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赤橙黃綠青藍紫 誰持彩練當空舞——激酶活性HTRF檢測的熒光酶標儀方案-下

基于應用的多功能熒光酶標儀選型-3

(續:激酶活性HTRF檢測的熒光酶標儀解決方案-中)

三、HTRF檢測應用中多功能熒光酶標儀的選型要略

        常規FI、TRF、FRET檢測,人們可能只關注到熒光酶標儀的激發、發射波長配置與測試要求契合的問題。而當了解HTRF標準流程后,對優化熒光檢測條件的意義和其中涉及的技術要素之復雜,興許會有感悟。

       開展HTRF測試前,實驗者首先須確認是否有可利用的HTRF認證多功能熒光酶標儀資源。

表2  Cisbio HTRF認證部分品牌多功能熒光酶標儀列表

Manufacturer

BMG

PerkinElmer

MD

BioTek

Tecan

可供型號

VantaStar

FluoStar Omega

PolarStar Omega

ClarioStar Plus

PheraStar FSX

EnVision XCite

EnVision HTS

SpectraMax iD5

SpectraMax Paradigm

SpectraMax i3x

SpectraMax M5e

Flexstation 3

Synergy H1

Synergy NEO2

Cytation 1

Cytation 5

Infinite F Nano+

Infinite F Plex

Spark

停產型號

Clariostar

Pherastar Plus

Pherastar FS

/

SpectraMax i3

Cytation 3

Synergy 4

Synergy H4

Synergy NEO

Synergy 2

Infinite F200 PRO

Infinite F500

Infinite M1000

Infinite M1000 PRO

Safire 2

                                                    (資料來源:PerkinElmer&Cisbio官網信息,入選時有刪減)

       確認多功能熒光酶標儀屬HTRF認證型號后,還應核實是否配置有HRTF檢測規定的專用光學元件。不同品牌機型,制造商提供的HTRF功能組件有區別。

        如VantaStar、Folarstar Omega、SpectraMax iD5、Synergy NEO2均須添加HTRF專用的具有Z軸調節功能的增強型時間分辨熒光模塊(Enhanced TRF module)、320/340nm紫外激發濾片、520/620/665nm發射熒光濾鏡這三件套。

       相對而言,MD SpectraMax i3、SpectraMax Paradigm、SpectraMax i3x三款熒光酶標儀,由于采用的是檢測功能模塊化設計,必要時直接將選配的HTRF檢測卡盒插入主機卡槽就可以啟動測試。實驗者無須為激發與發射濾光片的波長與帶寬、二向分色鏡截止波長范圍這類問題糾結。
        為方便查詢,表3列出了部分品牌制造商的HTRF認證機型系統配置,可供參考。

表3 品牌多功能熒光酶標儀HTRF檢測所需配置

Brand

Instrument Model

HTRF Configurations

MD

SpectraMax iD5

HTRF Detection System (6590-0144), includes:

Enhanced TRF module (0200-7030)

Filter slides

EX: 340nm BW 70nm (6590-0080)

EM2: 520nm BW 15nm (6590-0098)

EM1: 616nm BW 10nm (6590-0118)

EM2: 665nm BW 10nm (6590-0121)

SpectraMax Paradigm

Cisbio HTRF detection (0200-7011) , includes:

Xenon light source

616nm EM1 filter

665nm EM2 filter

SpectraMax i3X

BioTek

Synergy H1/H4

HTRF europium/Red filter cubes (8040505):

EX 330/80nm;

EM1 620/10nm, EM2 665/8nm; Dichroic 365nm

Synergy Cytation 1/3/5

Synergy NEO2

1) HTRF Europium/Red Kit, includes:

HTRF-europium Excitation cubes(1035011):

330/80nm; Dichroic 365nm

HTRF/Lance red Emission cubes (dual   emission)(1035041):

EM1 620/10nm, EM2 665/8nm; Dichroic 650nm

2) HTRF Europium/Green Kit,   includes:

HTRF terbium Excitation (1035012):

340/30nm; Dichroic 400nm

HTRF green Emission cubes (dual emission)(1035049):

EM1 520/10nm, EM2 620/10nm; Dichroic   550nm

BMG


VantaStar
Folarstar Omega
Polarstar Omega
ClarioStar series

?1) HTRF Europium/Red Kit, includes:

?Advanced Optic Head with   z-adjustment for TRF/TR-FRET

Excitation filter:337nm (TR-Ex H)(001-337TR)

Emission filter:620/8.5nm(001-615TR); 665/10nm(001-665TR)

2) HTRF Terbium/Green Kit, includes:

Advanced Optic Head with   z-adjustment for TRF/TR-FRET

?Excitation filter:337nm (TR-Ex H)(001-337TR)

Emission filter:620/8.5nm(001-615TR); 520/10nm(001-520TR)

Tecan

Infinite F Nano+

1) HTRF Europium/Red Kit, includes:

?Excitation filter:320/25nm (8C138)

Emission filter:620/10nm;Dichroic 510nm(6F041)

2) HTRF Terbium/Green Kit, includes:

?Excitation filter:320/25nm (8C138)

Emission filter:665/8.5nm;Dichroic 510nm(9E336)

nfinite F Plex

Spark

PE

EnVision XCite (TSM)

1) HTRF Europium/Red Kit, includes:

Excitation filter:UV2(TRF) 320 (#111)

Emission filter:APC 665 (#205)

2nd emission filter:CY5 620 (#118) or 615 (#203)

Top Mirror:TRF advanced single mirror (#615) or Lance/Delfia D400 single mirror (#412)

2) HTRF Terbium/Green Kit, includes:

Excitation filter:TRF UV2 340 (#101)

Emission filter:TRF 520(#275) or (#226)

2nd emission filter:495 (#276) for simultaneous readout

Top Mirror:TRF advanced single mirror (#615) or Lance/Delfia D400 single mirror (#412)

EnVision HTS (SDE)

1) HTRF Europium/Red Kit, includes:

Excitation filter:UV2(TRF) 320 (#111)

Emission filter:APC 665 (#205)

2nd emission filter:CY5 620 (#118) or 615 (#203)

Mirror:TRF Lance D407/D630 advanced dual mirror (#666) or Lance/Delfia D400/D630 dual mirror (#662)

2) HTRF Terbium/Green Kit, includes:

Excitation filter:TRF UV2 340 (#101)

Emission filter:TRF 520(#275) or (#226)

2nd emission filter:495 (#276) for simultaneous readout

Top Mirror:TRF D407/D505 advanced dual mirror(#664) or D400/D505 dual mirror (#657)

 

        擁有HTRF認證資質的熒光酶標儀、配套光學元件支持,再加上HTRF檢測試劑,開展HTRF技術研究方可謂萬事俱備、水到渠成。

四、HTRF檢測程序與同類TR-FRET方法間的兼容性

       目前,同樣是基于TR-FRET技術開發的檢測試劑, HTRF之外還有MD IMAP Assays、BellBrook Transcreener assay 、Invitrogen LanthaScreen TR-FRET Kinase Assay、Bioauxilium Thunder Enhanced TR-FRET assay、PE Lance TR-FRET Assays等。

       單論測試光譜數據,這些TR-FRET試劑的檢測光譜設置基本是通用的。那實際工作中,其他同類產品能否用HTRF的檢測標準程序測定?

       分析結果是:不行。

表4 市面TR-FRET類試劑產品測試光譜特征

TR-FRET Assay

TRF Cage Type

Excitation

2nd Emission

FRET   Acceptor

1st   Emission

HTRF-Eu/red assay

Europium cryptate

320 nm

620 nm

XL665/d2

665 nm

HTRF-Tb/red assay

Terbium cryptate

340 nm

620 nm

XL665/d2

665 nm

HTRF-Tb/green assay

Terbium Cryptate

340 nm

620 nm

Fluorescein/GFP

520 nm

LanthaScreen TR-FRET assay

Europium Chelate

320 nm

620 nm

AlexaFluor 647

665 nm

LanthaScreen TR-FRET assay

Terbium Chelate

340 nm

490 nm

Fluorescein/GFP

520 nm

Transcreener   TR-FRET assay

Terbium Chelate

340 nm

620 nm

HiLyte647

665 nm

LANCE assay

Europium Chelate

320 nm

620 nm

ULight

665 nm

LANCE  assay

Europium Chelate

320 nm

620 nm

Surelight APC

665 nm

THUNDER TR-FRET assay

Europium Chelate

320 nm

620 nm

Far-red dye

665 nm

       PE-Cisbio強調的是,HTRF認證測試流程、方法的應用范圍嚴格限于HTRF試劑和技術使用,不適用于用于其他TR-FRET產品的測定。

       TRF-FRET檢測具有時間高度靈敏的特性。在各種不同來源樣品測試條件下,不同TRF-FRET試劑盒的熒光光譜穩定性、半衰期特征的差異,注定了檢測過程的積分時間、脈沖數量、時間延遲幾個關鍵參數難以完全匹配。盡管兩種試劑盒測定的激發波長、發射波長峰值特征一致,但不代表TRF-FRET過程中供體、受體兩種熒光信號的動力曲線相同。

       HTRF試劑采用有大環結構的穴狀態鑭系配合物,其光譜穩定性優異,與特殊優化的緩沖液配合,可以實現0.6-1ms的超長熒光壽命。其他的TR-FRET技術中所用的鑭系配合物、FRET能量受體釋放的FRET熒光信號動力學特征除非能高度還原HTRF產品FRET過程的熒光表現。否則,在同一型號熒光酶標儀上用HTRF提供的標準測試程序測定其他廠商試劑,難免弄巧成拙、東施效顰。

表5  Ciosbio HTRF VS LanthaScreen檢測參數設置對比 

Assays

Ciosbio HTRF-Eu/red Assay

LanthaScreen Eu Kinase   Activity Assay

Excitation filter

337nm

Ex 340/30 nm

Emission filter

620/8.5nm,665/10nm

Em 665/10 nm,Em 620/10 nm; Dichroic LANCE/TRF

Delay Time

60 μs

100 μs

Integration Time

400 μs

200 μs

      對比Ciosbio HTRF-Eu/red Assay、LanthaScreen Eu Kinase Activity Assay在Perkin Elmer EnVision多功能熒光酶標儀上檢測所用的測試參數,可發現:盡管兩個試劑盒規定的激發波長、雙熒光發射波長設置基本一致(測試年份不同,同一型號儀器出廠預裝濾光片規格略有變動),但脈沖信號的積分時間延遲(HTRF-60μs VS LanthaScreen-100μs)、積分時間長度(HTRF-400μs VS LanthaScreen-200μs)的設定迥異。

        這證實了我們分析的結論:不同TR-FRET試劑盒在熒光多功能酶標儀上的規定的測試條件設置存在較大區別,其標準檢測程序相互難以有效兼容。
        表5的信息還表明:與Ciosbio HTRF-Eu比,LanthaScreen Europium激發的背景熒光衰減慢、而FRET供體和受體的熒光壽命短;LanthaScreen Eu檢測時間延遲拉長,極大地限制了檢測積分窗口的選擇余地和測試靈敏度的提升空間。

 

參考文獻:

[1]G Mathis. Rare earth cryptates and homogeneous fluoroimmunoassays with human sera. Clin Chem. 1993 Sep;39(9):1953-9.

[2]Mathis G. HTRF(R) Technology. J Biomol Screen. 1999;4(6):309–14.

[3]Bazin H, Trinquet E, Mathis G. Time resolved amplification of cryptate emission: a versatile technology to trace biomolecular interactions. J Biotechnol. 2002; 82(3):233–50.

[4]Degorce F, Card A, Soh S, Trinquet E, Knapik GP, Xie B. HTRF: A technology tailored for drug discovery — a review of theoretical aspects and recent applications. Curr Chem Genomics. 2009;3:22–32.

[5]HTRF Europium cryptate donor/Red acceptor readout Setup recommendations for Polarstar Omega (PerkinElmer & Cisbio).

[6]HTRF Europium cryptate donor/Red acceptor readout Setup recommendations for SpectraMax iD5 (PerkinElmer & Cisbio).

[7]HTRF Europium cryptate donor/Red acceptor readout Setup recommendations for Infinite F200 PRO (PerkinElmer & Cisbio).

[8]HTRF Europium cryptate donor/Red acceptor readout Setup recommendations for Envision equipped in Flash lamp (PerkinElmer & Cisbio).

[9]HTRF Europium cryptate donor/Red acceptor readout Setup recommendations for Synergy NEO2 equipped in Flash lamp (PerkinElmer & Cisbio).

[10]SpectraMax iD5 Multi-Mode Microplate Reader User Guide (5059784H,01/2022).

[11]Omega Family of Readers Operating Manual (Rev G).

[12]柳蘇月, 田晶晶, 田洪濤等. 鋱(Ⅲ)離子及其復合物:從發光特性到傳感應用. 生物技術通報, 2020, 36(4): 192-207.

[13]Agilent BioTek Detection Accessories Brochure (5994-4172EN).

[14]LanthaScreen TR-FRET Phospho-specific Antibody Reagents and Substrates User Guide (14 April 2009).

[15]EnVision Multi-label Plate Reader Brochure (007309D_01).
[16]Franka Maurer, Thomas Roux, Fanny Plénière, et al. Cellular and biochemical HTRF assays measured in 1536-well microplates.(BMG LABTECH GmbH, Cisbio Bioassays).

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