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INHIBITION ELISA
1. Prepare target plate :
a) Design the experiment to leave at least one well, row or column blank for the appropriate controls , including standards.
b) Add target antigen ( e. g purified soluble CD 4 ) diluted in buffer
( e. g. PBS / NaN3 ) at 50 µg/ml to an ELISA plate, 50 µl/well. Leave 60 ' at room temperature.
Meanwhile:
2. While the target plate is being prepared, prepare the inhibitor plate by preincubating supernatants with Antibody:
a) In a round bottomed plate add 80/100 µl supernatant to each well.
b) Make a dilution series of a standard (e.g. purified CD 4 ) and place it in one column. (A suitable initial concentration is 2 µg/ml for 2 fold dilution's and 10µg/ml for 5 fold dilution's) .
c) Add 40 µl /50 µl Antibody diluted in 0.1-0.5% BSA in PBS / NaN3. (Determining the antibody concentration to use.) Seal round bottomed (inhibitor ) plate and incubate for 60' on shaker in cold room.
After 60 minutes:
3. Remove Antigen from the target plate for re-use. Wash plate 1 x with PBS/NaN3.
4. Block remaining sites on the target plate with 0.5% BSA in PBS/ NaN3 for 30 minutes at room temperature ( or 10 minutes at 37 oC).
5. Wash the target plate twice with PBS / NaN3.
When the target plate is ready:
6. Transfer 50 µl from each well of the inhibitor plate to duplicate wells on the target plate. Seal target plate and incubate for 60' on a shaker in cold room.
7. Wash target plate 3 x PBS / NaN3 and add 50µl /well of Alkaline - Phosphatase coupled anti-mouse IgG or appropriate species diluted 1 : 1000 (Sigma ). Seal target plate and incubate for 60' on shaker in cold room. Whilst waiting for this take the substrate (Sigma 104 phosphatase substrate) from the freezer and warm to room temperaturebefore opening.
8. Make up enough substrate for 100 µl to be added to all the wells of the target plate by dissolving 50 mg of substrate in 10 ml of Diethanolamine buffer pH 9.8.
Diethanolamine buffer pH 9.8
Add 97 ml of Diethanolamine + 800 ml H2O.
Add 1 ml 0.5 M MgCl2.
Adjust pH to 9.8 with 1M HCl.
Make up to 1L and store at 4 oC in the dark.
9. Wash the plate 3 x PBS / NaN3 and add 100µl of substrate to each well. Put the plate at 37 oC. In a standard CD 4 assay , colour should develop in 30 - 60 min.
10. Read at 405 nm. The maximum absorbence for linear readings is 2. In a standard CD 4 assay 50% inhibition is achieved with 50 ng CD 4 / ml.
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The steps of the general, "indirect," ELISA for determining r antibodies,serum antibody concentrations are:
1. Apply a sample of known antigen of known concentration to a surface, often the well of a microtiter plate. The antigen is fixed to the surface to render it immobile. Simple adsorption of the protein to the plastic surface is usually sufficient. These samples of known antigen concentrations will constitute a standard curve used to calculate antigen concentrations of unknown samples. Note that the antigen itself may be an antibody.
2. A concentrated solution of non-interacting protein, such as bovine serum albumin (BSA) or casein, is added to all plate wells. This step is known as blocking, because the serum proteins block non-specific adsorption of other proteins to the plate.
3. The plate wells or other surface are then coated with serum samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards. Since antigen immobilization in this step is due to non-specific adsorption, it is important for the total protein concentration to be similar to that of the antigen standards.
4. The plate is washed, and a detection antibody specific to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins.
5. Secondary antibodies, which will bind to any remaining detection antibodies, are added to the wells. These secondary antibodies are conjugated to the substrate-specific enzyme. This step may be skipped if the detection antibody is conjugated to an enzyme.
6. Wash the plate, so that excess unbound enzyme-antibody conjugates are removed.
7. Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or electrochemical signal.
8. View/quantify the result using a spectrophotometer, spectrofluorometer, or other optical/electrochemical device.
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