A Universal DNA-Based Protein Detection System

一種基于DNA的通用型蛋白檢測系統

摘要：基于抗體的蛋白質檢測方法，主要有western blot、ELISA、點雜交以及免疫組化等，這些方法被廣泛地應用于科研和診斷領域。在蛋白質的免疫檢測過程中，樣品蛋白首先結合與特異性一抗上，然后再用攜帶標簽（諸如：熒光染料，放射性元素，酶等）的二抗進行檢測。然而，為了避免種間內的交叉反應，必須謹慎地選擇二抗。同時，有限的、可利用的一抗和二抗抗體對，進一步限制了對蛋白質的免疫檢測。而本文主要介紹一種不需要二抗的、基于DNA的蛋白質檢測系統，該系統主要是應用一種既和DNA修飾的報告分子結合，又與IgG抗體結合的通用型適配子，替代了標記二抗，可以對單一實驗進行多重檢測，并實現了功能的模塊化。文中以DNA修飾的Nanobarcodes、quantum dots（QDs）、horseradishperoxidase（HRP）為例，通過點雜交、western blot、免疫染色以及微球的方式，來闡述基于DNA的通用型蛋白檢測平臺。

 

1.基于DNA的蛋白檢測系統總體思路

Figure 1. (Middle) UA, a bifunctional protein−DNA hybrid molecule, which binds to most types of IgG antibodies (left) and any DNAmodified reporter molecules (right) to generate a modular library of pre-labeled primary IgG antibodies for any applications of protein detection in place of secondary antibodies.

2.UA的合成與鑒定

(a) Scheme of forming the universal adapter (UA) using a self-catalyzing enzyme (SNAP). Thiol-modified DNA tag was first conjugated to maleimide-benzyl guanine (BG) that served as the substrate for the SNAP enzyme. This BG-modified DNA tag was then linked to EZZ protein through SNAP catalysis to create the universal adapter (UA), a bifunctional protein-DNA hybrid molecule. (b) Confirmation of UA on 4-20% SDS-PAGE gel electrophoresis. The same gel was first stained with GelRed dye (left) to visualize DNA products and then stained with Comassie Brilliant Blue (right) to visualize protein products. Only UA with both DNA and protein components is visualized in both cases. Lane 1. UA; Lane 2. thiol-modified DNA only; 3. EZZ-SNAP protein only.

 

3.UA的功能驗證---既能結合IgG，又能結合DNA修飾的標記分子

(a) Scheme to confirm the binding of UA against DNA nanobarcodes and IgG primary antibodies. DNA linker of UA was hybridized with DNA nanobarcodes to form UA-DNA nanobarcodes. UA-DNA nanobarcodes bound to IgG antibodies to form IgG nanobarcodes. (b) Characterizing of the hybridization of UA and DNA nanobarcodes on native PAGE gel electrophoresis. The same gel with no staining (left) showed signals of distinct color ratios from DNA nanobarcodes, and with GelRed nucleic acid stain (right) showed signals from all DNA products. Lane 1. UA; Lane 2. R2G0-DNA nanobarcode; Lane 3. EZZ protein mixed with R2G0-DNA nanobarcode; Lanes 4-6. UA hybridized with R2G0-, R1G1-, and R0G2-DNA nanobarcodes, respectively. The shifted bands in lanes 4-6 showed that DNA nanobarcodes were linked to UA through DNA hybridization. (c) Characterizations of the binding of UA-DNA nanobarcodes to IgG antibodies using dot blot technique. Different IgG antibodies were spotted on PVDF membrane and incubated with the UA-DNA nanobarcodes. Dots 1-3. Rabbit polyclonal antibodies (anti-glucagon, anti-PDX-1, and anti-insulin) labelled with R2G0-, R1G1-, and R0G2-nanobarcodes, respectively. Fluorescence signals detected from dots 1-3 showed strong binding between the IgG antibodies and EZZ protein of UA. Dot 4. Goat polyclonal antibody (anti-PDX-1) labelled with R1G1-nanobarcode. When using a non-specific IgG that cannot bind with EZZ protein, there was no fluorescence signal detected from dot 4. Dot diameters were 250 Km for all images.

 

4.蛋白檢測---利用IgG nanobarcodes，通過點雜交、微球和免疫染色的方式對相應蛋白進行檢測

Figure 2. Using IgG nano-barcodes for protein detection. (a) Multiplexed protein detection with dot blot. Proteins were spotted on a PVDF membrane and then incubated with a detection solution, which contained a mixture of IgG nano-barcodes. The dots with fluorescence signals showed specific binding between protein targets and the corresponding IgG nano-barcodes. From left to right: GFP, RL, HSF, and sortase (negative control) proteins were labeled with R2G0-anti-GFP, R1G1-anti-RL, and R0G2-anti-HSF IgG antibodies, respectively. Dot diameters were 250 μm for all images. Background fluorescence from GFP alone was subtracted from the measurement with IgG nano-barcodes. (b) Multiplexed bead-based protein detection. PS beads were tagged with the first IgG antibody which captured the antigen and then sandwiched with IgG nano-barcodes. GFP, RL, and HSF protein targets were specifically detected on microbeads with R2G0-anti-GFP (red), R1G1-anti-RL IgG antibodies  (orange), and R0G2-anti-HSF (green), respectively. (c) IgG nanobarcodes for immunostaining of insulin and glucagon proteins in a diabetic mouse pancreas tissue. Insulin was stained with R0G2-antiinsulin (green) antibody, and glucagon was stained with R2G0-antiglucagon (red) antibody. Intense yellow regions are the autofluorescence of contaminated red blood cells.

 

5.蛋白檢測---利用IgG-UA-QDs和IgG-UA-HRP，通過點雜交和western blot方式對HSF蛋白進行檢測

Figure 3. DNA-based protein detection system with different reporter molecules. (a) Protein detection using dot blot technique with quantum dots. Quantum dots were used to replace DNA nanobarcodes in our IgG nano-barcodes. HSF protein on PVDF membrane was recognized by its specific antibody and then labeled with UA-QDs. (b) Comparison of WB detection of HSF protein using DNA-modified HRP with IgG-UA (IgG-UA-HRP) (left) and traditional HRPmodified secondary antibody (right). Lanes 1−5, HSF protein detection using our IgG-UA-HRP at 0.25, 0.5, 0.75, 1, and 2 pmol; lanes 6−9, HSF protein detection using traditional WB method which utilized the HRP-modified secondary antibody at 0.25, 0.5, 0.75, and 1pmol.