Indirect ELISA
实验概要
In the indirect ELISA, the enzyme-antibody conjugate uses an antibody against the type of antibody that is used to detect the antigen, kind of like a sandwich. For instance, if the antigen is HIV, then an anti-HIV antibody is prepared (let's say from a mouse). Then, in a separate reaction, an enzyme is attached to an anti-mouse antibody. So, in order to detect the HIV in the assay, a mouse anti-HIV antibody is used to detect the antibody attached to the antigen.
The advantages are:
1. There are more binding sites for the anti-mouse antibody and so, more enzyme can attach and be detected as opposed to the direct ELISA where the antibody will attach to fewer sites and less enzyme is available in the assay;
2. The enzyme-antibody conjugate can be prepared against (in our example, mouse) antibodies and so can be used to detect any mouse antibody in any reaction. So, you can prepare a large stock and then use it in lots of different ELISAs detecting lots of different antigens (as long as you have a mouse antibody as the initial detecting antibody).
主要试剂
Phosphate buffered saline (PBS) tablet: 10 mM phosphate buffer, pH 7.4, 150 mM NaCl [Details:Product No. P4417] and 0.1% sodium azide [Details:Product No. S2002].
Carbonate-Bicarbonate buffer capsule, pH 9.6 [Details:Product No. C3041].
Washing buffer (PBS-T): 10 mM phosphate buffer pH 7.4, 150 mM NaCl, 0.05% Tween 20 [Details:Product No. P3563].
Monoclonal primary antibody.
Antibody controls: species- and isotype-matched, non-specific immunoglobulin [Details:e.g., mouse myeloma proteins as a control for a mouse monoclonal primary antibody]
Alkaline phosphatase-conjugated secondary antibody or peroxidase-conjugated secondary antibody
Substrate for alkaline phosphatase conjugated secondary antibody[Details:such as SIGMAFAST™ pNPP tablets, Product No. N1891] or substrate for peroxidase conjugated secondary antibody [Details:such as SIGMAFAST™ OPD tablets, Product No. P9187].
Stopping reagent for alkaline phosphatase: 3 M NaOH (optional) or stopping reagent for peroxidase: 3 M HCl or 3 M H2SO4 (optional).
实验材料
Microtiter plates
Microtiter plate reader equipped with a 405 nm or 450/492 nm filter [Details:for pNPP or OPD, respectively].
实验步骤
1. Antigen Coating
1) Prepare an antigen solution at the appropriate concentration in carbonate-bicarbonate buffer or PBS.
2) Pipette 0.2 ml of the above solution to each well of the microtiter plate.
3) Incubate at 37 °C for 30 min., or incubate (covered) overnight at 4 °C.
4) Remove the coating solution. Wash three times with PBS-T.
Note: If problems with non-specific binding occur, an additional blocking step (30 min. 5% BSA-PBS) may be required. For further information see: Vogt, R.F., et al., J. Immunol. Meth., 101, 43 (1987).
2. Primary Antibody Reaction
1) Dilute the monoclonal primary antibody in PBS-T. The optimal dilution should be determined using a titration assay.
2) Add 0.2 ml of the diluted monoclonal antibody to each well. The negative control should be species- and isotype-matched, non-specific immunoglobulin diluted in PBS-T.
3) Incubate at room temperature for 2 hours.
4) Wash as in step 4 of Antigen Coating.
3. Application of Secondary Antibody
1)Dilute the enzyme-conjugated secondary antibody in PBS-T. Add 0.2 ml of this solution to each well. The optimal dilution should be determined using a titration assay.
2)Incubate at room temperature for 2 hours.
3)Wash as in step 4 of Antigen Coating.
4. Substrate Preparation
During the last incubation and immediately before use, prepare the enzyme substrate or bring the premade liquid substrate to room temperature.
5. Development
1) Add 0.2 ml of the freshly prepared substrate to each well.
2) Color should develop in positive wells after 30 minutes (yellow or orange, for pNPP or OPD, respectively).
3) Absorbance may be read directly in a microplate reader (at 405 nm or 450 nm, for pNPP or OPD, respectively) or the reaction may be stopped with 50 µl per well of the appropriate stopping reagent and absorbance read later (at 405 nm or 492 nm, for pNPP or OPD, respectively).
