A.Sensitize a 96-well microtiterplate with purified antigen. 1.Prepare a solution of the purified antigen of interest inphosphate buffer (see recipe below) such that a concentration ofapproximately 10 µg/ml is achieved. Pipette 100 µl of this solutionto all wells of the plate with the exception of those in theoutermost rows and columns (these areas of the plate are prone toevaporation). 2.Cover the plate with Parafilm and incubate for 2 hours at37°C, followed by an overnight incubation at 4°C. Cover the platetightly to avoid evaporation. Store at 4°C. The plate(s) willremain usable for about one month. B.Block the sensitizedplate. 1.When ready to use, discard the antigen solution contained inthe wells of the sensitized plate. Pat the inverted plate sharplyseveral times against a pad of paper towels to insure completeremoval of the solution. 2.Wash the wells three times with 1x PBS/0.05%-Tween 20. Pat todry completely after the final wash, as above. 3.Pipette 100 µl of 5% skim milk/PBS/0.005% thimerasol (added toretard bacterial growth) to each well. Incubate the plate, coveredwith Parafilm, at 37°C for 2 hours to overnight. It is convenientduring this incubation step to begin preparation of the samples tobe tested, outlined below. 4.Wash the wells three times with PBS/Tween, dry as before afterthe last wash. C.Prepare the samples to betested. 1.Using a Sharpie or other suitable permenant marker, mark asecond, unsensitized microtiter plate to identify the intendedlocation of all samples to be tested, and the location of thepositive and negative controls (the positive control in this assayis a purified sample of the antigen of interest; the same antigenwith which the previous plate has been sensitized. The negativecontrol is PBS). Do not use the outermost wells of the plate, asthese have not been sensitized in the previous plate. See diagram. 2.Pipette 60 µl of 5% skim milk/PBS/Tween to all experimentalwells except the first column of these wells, which will containundiluted aliquots of the samples to be tested. 3.Add 60 µl of the samples to be tested to both the first(empty) and second wells of the experimental area of the plate. Mixthe 1:2 dilution (second well) by pipetting up and down severaltimes. 4.Using a fresh pipette tip, transfer 60 ul from well 2 (1:2dilution) to well 3, creating a 1:4 dilution in this well. Mix bypipetting several times. Serially dilute the samples in this manneruntil all desired dilutions are achieved. It is criticallyimportant to use a fresh pipette tip for each transfer. 5.Place 60 µl of PBS in the negative control wells and 60 µl ofthe purified antigen sample in the positive control wells. 6.Dilute antisera to the antigen of interest appropriately in 5%skim milk/PBS/Tween (i.e. 1:1000, optimal dilution may varydepending on antigen-antisera. Final dilution under experimentalconditions will be doubled) and pipette 60 µl of the dilutedantisera to all wells (experimental and control). 7.Incubate the plate, covered with Parafilm to minimizeevaporation, at 37°C for 2 hours to overnight. D.Testing thesamples. 1.Retrieve the sensitized and blocked microtiter plate preparedin sections A & B, above. Duplicate exactly the pattern drawnon the non-sensitized plate on this plate. 2.Transfer 100 µl of the antisera-antigen solutions from thenon-sensitized plate to their corresponding wells on thesensitized, blocked plate. Incubate this plate, covered, at 37°Cfor 1.5 hours. 3.Discard the solutions from the wells of the plate afterincubation. Wash all wells three times with PBS/Tween as before.Dry after last wash. 4.Add to each well 100 µl of conjugate antibody (i.e. goatantirabbit alkaline phosphatase, if antisera was raised in rabbits)diluted appropriately in skim milk/PBS/Tween (i.e. 1:2000).Incubate the covered plate at 37°C for 1.5 hours. 5.Discard the conjugate antibody solution from the wells. Washthree times with PBS/Tween as before. 6.Add to each well 100 µl of substrate solution appropriate forthe conjugate antibody used. For alkaline phosphatase conjugate,use p-nitrophenyl phosphate disodium (Sigma 104) dissolved insubstrate buffer (795 mg Na2CO3, 1.456 g NaHCO3, 100 mg NaN3, 50 mgMgCl2 in a final volume of 500 ml sdH2O). 7.Read absorbence values at appropriate time intervals (usuallyevery 15 minutes, a shorter interval is required if the colorreaction is quicker; watch color development).  Phosphate Buffer Skim Milk SolutionsSolution 1 Solution 2 50.0 g skim milk powderNa2HPO4 2.84 g NaH2PO4 2.76 g 100.0 ml 10x PBSdH2O to 100 ml dH2O to 100 ml 1.0 ml 5% thimerosal sterile dH2O to final volume of 1LCombine 6.1 ml of solution 1 and3.9 ml of solution 2. Add 40 ml dH2O. Divide solution into 500 ml aliquots. To one aliquot, add 250 µl Tween-20. Pasturize at 65°C for 30 minutes. 10x Phosphate Buffered Saline (PBS)NaCl 80.0 gKCl 2.0 gNa2HPO4 14.4 gKH2PO4 2.4 gDissolve reagents in 800 ml dH20.Adjust pH to 7.4 with HCl. Autoclave to sterilize. Dilute to 1x before use.Add 250 µl Tween to 500 ml 1x PBS for the PBS/Tween wash solution. | 