using HTRF one-step method screening PD1/PD-L1 the present invention provides a kind of
The method of agent, through the invention, it can be achieved that high flux screening in the short time, provides the selection result of more economic and reliable, be fast
Fast efficient screening PD1/PD-L1 blocking agents provide a kind of succinct effective method.
A method of PD1/PD-L1 blocking agents being screened using HTRF one-step method, are included the following steps：
Step 1 configures PD1 the and PD-L1 albumen with different labels built, sample to be screened；
Step 2, sample to be screened, PD1, PD-L1 are sequentially added in microwell plate and be coupled respectively Donor and
The tag antibody of Acceptor；
Step 3, incubation at room temperature read detection fluorescence with microplate reader, and the ratio of 665nm/620nm is initial data；
Two kinds of tag antibodies of wherein above-mentioned coupling Donor and Acceptor detect respectively interaction albumen PD1 and
PD-L1, to form the compound of four polymerizations, the distance of further Donor and Acceptor, energy can be from Donor
On be transferred on Acceptor, make Acceptor generate fluorescence；If sample can block the two to combine, with sample concentration
Increase, the ratio of 665nm/620nm reduces；The variation of fluorescent value is measured after stablizing can quantify the potency IC50 of blocking agent；
Detection is two fluorescence 665nm and 620nm of HTRF, i.e. time-resolved fluorescence, when the ratio of 665nm/620nm is lower, is blocked
The effect of agent is higher.
Further, above-mentioned PD1 and PD-L1 is recombinant protein, carries different labels.
Further, above-mentioned microwell plate includes 96 orifice plates, 384 orifice plates and 1536 orifice plates.
Further, the reaction system of the screening technique is 8ul-20ul.
Further, the blocking agent that can be used for screening includes antibody, albumen, polypeptide and the small molecule in various sources.
HTRF is the abbreviation of homogeneous phase time discrimination fluorescence technology, and technology is the further improvement to TR-FRET technologies.HTRF
Based on time-resolved fluorescence (TRF) itself and two big technology of fluorescence resonance energy transfer (FRET).TRF is partly declined using rare earth element
The characteristic of phase length (Millisecond has 6 magnitude differences compared with common fluorescent nanosecond).So can be micro- by postponing 50-100
Second excludes background.
FRET utilizes the energy transfer of Liang Zhong fluorescence group, both fluorescence groups to be referred to as energy donor (Donor)
With energy acceptor (Acceptor).Donor is by external light source activation, if it is close with Acceptor, can turn energy resonance
It moves on on Acceptor, it is made to be excited, launch the transmitting light of specific wavelength 665nm.
The energy donor of more traditional TR-FRET is wrapped in chelate, two kinds of Donor europiums (Eu3 of the fluorescent energy of HTRF
+ cryptate) and terbium (Lumi4TMTb), they are for good and all entrenched in cryptate, sensitiveer and stable.Europium and terbium
After stimulated light excitation, emission spectrum has a wave crest at 620nm.Acceptor also there are two types of, one is APC hinges are compound
Body, trade name XL665, another kind are d2, are micromolecular compound, molecular weight about 1kD.The exciting light of XL665 and d2 with
The transmitting light of Donor has certain overlapping, a special wave crest can be formed at 665nm after being excited.
HTRF technical characterstics have：
1. easy to operate：It need to only be loaded and detection reagent, i.e. detection after incubation are not necessarily to wash.It is completed in 1 hour real
It tests；
2. flux is high, easily minimize：384 or 1536 orifice plates operate, and reaction system minimum is up to 8uL；
3. signal stabilization：Can continuous several times detection, it is reproducible；
4. false positive, false negative rate are low：Homogeneous detection, does not destroy protein conformation；It can exclude natural products autofluorescence
Background.
The advantage of the invention is that：1. providing new method：Screening PD1/PD-L1 blocking agents on biochemistry level are realized,
It is more directly quick without building cell；2. being used in all blocking agent screenings, either antibody, albumen, polypeptide or small point
Sub- compound；3. experimental work amount and experimental period is greatly saved：Experimental implementation from original coating, closing, multiple board-washing,
Till now only sample and detection reagent need to be added, experimental period shortens to 1-2 hour by the 1 day original time；4. greatly
Improve the flux of experiment：Column is crossed by 96 holes or FACS individual cells of original ELISA, is increased to 384 holes even 1536 holes,
To realize the purpose for carrying out batch samples screening in a short time；5. result is more reliable：Homogeneous detection, antigen protein is in liquid
Nature conformation is presented in phase system, effectively avoids generating false positive and false negative；6. signal is more stable：Fluorescence duration time is long,
Can overnight after be detected again, do not influence testing result, and the testing result of ELISA is by time restriction, overlong time or
Person is too short all to influence testing result.
Description of the drawings
Fig. 1 is the schematic diagram of HTRF screening PD1/PD-L1 blocking agents.
Fig. 2 is the experimental data that HTRF methods establish PD1/PD-L1.
Fig. 3 is the data of the positive blocking agent of HTRF methods detection.
Specific implementation mode
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
The every other embodiment that technical staff is obtained without making creative work belongs to the model that the present invention protects
It encloses.
It should be noted that in the absence of conflict, the feature in embodiment and embodiment in the present invention can phase
Mutually combination.
The method for screening PD1/PD-L1 blocking agents using HTRF one-step method in the present invention, includes the following steps：
Step 1 configures PD1 the and PD-L1 albumen with different labels built, sample to be screened；
Step 2, sample to be screened, PD1, PD-L1 are sequentially added in microwell plate and be coupled respectively Donor and
The tag antibody of Acceptor；
Step 3, incubation at room temperature read detection fluorescence with microplate reader, and the ratio of 665nm/620nm is initial data；
Two kinds of tag antibodies of wherein above-mentioned coupling Donor and Acceptor detect respectively interaction albumen PD1 and
PD-L1, to form the compound of four polymerizations, the distance of further Donor and Acceptor, energy can be from Donor
On be transferred on Acceptor, make Acceptor generate fluorescence；If sample can block the two to combine, with sample concentration
Increase, the ratio of 665nm/620nm reduces；The variation of fluorescent value is measured after stablizing can quantify the potency IC50 of blocking agent；
Detection is two fluorescence 665nm and 620nm of HTRF, i.e. time-resolved fluorescence, when the ratio of 665nm/620nm is lower, is blocked
The effect of agent is higher.
As preferred and non-limiting, above-mentioned PD1 and PD-L1 are recombinant protein, carry different labels.
As preferred and non-limiting, above-mentioned microwell plate includes 96 orifice plates, 384 orifice plates and 1536 orifice plates.
As preferred and non-limiting, the reaction system of the screening technique is 8ul-20ul.
As preferred and non-limiting, the blocking agent that can be used for screening includes the antibody in various sources, albumen, polypeptide and small point
Son.
As shown in Figure 1, Fig. 1 is the schematic diagram of HTRF screening PD1/PD-L1 blocking agents.FRET utilizes Liang Zhong fluorescence group
Energy transfer, both fluorescence groups are referred to as energy donor (Donor) and energy acceptor (Acceptor).Donor quilts
External light source activation can be such that it is excited if it is close with Acceptor by resonance energy transfer to Acceptor,
Launch the transmitting light of specific wavelength 665nm.
Embodiment 1
1) albumen is prepared, PD1 2 times of gradient dilutions since 400nM obtain the various concentration of 400nM to 0nM；PD-L1
2 times of gradient dilutions since 50nM, obtain the various concentration from 50nM to 0nM；
2) each 5ul of prepared albumen is added in 384 hole detection plates, after being incubated at room temperature 15 minutes, coupling Donor is added
With the tag antibody (or adding 10ul after equivalent mixing) of Acceptor, it is incubated 1 hour and reads；
According to HTRF Method And Principles, the fluorescence ratio of 665nm/620nm is detected, according to the ratio, judges the combination of albumen,
Signal is higher, in conjunction with more, as a result sees Fig. 2.
Embodiment 2
1) positive blocking agent is configured, by people source anti-human PD1 blocking antibody and mouse source anti-
Human PD1 blocking antibody carry out gradient dilution, since 100nM, 3 times of dilutions；Meanwhile by positive chemical combination
Object is from 3000nM gradient dilutions；
2) albumen is prepared, the screening of follow-up blocking agent, PD1 50nM, PD-L1 are according to 1 suitable albumen concentration of example
For 5nM；
3) prepared blocking agent and albumen sequentially add in 384 hole detection plates, after being incubated at room temperature 15 minutes, are added even
The tag antibody (or adding 10ul after equivalent mixing) for joining Donor and Acceptor, is incubated 1 hour and reads；
According to HTRF Method And Principles, the fluorescence ratio of 665nm/620nm is detected, according to the ratio, judges the combination of albumen,
Signal is higher, in conjunction with more, as a result sees Fig. 3.
By Fig. 2 and Fig. 3 it is found that the present invention is suitable for the screening of a variety of PD1 or PD-L1 blocking agents, and albumen dosage is less,
Method high sensitivity.Experiments have shown that method using the present invention, which carries out PD1/PD-L1 blocking agents, screens simple possible.
In conclusion the method for current main screening PD1/PD-L1 is based on traditional EL ISA, FACS, and it is mostly
Cell experiment needs complicated cell line structure.And HTRF is applied screened in biochemistry level for the first time by the present invention, solves tradition
Method is cumbersome, and the time is long, and the low problem of flux realizes low cost, high-throughput, highly sensitive and stability, operation letter
The method for singly screening blocking agent provides more stable practical scheme to accelerate drug discovery progress.
It these are only the embodiment of the present invention, be not intended to limit the scope of the invention, it is every to be said using the present invention
Equivalent structure or equivalent flow shift made by bright book content is applied directly or indirectly in other relevant technical fields,
Similarly it is included within the scope of the present invention.
Claims (5)
1. a kind of method for screening PD1/PD-L1 blocking agents using HTRF one-step method, it is characterised in that include the following steps：
Step 1 configures PD1 the and PD-L1 albumen with different labels built, sample to be screened；
Step 2 sequentially adds sample to be screened, PD1, PD-L1 in microwell plate and has been coupled Donor and Acceptor respectively
Tag antibody；
Step 3, incubation at room temperature read detection fluorescence with microplate reader, and the ratio of 665nm/620nm is initial data；
Two kinds of tag antibodies of wherein above-mentioned coupling Donor and Acceptor detect the albumen PD1 and PD- of interaction respectively
L1, to form the compound of four polymerizations, the distance of further Donor and Acceptor, energy can turn from Donor
It moves on on Acceptor, Acceptor is made to generate fluorescence；It is combined if sample both can block, with the increase of sample concentration,
The ratio of 665nm/620nm reduces；The variation of fluorescent value is measured after stablizing can quantify the potency IC50 of blocking agent；Detection
For HTRF two fluorescence 665nm and 620nm, i.e. time-resolved fluorescence, when the ratio of 665nm/620nm is lower, the effect of blocking agent
Ying Yuegao.
2. the method method according to claim 1 for being screened PD1/PD-L1 blocking agents using HTRF one-step method, feature are existed
In：Above-mentioned PD1 and PD-L1 is recombinant protein, carries different labels.
3. the method method according to claim 1 for being screened PD1/PD-L1 blocking agents using HTRF one-step method, feature are existed
In：Above-mentioned microwell plate includes 96 orifice plates, 384 orifice plates and 1536 orifice plates.
4. the method method according to claim 1 for being screened PD1/PD-L1 blocking agents using HTRF one-step method, feature are existed
In：The reaction system of the screening technique is 8ul-20ul.
5. the method method according to claim 1 for being screened PD1/PD-L1 blocking agents using HTRF one-step method, feature are existed
In：The blocking agent that can be used for screening includes antibody, albumen, polypeptide and the small molecule in various sources.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109799353A (en) * | 2019-02-15 | 2019-05-24 | 浠思（上海）生物技术有限公司 | The method for detecting multiple cell factors simultaneously using HTRF technology |
| CN109813916A (en) * | 2019-02-15 | 2019-05-28 | 浠思（上海）生物技术有限公司 | Utilize the method for the blocking agent combined between HTRF one-step method screening Bcl-2 family member |
| CN109839509A (en) * | 2019-02-15 | 2019-06-04 | 浠思（上海）生物技术有限公司 | Utilize the method for the HTRF binding analysis experimental technique screening potential agonist of OX40/OX40L |
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