IndirectELISA

實驗概要

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).

主要試劑

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).

實驗材料

Microtiter plates

Microtiter plate reader equipped with a 405 nm or 450/492 nm filter  [Details:for pNPP or OPD, respectively].

實驗步驟

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).