An easy and rapid method to screen large numbers of antibodies against internal cellular determinants.
A rapid and easy method is described to screen great numbers of antisera or antibodies against internal cellular antigens including viral antigens or to screen various target cells for proper expression of antigens; the method can also be applied to determine fluorescent foci to enumerate non-cytopathic viruses. Cells, infected with a particular virus or uninfected, adhering to flat-bottomed 96-well microtiter plates were fixed with conventional phosphate-buffered saline containing 4% formaldehyde for 10 min, alternatively, cells were first fixed with 3% paraformaldehyde for 10 min and were then treated with Trition X-100 for another 10 min. After two washes, either fluorescein-labelled antiviral antibodies or first antiviral antibodies followed by labelled anti-species antibodies were applied, incubated and washed off as usual. A few drops of a balanced salt solution were kept in the well and were drained off gently just before the plates were examined. The plates were viewed directly in an inverted UV microscope or were inspected and photographed bottoms up with a conventional UV microscope mounted with an old-fashioned uncorrected objective (20 X) which, because of its shorter length, permitted proper focussing. For most cases studied, sensitivity was comparable to the fluorescence analysis method of cells on slides. The plates could be stored for several months in a dark refrigerator if kept moist. The method is rapid because it avoids individual handling of samples for the washing procedures and does not need growing and mounting of cells on slides; up to 1000 samples can be tested by one person in a day.
Stitz
L
L
Institute of Pathology, University Hospital, Zürich, Switzerland.
Hengartner
H
H
Althage
A
A
Zinkernagel
R M
RM
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000914:Antibodies, Viral / Q000032:analysis*; D000956:Antigens, Viral / Q000032:analysis; D002478:Cells, Cultured; D003593:Cytoplasm / Q000276:immunology*; D007158:Immunologic Techniques; D008856:Microscopy, Fluorescence / Q000379:methods*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral