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Various compounds are recognized by antibodies as an antigen. Antibodies are widely applied not only for separation or analysis of cells in immunology and cell biology but also for identification of environmental pollutants, etc. Antibodies, therefore, are essential reagents for life science research. This rabbit antiserum was obtained by immunization of DTBTA-Eu3+-labeled KLH (keyhole limpet hemocyanin) as an immunogen and can be recognized with DTBTA-Eu3+ as a hapten molecule. DTBTA-Eu3+ is a fluorescent label for proteins and nucleic acids. With the combination of the antiserum, DTBTA-Eu3+ is applicable as a tag molecule and DTBTA-Eu3+-labeled molecules can be immunologically detected with ELISA and Western-blotting via the tag. In addition, the tagged molecule can be monitored and quantified by the fluorescence from DTBTA-Eu3+ (λex. max 335nm, λem. max 616nm). As shown in Figure 1, the antiserum can also bind to ATBTA-Eu3+ (a), which is a precursor of DTBTA-Eu3+, and the binding of the antibody to DTBTA-Eu3+-labeled protein is inhibited by ATBTA-Eu3+ (b).
Fig.1. Reactivity of Anti-DTBTA-Eu3+ Antiserum and Protein A purified antibody to antigen.
For ELISA, a microtiter plate was coated with DTBTA-Eu3+-labeled OVA or ATBTA-Eu3+, which is a pre-compound of DTBTA-Eu3+. a: ATBTA-Eu3+ was coated onto the wells and then reacted with anti DTBTA-Eu3+ serum (TCI code: A2181). Anti-rabbit IgG-HRP conjugate was used for detecting the antiserum bound to ATBTA-Eu3+. b: DTBTA-Eu3+-labeled OVA was coated onto the wells. Inhibition of Protein A-purified anti DTBTA-Eu3+ antibody (TCI code: A2239) to DTBTA-Eu3+-labeled OVA was observed in the presence of ATBTA-Eu3+ (50μM). Anti-rabbit IgG-HRP conjugate was used for detecting the bound IgG. Absorbance at 405nm was determined for HRP with ABTS as a substrate.
[Application] •With the combination of the antiserum, DTBTA-Eu3+ is applicable as a tag molecule. •DTBTA-Eu3+-labeled molecules can be immunologically detected with ELISA and Western-blotting via the Tag.
?About DTBTA-Eu3+ The DTBTA-Eu3+ is a europium ? uorescent labeling reagent, whereas ATBTA-Eu3+ (TCI code: A2083) is used as a label after conversion to DTBTA-Eu3+ by reacting with trichlorotriazene. DTBTA-Eu3+ can be labeled onto proteins and nucleic acids. Di? erent from other lanthanide chelate reagents, the DTBTA-Eu3+ complex has a high stability constant, and therefore, the problem of ? uorescence intensity change in various bu? ers has been greatly reduced. DTBTA-Eu3+ also has several advantages such as intensity stability in water for a long period, and the stability against photo-bleaching. If further detailed information about DTBTA-Eu3+ is required, please refer to “New Europium Fluorescent Labeling Reagent”.
¦Typical Procedure ELISA using DTBTA-Eu3+-labeled antibody We demonstrate the immunological technique to detect an antigen with an antibody labeled with a hapten tag. The antibody that recognizes a certain protein, was labeled with DTBTA-Eu3+ and a secondary-antibody (anti-DTBTA-Eu3+ antibody) binds to the tag molecule (DTBTA-Eu3+).
Reagents •Antigen protein (10μg/mL) •DTBTA-Eu3+-labeled antigen-specific antibody (1mg/mL):Antigen specific primary antibody, which has been labeled with DTBTA-Eu3+ •Purified anti DTBTA Eu3+ antibody:purified anti-DTBTA-Eu3+ rabbit antiserum with Protein A column •Alkaline phosphatase-conjugated anti rabbit IgG :commercially available •10mM diethanolamine buffer (pH9.5) containing 0.5mM MgCl2 •Chromogenic substrate: 1 mg/mL pNPP in the diethanolamine buffer •Stop solution:0.1M EDTA, pH7.5 •TBS:25mM Tris-HCl (pH7.2) containing 0.15M NaCl •TBST:TBS containing 0.05% Tween-20 •Blocking solution:4% skim milk in TBST •Antibody diluting solution:1% skim milk in TBST
Procedure 1. Place antigen protein into a 96-well plate (50μL/well) and incubate for 2 hours at room temperature 2. Remove the antigen solution, add blocking solution (150μL/well) to the wells and incubate for 2 hours at room temperature 3. Wash the wells with 200μL TBST, twice 4. Add purified DTBTA-Eu3+-labeled antigen-specific antibody , which has been diluted at 1, 0.5, 0.25, and 0.125μg/mL with antibody diluting solution, to the well (100μL/well) and incubate for 1 hour at room temperature 5. Remove the antibody solution and wash the well with TBST, 3-times 6. Add purified anti-DTBTA-Eu3+ antibody (diluted at 1/1000, 100μL/well) to the wells and incubate for 1 hour at room temperature 7. Remove the antibody solution and wash the well with TBST, 3-times 8. Add alkaline phosphatase-conjugated anti rabbit IgG (100μL/well) to the wells and incubate for 1 hour at room temperature 9. Remove the antibody solution and wash the well with TBST, 3-times and further wash with 10mM diethanolamine buffer (pH9.5) containing 0.5mM MgCl2 10. Add the chromogenic substrate (100μL/well) to the wells and add stop solution (100 μL/well) after the incubation for 5-20 minutes at room temperature 11. Determine absorbance at 405nm
The increase of absorbance observed depending on the antibody concentration. On the other hand, antigen-uncoated wells gave low background signal. These observations have shown that the anti-DTBTA-Eu3+ antibody recognizes DTBTA-Eu3+ with higher specificity.
*This data is an application example of the products. Product performances are not guaranteed by this data.