To verify its function as a signal amplifier in ELISA and Western blot, we used and optimized mAP-mSA2 for indirect and sandwich ELISA with different antigens such as TNFα, HA-tag and Flag-tag and validated its function. We then verified the function of mAP-mSA2 in Western blot by treating mAP-mSA2 with biotinylated antibodies, which were conjugated with different antigens in different molecular weights. In both ELISA and Western blot results, mAP-mSA2 had similar performance in linear range and limit of detection compared to commercial streptavidin-conjugated alkaline phosphatase.
Additionally, we tested a potential applicability of mSA2 fusion proteins to other experimental methods, such as cell targeting and indirect ELISA using HRP. Taken together, we developed mAP-mSA2 which acts as a signal amplifier in the enzyme immunoassay detection system with the advantages of enabling mass and inexpensive production because it does not need chemical conjugation and can be continuously overexpressed in E.coli. The function of mAP-mSA2 was confirmed to have a robust level of performance comparable to the commercially available product by testing different biotinylated molecules.
29 Figure 2.8 (A) Schematic illustration of indirect ELISA with flag (B) Absorptions, relative signals of indirect ELISA and linear ranges of mAP-mSA2 and SA-AP. 30 Figure 2.9 (A) Schematic illustration of HA-tagged sandwich ELISA (B) Absorbance, sandwich ELISA relative signals and linear ranges of mAP-mSA2 and SA-AP. 32 Figure 2.10 (A) Schematic illustration of sandwich ELISA with flag (B) Absorbance, relative signals of sandwich ELISA and linear ranges of mAP-mSA2 and SA-AP.
33 Figure 2.11 (A) Schematic representation of Western blot (B) Purification of target proteins (C) Quantitative analyzes of band signal intensities.
Introduction
Biotin-Streptavidin binding system
The biotin-streptavidin binding system has been widely used due to the high affinity of biotin for non-covalent binding to streptavidin (KD = 10-14 M). This system is widely used in many ways, such as nucleic acid detection, protein purification, protein enrichment or protein detection in immunoassay4,5. Especially in immunoassay, a biotin-binding protein conjugated to an enzyme such as HRP or AP can be used to produce biotinylated antibodies in ELISA or western.
Also, the biotin-streptavidin system has the advantage of flexibility, as streptavidin can be conjugated to different molecules.
Monomeric proteins
Alkaline phosphatase (AP), which is widely used in immunoassays, has a homodimeric structure and a molecular weight of about 140 kDa. In this case, a metagenome library generated from flat oceanic tidal sediments from the west coast of Korea revealed monomeric alkaline phosphatase (mAP) in the research group of Seung-Goo Lee10. In addition, mAP was genetically fused to the TNFα retetropin and showed higher enzymatic activity than dimeric AP from E.coli recently11 (Figure 1.4).
Thus monomeric form of AP can overcome the steric hindrance of genetic fusion of a large-sized dimeric form of AP.
Research Outline
Biotinylated Target-specific Signal amplifier
Materials and Methods
- Protein purification
- Catalytic activity test
- Indirect ELISA
- Sandwich ELISA
- Western blot
- Cell culture and fluorescence cell microscopy
To catalyze the hydrolysis of the bacterial cell wall, lysozyme (50 ug/ml) was added after resuspension of the solution and incubated at 30 min at room temperature. The free imidazole was washed out by dialysis with PBS buffer and the purified mAP-mSA2 by IMAC was characterized by SDS-PAGE. Enzyme activity assay mAP and mAP-mSA2 were performed in MicroWell 96-well plates (Nunc Maxisorp, flat bottom).
Protein antigens (GST-TNFα, Flag-GST-HA) were serially diluted and immobilized in each well and incubated at 4°C overnight. Blocking buffer (5% goat serum in TBST) was loaded and incubated on a shaker for 2 hours at room temperature. Then, solutions of primary antibodies targeting TNFα (Invitrogen), Flag-tag (Genescript), and HA-tag (Abcam) were loaded into the wells and incubated for 1 h with shaking at room temperature.
All wells were then washed four times and mAP-mSA2 or Streptavidin-AP (Thermo-Fisher) and incubated for 1 hour with shaking at room temperature. After incubation, all wells were washed four times and a solution of 1-Step™ PNPP Substrate Solution (Thermo-fisher) and 2.5mM CaCl2 was added. Capture antibodies targeting Flag-tag (Thermo-fisher) and HA-tag (Thermo-fisher) were immobilized in each well and incubated for 2 h at 37 °C.
Protein antigens (Flag-GST-HA) were serially diluted in TBS and loaded into each well and incubated for 1 hour at room temperature. Then, all wells were washed three times and solutions of detection antibodies targeting Flag-tag (Genescript) and HA (Abcam) were loaded into the wells and incubated for 1 hour at room temperature with shaking. All wells were then washed four times with mAP-mSA2 or Streptavidin-AP (Thermo-fisher) and incubated for 1 hour at room temperature with shaking.
Then washed three times and added a solution of mAP-mSA2 in TBS and incubated for 1 hour at room temperature. Then washed three times with PBS and blocking is performed with blocking buffer (5% BSA, 5% FBS, 0.5% Triton X-100 in PBS) was added and incubated for 1 hour at room temperature. Then washed and prepared three times. HER2/AaLS/TMR was treated with cells for 1 hour at room temperature.
Results and Discussion
- Characterization of Enzyme-linked recombinant protein
- Catalytic activity test
- Indirect ELISA
- Sandwich ELISA
- Western blot
- Expanded experiments using mSA2
At the beginning of the experiment, we found that TNFα immobilization on the plate was difficult, and we tested various TNFα antigens (purchased TNFα(Biolegend), recombinant TNFα and GST-conjugated TNFα, Figure 2.4A). Then we loaded the substrate solution into all wells in the plate and measured the absorbance at 405nm (Figure 2.4B). We sequentially tested mAP-mSA2 as a biotinylated target-specific signal enhancer and compared it to commercial Streptavidin-AP(SA-AP) (Figure 2.5A).
We then loaded the substrate solution into all wells in a plate and measured the absorbance at 405 nm (Figure 2.4B). For concentration optimization of the treatment of mAP-mSA2, we tested different concentrations ranging from the concentration of 1/27 to 9 times the concentration based on 10 µg/ml mAP-mSA2 (Figure 2.6A). We then loaded the substrate solution into all wells in a plate and measured the absorbance at 405 nm (Figure 2.7A).
We obtained sigmoidal curves between the linear absorption range of nM and 3.55 nM LOD using mAP-mSA2. We also tested the Flag tag as an antigen by indirect ELISA to verify that mAP-mSA2 could be a biotinylated target-specific signal enhancer. We then loaded the substrate solution into all wells in a plate and measured the absorbance at 405 nm (Figure 2.8A).
Next, we loaded the substrate solution into all the wells in a plate and measured the absorbance at 405 nm (Figure 2.9A). We obtained sigmoidal curves between the linear absorbance range of 1.16~93.75nM and 1.23nM LOD using mAP-mSA2. Next, we loaded the substrate solution into all the wells in a plate and measured the absorbance at 405 nm (Figure 2.10A).
Thus, we performed a Western blot with mAP-mSA2 to verify the ability of the biotinylated target-specific signal enhancer (Figure 2.11A). Target proteins were selected based on different molecular weights and purified (HaloTag-EGFR aphibody, HaloTag-T-EGFR aphibody, HaloTag-DTA-EGFR aphibody) and biotinylated (Figure 2.11B). As shown in Figure 2.11C, the signal intensities gradually increased as the concentrations of biotinylated target proteins increased.
As shown in Figure 2.12, HER2/AaLS/TMR construct, which used mSA2-HaloTag TMR tagging, could stain NIH3T6.7 cells. SDS-PAGE analysis showed a soluble fraction of mSA2-RNb, and unwanted protein was removed through anion exchange chromatography (Figure 2.13B).
Conclusion
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