ELISA for Legionnaires' Disease - Antibody and Antigen Detection Systems: An Interim Report 



2. Working in a microbiological safety Jiood, harvest growth by adding 3 ml of sterile 

 phosphate buffered saline (PBS), pH 7.2, to each agar plate and gently scrapping the agar 

 with a bent capillary pipette. 



3. Transfer the cell suspension to a sterile screw-capped flask, and steam in an Arnold 

 sterilizer for 1 h at lOTC. 



4. Centrifuge the killed-cell suspension in sterile 1 5-ml conical centrifuge tubes for 30 min 

 at 1600 X g. Decant the supernatant, and wash packed cells twice in sterile PBS, pH 7.2. 



5. Prepare a stock suspension of the cells by adding 2 ml of sterile PBS, pH 7.2, to each 0.1 

 ml of packed, centrifuged cells. Store stock antigen suspension at 4°C for 10 days to allow 

 the release of soluble antigen from the cells. 



6. Centrifuge the suspension at 1600 X g. Save the supernatant to be used as the stock 

 soluble antigen. (By doing block titrations, we found that a 1 :40 dilution was adequate with 

 our system.) 



B. Test Procedure 



1. Dispense 0.1-ml volumes of diluted antigen into each well of as many Cooke micro- 

 ELISA plates as are needed (protein-binding polystyrene no. 1-223-29). Seal plate with 

 Cooke Microtiter plate sealers, incubate at 37°C for 3 h, and store at 4°C until used. 



2. Aspirate excess antigen from wells, and wash each plate at least three times with PBS, 

 pH 7.2, containing 0.05% Tween 20. Leave PBS in the plate for at least 3-4 min per wash. 



3. If numerous sera are to be tested, screen them at a 1;8 or 1:16 dilution. Set up two 

 wells per specimen. 



4. Detemiine the end points of the positive sera by making further 2-fold dilutions. Assign 

 each specimen to be tested a row of wells on a plate. Also assign one row to a known 

 negative serum and one row to a known positive serum for each test run. Include PBS 

 control. 



5. With an automatic pipette, dispense 0.1 ml of a 1:8 or 1:16 dilution of the specimen to 

 be tested into well 1 of the row assigned to it. Prepare serial 2-fold dilutions as follows. To 

 wells 2 through 8 assigned to a specimen, with a Cooke microdropper add 0.05 ml of PBS, 

 pH 7.2, containing 0.05% Tween 20. Then, make 2-fold dilutions with Cooke microdiluters 

 (0.05 ml). (See Figure 1.) Finally, add 0.05 ml of PBS, pH 7.2, with 0.05% Tween 20, to all 

 wells on each plate, and vibrate or gently mix. 



6. Incubate plates at 37°C for 1 h. 



7. After they are incubated, wash plates as in Step 2. 



8. Predetermine appropriate conjugate dilution by block titration. Dispense 0.1 ml of the 

 appropriate dilution of enzyme-labeled conjugate [horseradish peroxidase(HRPO)-labeled, 

 antihuman immunoglobulin] into each well of every plate. 



NOTE: Assaying and adding the conjugate are perhaps the most critical steps in the proce- 

 dure. Impure and nonspecific conjugates adversely affect both specificity and sensitivity. 



9. Cover plates, and incubate them at 37°C for 1 h. 



10. Wash as described in Step 2 using five washes. 



1 1. Add 0.1 ml substrate to each well. The substrate for HRPO-labeled conjugate is ortho- 

 phenylenediamine (OPD). Stock contains 1% (wt/vol) OPD in methanol (100 mg OPD/10 



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