Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 93, Num. 1, 1998, pp. 111-112

RESEARCH NOTE

Rapid Elisa for Plague

AM Araujo/*, ATS Petribu, GHTS Barbosa, JRP Diniz, AMP Almeida/**, LB Carvalho Jr/*/^+

Laboratorio de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Cidade Universitaria, 50670-420 Recife, PE , Brasil
* Fundaçao de Hematologia e Hemoterapia de Pernambuco, Recife, PE, Brasil
** Centro de Pesquisas Aggeu Magalhaes-Fiocruz, Recife, PE, Brasil
^+ Corresponding author. Fax: +55-81-271.8485. E-mail: lbcj@npd1.ufpe.brnbsp;

Financial support: FINEP (grant no. 66.92.0454.000), CNPq, FACEPE and JICA.

Received 14 April 1997; Accepted 1 October 1997

Code Number:OC98020
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    Graphics: Tables (jpg) - 32.6K

RESEARCH NOTE

Key words: polyvinyl alcohol-glutaraldehyde - Yersinia pestis - ELISA - Brazil

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 ul of diluted F1 antigen (1.3 ug/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, Nestle) for 1 hr (instead of overnight as in the original method) at 28 C and washed with PBS/Tween once.

Diluted serum (100 ul 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 ul 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.

Table I and Table II show the results obtained using this rapid ELISA in human sera from patients and health individuals living at different plague foci in the northeast Brazil, respectively.

These subjects were clinically selected by the Centro de Pesquisas Aggeu Magalhaes-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.

Copyright 1998 Fundacao Oswaldo Cruz - Fiocruz

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