Thunder-Link® Oligonucleotides 寡核酸標誌套組 貨號425-0000 

快速標誌抗體與寡核苷酸

抗體(antibodies) -寡核苷酸 (oligonucleotides)標誌，是新一代生物標誌物檢測方法，可克服靈敏度和線性範圍問題，可能成為多重蛋白質診斷分析中之平台。Thunder-Link ® PLUS 抗體標誌套組，快速將抗體(antibodies)與寡核苷酸(oligonucleotides)以共價(Covalent bond)穩定性結合， 95-100%回收率高，可應用於免疫PCR（Immuno-PCR）、接近連接測定(Proximity Ligation Assay)、電化學鄰近測定 (Electrochemical Proximity Assay) CITEseq 及 REAPseq。與傳統ELISA方法相比，免疫PCR（Immuno-PCR）大幅提高偵測極限。

Thunder-Link ® PLUS 操作流程:

此產品可將oligo 標誌於抗體/Protein/peptide分子。首先將抗體及胺基化之oligo，各別加入activation buffer 後，30分後各別形成Activation型式，進行1小時反應。經由desalt多餘離子後，將抗體與oligo混合在一起進行結合，最後經由低速離心及沉澱步驟，純化抗體-oligo複合物。寡核苷酸(oligonucleotides)的3’或5’端進行-NH2修飾設計，長度為10-120個鹼基的單鏈寡核苷酸/80個鹼基對 (bases) 的雙鏈寡核苷酸(double stranded oligos )，pH6和pH8，HPLC純化，60-100µM (high)濃度。緩衝液不得含有胺基團 (primary amine groups)， 應避免添加BSA和疊氮化物(azide) 等添加劑 (可將寡聚物溶解在pH7的磷酸鹽緩衝液( phosphate buffer )中。 抗體每次反應為100 ul (1mg/ml) 。30分活化抗體(antibodies)與寡核苷酸(oligonucleotides)，進行1小時反應，使用內附Separation column快速移去未結合的寡核苷酸的干擾。套組內附有陽性對照抗體(control antibody)和寡核苷酸(control oligo)、 Oligo Activation reagent、 Antibody Activation Reagent、 Oligo Activation reagent、separation column/Wash buffer/ Conjugate Clean Up Reagent/Antibody Suspension Buffer。(請參考操作手冊)

Immuno-PCR應用

免疫PCR結合oligonucleotide於Immunoassay中，大幅提高偵測極限。在測試樣品例如全血和血清中，可精確和靈敏地檢測稀有細胞或低濃度的蛋白質。免疫PCR擴增，常規檢測單個核酸分子。與其模板雜交的DNA探針設計，提供高靈敏度和高特異性之方法。

INNOVA提供兩種商品，可將寡核苷酸連接到不同組的分子（例如抗體或金納米顆粒），讓免疫測定更多元化。Immuno-PCR靈敏度增加10至1000倍。亦可做為多重蛋白質診斷測定的平台。下圖顯示了在特定抗原濃度下由SYBR Green產生的PCR探針產生的PCR循環次數與螢光強度的關係圖。 下圖顯示了轉換為抗原量與循環次數後的數據，以便能夠計算標準曲線。 結果顯示該測定使用的捕獲抗體少1000倍，檢測抗體少100倍，靈敏度比同等ELISA高1000倍。

Proximity ligation assay-Used to identify proteins co-located within cells.

Proximity extension assay-Variation on ImmunoPCR using two antibodies specific for different epitopes.

ImmunoPCR Data

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ImmunoPCR data generated using Thunder-Link^®. A mouse monoclonal antibody specific for human CRP (clone C7) was purchased in unconjugated format from HyTest. The antibody was conjugated to an oligonucleotide using a Thunder-Link^® kit, and was used as a detection antibody in a sandwich immunoPCR assay with a polyclonal antiCRP antibody as the capture reagent.

The top graph shows a plot of the number of PCR cycles undertaken vs. fluorescence intensity generated by SYBR Green containing PCR probes at particular antigen concentrations. The bottom graph shows this data following conversion to antigen amount vs. cycle number to enable the calculation of a standard curve. The results show that the assay utilizes 1000-fold less capture antibody and 100-fold less detection antibody, and provides 1000-fold greater sensitivity than the equivalent ELISA

Features	Benefits
Quick and easy to use	Save time, no specialist knowledge required
High levels of antibody and oligo recovery	Save precious reagents
Use your own oligo and antibody, at your desired ratio	Flexible
Freeze dried	Ships at ambient temperature, long shelf-life
Stringently QC tested	Consistent high quality, excellent batch-to-batch reproducibility
Unidirectional chemistry	No risk of cross-linking
Covalent bond	Highly stable conjugates
Suitable for single-stranded oligos of 10-120 bases, double-stranded oligos up to 80 base pairs	Covers the majority of sequences
Linking chemistry works at both 5’ and 3’ end	Provides ability to combine with other modifications
Post-conjugation clean-up step	No interference from unbound oligo
Positive control antibody and oligo included	Enables confirmation of protocol success
Wide range of target proteins	Also applicable to antibody fragments and small proteins

產品編號	產品名稱	包裝
425-0000 425-0300	Thunder-Link® oligo conjugation system	1 reaction 3 reaction