Mem Inst Oswaldo Cruz, Rio de Janeiro, 93(1) Jan/Feb 1998
Rapid Elisa for Plague
Laboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Cidade Universitária, 50670-420 Recife, PE , Brasil
IFundação de Hematologia e Hemoterapia de Pernambuco, Recife, PE, Brasil
IICentro de Pesquisas Aggeu Magalhães-Fiocruz, Recife, PE, Brasil
Recently, an enzyme linked immunosorbent assay (ELISA) for plague was proposed in our laboratories using a modified polymer, polyvinil alcohol (PVA) - glutaraldehyde, as an alternative solid phase (AM Araujo et al. 1996 Mem Inst Oswaldo Cruz 91: 195-198). An antigen (F1) obtained from Yersinia pestis was covalently fixed onto PVA-glutaraldehyde discs.
The synthesis of these discs is simple and the low prices of the employed reagents are economically attractive. The present study describes a modification of this method, aiming to reduce the time of procedure from 36 hr to 3 hr.
The discs were introduced into flat bottomed microplates covered with 100 ml of diluted F1 antigen (1.3 mg/well) and left at 28°C for 1 hr (instead of overnight as in the original method). These treated discs were washed twice with PBS, containing 0.05% Tween 20 (Labsynth); blocked with skimmed milk (Molico, Nestlé) for 1 hr (instead of overnight as in the original method) at 28°C and washed with PBS/Tween once.
Diluted serum (100 ml of a 1:200 dilution in PBS) was incubated with the antigen-disc into clean microplates at 37°C for 30 min (instead of 1 hr as previously). After washing the antigen-antibody-disc complex five times with PBS/Tween, 100 ml of goat anti-human IgG (Sigma) conjugated to peroxidase diluted 1,500 times in 3% w/v skimmed milk were added and incubated at 37°C for 30 min (instead of 1 hr as previously). Afterwards, five washings with PBS/Tween were carried out. Then, the substrate solution (100 ml), composed of 0.325% w/v orthophenylenediamine dihydro-cloride (OPD-Sigma) and 0.085% H2O2 prepared in 0.3M Tris-citrate buffer, pH 6.0, was added. After incubation at room temperature (28°C) for 15 min, in the dark, the reaction was stopped with 2.5M H2SO4 (25 ml), the discs removed and the plates read in ELISA reader (Bio-rad) at 492 nm.
These subjects were clinically selected by the Centro de Pesquisas Aggeu Magalhães-Fiocruz, Research Institute responsible to monitor plague in the northeast Brazil. The mean value of the optical densities (OD) in Table I was equal to 1.289. From the results shown in Table II one can establish a "cut-off" value of 0.113. This method also gave OD equal to 0.004 for the blank controls. Furthermore, the ELISA OD positive values obtained using the 3 hr procedure plotted against those from the 36 hr presented a linear relationship (r and p values equal to 0.87 and 0.01, respectively).
Therefore, the ELISA procedure for plague, employing PVA alcohol glutaraldehyde as solid-phase, was successively shorted from 36 hr to 3 hr.