Specific antibody-alkaline phosphatase conjugate (second support protocol)
Standard antigen solution
Test antigen solutions
1. Determine the optimal concentration of coating reagent and antibody-alkaline phosphatase conjugate by criss-cross serial-dilution analysis in which the concentrations of both the antigen (coating reagent) and the conjugate (developing reagent) are
coat well with antigen incubate with antibody-enzyme conjugate with or without inhibitor antigen add substrate and measure inhibition of color change or fluorescence g> = detected AG
coat well with antigen incubate with antibody-enzyme conjugate with or without inhibitor antigen add substrate and measure inhibition of color change or fluorescence
Figure 2.1.2 Direct competitive ELISA to detect soluble antigens. Ag = antigen; Ab = antibody; E = enzyme.
varied (see first support protocol). Prepare a 2x conjugate solution by diluting the specific antibody-alkaline phosphatase conjugate in blocking buffer to twice the optimal concentration.
The final concentration is usually 25 to 500 ng antibody/ml. Prepare 3 ml antibody-alkaline phosphatase conjugate for each plate.
2. Coat and block wells of an Immulon microliter plate with 50 ^l antigen solution as in steps 2 to 7 of the basic protocol.
3. Prepare six 1:3 serial dilutions of standard antigen solution in blocking buffer (see first support protocol for preparation of serial dilutions)—these antigen concentra tions will be used in preparing a standard inhibition curve (see step 10).
Antigen concentrations should span the dynamic range of inhibition. The dynamic range of inhibition is defined as that range of inhibitor concentrations wherein changes of inhibitor concentration produce detectable changes in the amount of inhibition. The dynamic range of inhibition is empirically determined in an initial assay in which antigen concentration is typically varied from the micromolar (10-6 M) to the picomolar (10~12 M) range. For most protein antigens, initial concentration should be ~10 p g/ml, followed by nine 1:4 serial dilutions in blocking buffer. These antigen dilutions are assayed for their ability to inhibit the binding of conjugate to antigen-coated plates under standard assay conditions. From this initial assay, six 1:3 antigen dilutions spanning the dynamic range of inhibition are selected for further use as standard antigen-inhibitor dilutions. Prepare >75 p l of each dilution for each plate to be assayed.
Inhibitor curves are most sensitive in the region of the curve where small changes in inhibitor concentrations produce maximal changes in the amount of inhibition. This region of the curve normally spans 15% to 85% inhibition. In most systems, this range of inhibition
Induction of Immune
Responses is produced by concentrations of inhibitor between 1 and 250 ng/ml.
4. Mix and incubate conjugate and inhibitor by adding 75 |l of 2x conjugate solution (from step 1) to each well of a round- or cone-bottom microtiter plate, followed by 75 |l inhibitor—either test antigen solution or standard antigen solution (from step 3). Mix the conjugate and inhibitor solutions by pipetting up and down in the pipet tip three times (see annotation to step 8 in the basic protocol) and incubate >30 min at room temperature.
For accurate quantitation of the amount of antigen in the test solutions, test antigen solutions should inhibit conjugate binding between 15% to 85%. It may be necessary to assay two or three different dilutions of the test solutions to produce inhibitions within this range.
5. Prepare uninhibited control samples by mixing equal volumes of 2x conjugate solution and blocking buffer.
6. Transfer 50 |l of the mixture of conjugate plus inhibitor (from step 4) or conjugate plus blocking buffer (from step 5) to an antigen-coated plate (from step 2) and incubate 2 hr at room temperature.
If samples are to be assayed in duplicate, the duplicates should be in adjacent columns on the same plate. Reserve column 11 for uninhibited control samples (step 5) and column 12 for substrate alone without any conjugate. If the concentration of antigen in the test samples is to be accurately quantitated, dilutions of homologous antigen solutions (step 3) should be included on each plate.
7. Wash plate as in steps 9 to 11 of the basic protocol.
8. Add 75 |l of MUP or NPP substrate solution to each well and incubate 1 hr at room temperature.
9. Read plates on the microtiter plate reader after >1 hr, at which time enough substrate has been hydrolyzed in the uninhibited reactions to permit accurate measurement of the inhibition.
10. Prepare a standard antigen-inhibition curve constructed from the inhibitions produced by the dilutions of the standard antigen solutions from step 3. Plot antigen concentration on the x axis, which is a log scale, and fluorescence or absorbance on the y axis, which is a linear scale.
11. Interpolate the concentration of antigen in the test solutions from the standard antigen-inhibition curve.
The dynamic range of the inhibition curve may deviate from linearity if the specific antibodies are heterogeneous and possess significantly different affinities or if the standard antigen preparation contains heterogeneous forms of the antigen. Antigen concentration in test samples can be accurately interpolated from the inhibition curve as long as the test antigen is antigenically identical to the standard antigen and the concentration of test antigen falls within the dynamic range of inhibition.
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