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Clinical and Vaccine Immunology, August 2008, p. 1265-1271, Vol. 15, No. 8
1071-412X/08/$08.00+0     doi:10.1128/CVI.00069-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Reverse Enzyme-Linked Immunosorbent Assay Using Monoclonal Antibodies against SAG1-Related Sequence, SAG2A, and p97 Antigens from Toxoplasma gondii To Detect Specific Immunoglobulin G (IgG), IgM, and IgA Antibodies in Human Sera{triangledown}

Fernando R. Carvalho,1 Deise A. O. Silva,1 Jair P. Cunha-Júnior,2 Maria A. Souza,3 Taísa C. Oliveira,1 Samantha R. Béla,1 Gabriele G. Faria,1 Carolina S. Lopes,1 and José R. Mineo1*

Laboratory of Immunoparasitology,1 Laboratory of Molecular Biology, Institute of Biomedical Sciences, Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil,3 Division of Microbiology and Immunology, Federal University of Tocantins, Palmas, Tocantins, Brazil2

Received 22 February 2008/ Returned for modification 2 April 2008/ Accepted 8 June 2008

The present study aimed to evaluate the performance of three monoclonal antibodies (MAbs) in reverse enzyme-linked immunosorbent assays (ELISAs) for detecting immunoglobulin G (IgG), IgM, and IgA antibodies against Toxoplasma gondii in 175 serum samples from patients at different stages of T. gondii infection, as defined by both serological and clinical criteria, as follows: recent (n = 45), transient (n = 40), and chronic (n = 55) infection as well as seronegative subjects (n = 35). The results were compared with those obtained by indirect ELISA using soluble Toxoplasma total antigen (STAg). Our data demonstrated that MAb A3A4 recognizes a conformational epitope in SAG1-related-sequence (SRS) antigens, while A4D12 and 1B8 recognize linear epitopes defined as SAG2A surface antigen and p97 cytoplasmatic antigen, respectively. Reverse ELISA for IgG with A3A4 or A4D12 MAbs was highly correlated with indirect ELISA for anti-STAg IgG, whereas only A4D12 reverse ELISA showed high correlation with indirect ELISA for IgM and IgA isotypes. To our knowledge, this is the first report analyzing the performance of a reverse ELISA for simultaneous detection of IgG, IgM, and IgA isotypes active toward native SAG2A, SRS, and p97 molecules from STAg, using a panel of human sera from patients with recent and chronic toxoplasmosis. Thus, reverse ELISA based on the capture of native SAG2A and SRS antigens of STAg by MAbs could be an additional approach for strengthening the helpfulness of serological tests assessing the stage of infection, particularly in combination with highly sensitive and specific assays that are frequently used nowadays for diagnosis of toxoplasmosis during pregnancy or congenital infection in newborns.

* Corresponding author. Mailing address: Laboratório de Imunoparasitologia, Universidade Federal de Uberlandia, Bloco 4C, Av. Pará, 1720, Campus Umuarama, 38400-902 Uberlandia, Minas Gerais, Brazil. Phone: 55-34-3218-2058. Fax: 55-34-3218-2333. E-mail: jrmineo{at}ufu.br

{triangledown} Published ahead of print on 18 June 2008.

Clinical and Vaccine Immunology, August 2008, p. 1265-1271, Vol. 15, No. 8
1071-412X/08/$08.00+0     doi:10.1128/CVI.00069-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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