This assay is used to detect or quantitate soluble antigens and is most useful when both a specific antibody and milligram quantities of purified or semipurified antigen are available. To detect soluble antigens, plates are coated with antigen and the binding of specific antibody-enzyme conjugates to antigen-coated plates is inhibited by test solutions containing soluble antigen.
The direct assay may also be adapted as an indirect assay by substituting specific antibody for specific antibody-enzyme conjugate. The amount of specific antibody bound is then detected using a species-specific or isotype-specific conjugate as a tertiary reactant.
Materials
| Developing reagent | Antigen solution |
| PBS (APPENDIX 2) containing 0.05% NaN3 (PBSN) | Water, deionized or distilled |
| Blocking buffer | Test antibody samples |
| substrate solution | 0.5 M NaOH (optional) |
| Multichannel pipet and disposable pipet tips | Immulon 2 , Immulon 4 , or equivalent microtiter plates |
| Plastic squirt bottles | Microtiter plate reader (optional) |
| Specific antibody–alkaline phosphatase conjugate | Standard antigen solution |
| Test antigen solutions | Round- or cone-bottom microtiter plates |
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 varied (see first support protocol). Prepare a 2× conjugate solution by diluting thespecific antibody–alkaline phosphatase conjugate in blocking buffer to twice the optimal concentration.
2. Coat and block wells of an Immulon microtiter plate with 50 µl 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).
4. Mix and incubate conjugate and inhibitor by adding 75 µl of 2× conjugate solution (from step 1) to each well of a round- or cone-bottom microtiter plate, followed by 75 µl 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.
5. Prepare uninhibited control samples by mixing equal volumes of 2× conjugate solution and blocking buffer.
6. Transfer 50 µl 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.
7. Wash plate as in steps 9 to 11 of the basic protocol.
8. Add 75 µl 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 pro duced 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.