This assay is useful for screening antisera or hybridoma supernatants for specific antibodies when milligram quantities of purified or semipurified antigen are available. Antibodies are detected by coating the wells of microtiter plates with antigen, incubating the coated plates with test solutions containing specific antibodies, and washing away unbound antibodies.

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)

Determine developing reagent and antigen concentrations

1. Determine the optimal concentration of the developing reagent (conjugate) by criss-cross serial dilution analysis

2. Determine the final concentration of antigen coating reagent by criss-cross serial dilution analysis. Prepare an antigen solution in PBSN at this final concentration. The final concentration of antigen is usually 0. 2 to 10. 0 µg/ml. Prepare 6 ml antigen solution for each plate.

Coat plate with antigen

3. Using a multichannel pipet and tips, dispense 50 µl antigen solution into each well of an Immulon microtiter plate. Tap or shake the plate to ensure that the antigen solution is evenly distributed over the bottom of each well.

4. Wrap coated plates in plastic wrap to seal and incubate overnight at room temperature or 2 hr at 37°C.

5. Rinse coated plate over a sink by filling wells with deionized or distilled water dispensed either from a plastic squirt bottle or from the tap. Flick the water into the sink and rinse with water two more times, flicking the water into the sink after each rinse.

Block residual binding capacity of plate

6. Fill each well with blocking buffer dispensed as a stream from a squirt bottle and incubate 30 min at room temperature.

7. Rinse plate three times in water as in step 5. After the last rinse, remove residual liquid by wrapping each plate in a large paper tissue and gently flicking it face down onto several paper towels laying on the benchtop.

Add antibody to plate

8. Add 50 µl antibody samples diluted in blocking buffer to each of the coated wells, wrap plate in plastic wrap, and incubate ≥2 hr at room temperature.

Wash the plate

9. Rinse plate three times in water as in step 5.

10. Fill each well with blocking buffer, vortex, and incubate 10 min at room temperature.

11. Rinse three times in water as in step 5. After the final rinse, remove residual liquid as in step 7.

Add developing reagent to plate

12. Add 50 µl developing reagent in blocking buffer (at optimal concentration determined in step 1) to each well, wrap in plastic wrap, and incubate ≥2 hr at room temperature.

13. Wash plates as in steps 9 to 11. Add substrate and measure hydrolysis.

14. Add 75 µl MUP or NPP substrate solution to each well and incubate 1 hr at room temperature.

15. Monitor hydrolysis qualitatively by visual inspection or quantitatively with a mi crotiter plate reader (see below). Hydrolysis can be stopped by adding 25 µl of 0. 5 M NaOH.

a. Visually, hydrolysis of NPP can be detected by the appearance of a yellow color. If using a microtiter plate reader to measure NPP hydrolysis, use a 405-nm filter.
b. Visually, hydrolysis of MUP can be monitored in a darkened room by illumination with a long-wavelength UV lamp. If using a microtiter plate spectrofluorometer to measure MUP hydrolysis, use a 365-nm excitation filter and a 450-nm emission filter.