Enzyme-Linked Immunosorbent Assay (Elisa) for Detection of Antibodies Against Pityrosporum Orbiculare

Abstract

Serum IgG antibodies against Pityrosporum orbiculare were detected by enzyme-linked immunosorbent assay (ELISA). Preliminary experiments with different microtiter plates, various techniques for coating, incubation and washing indicated that the following procedures were most suitable. Titertec P.V.C. Microplates, high activated (HAC) (Flow, England) were used. Wells were coated with whole P. orbiculare cells suspended in a 0.05 M carbonate buffer (10⁷ cells ml⁻¹), pH 9.6 for 3h at 37°C. After each incubation step the wells were washed 3 times with phosphate buffered saline (PBS) con-taining 0.05% Tween 20 (PBS-Tw), pH 7.4. The wells were filled with bovine serum albumin (BSA) 20 g L^-1 in PBS and left overnight at 4°C. The plates were incubated for 2.5h at 37°C with serum diluted in PBS-Tw. Alkaline phosphatase-conjugated rabbit anti-human IgG (Dako-Pads, Denmark) diluted in PBS-Tw was added and left overnight at 4°C. Finally 4-nitrophenyl phos-phate (Sigma, USA) 1 mg ml⁻¹ in 1 M diethanolamine buffer, pH 9.8 (Merck, West Germany) was allowed to react. The absorbance was read after 50 min with a Titertec-Multiscan MCC photometer (Flow, England). Ten sera from healthy adult volunteers were used. The experiment was done with 4 different samples from each of the sera and each sample was run in triplicate. The ELISA-method was compared with the indirect immunofluorescent technique.