Characterization of a clone from an adult worm cDNA library selected with anti-Schistosoma mansoni human antibodies dissociated from immune complexes: a preliminary report

Abstract

SUMMARYConsidering the scarcity of defined antigens, actually useful and reliable for use in the field studies, we propose an alternative method for selection of cDNA clones with potential use in the diagnosis of schistosomiasis. Human antibodies specific to a protein fraction of 31/32 kDa (Sm31/32), dissociated from immune complexes, are used for screening of clones from an adult worm cDNA library. Partial sequencing of five clones, selected through this strategy, showed to be related to Schistosoma mansoni: two were identified as homologous to heat shock protein 70, one to glutathione S-transferase, one to homeodomain protein, and one to a previously described EST (expressed sequence tag) of S. mansoni. This last clone was the most consistently reactive during the screening process with the anti-Sm31/32 antibodies dissociated from the immune complexes. The complete sequence of this clone was obtained and the translation data yielded only one ORF (open reading frame) that code for a protein with 57 amino acids. Based on this amino acid sequence two peptides were chemically synthesized and evaluated separately against a pool of serum samples from schistosomiasis patients and non-schistosomiasis individuals. Both peptides showed strong reactivity only against the positive pool, suggesting that these peptides may be useful as antigens for the diagnosis of schistosomiasis mansoni.

Antischistosomal Activity of Two Active Constituents Isolated from the Leaves of Egyptian Medicinal Plants

“…In general, the use of various specific epitopes, in the same diagnostic method, can improve the sensitivity without interfering with the specificity. 35 …”

Development and evaluation of an indirect ELISA using a multiepitope antigen for the diagnosis of intestinal schistosomiasis

“…( 2001 ), and the ORF of the clone ET03, by Valli et al . ( 2007 ), previously evaluated by Oliveira et al . ( 2008 ), were included and designated P5 ( Table 1 ).…”

“…The regions with a high antigenicity score were defined and 4 peptide regions, named P1, P2, P3 and P4 were selected (Supplementary Figs S1 and S2). In addition, peptides from the cathepsin B amino acid sequence, published by Noya et al (2001), and the ORF of the clone ET03, by Valli et al (2007), previously evaluated by Oliveira et al (2008), were included and designated P5 (Table 1). The selected peptides were backtranslated to their respective nucleotide sequences, using BioEdit 7.2 software and sent to GenScript (Piscataway, NJ, USA), where P1-P5 antigen were optimized, including cleavage sites for BamHI (5 ′ ), HindIII (3 ′ ) and stop codon (3 ′ ).…”

“…In experiments with clones of an adult worm cDNA library, the clone designated as ET03 was the most reactive during screening with anti-Sm31/Sm32 antibodies eluted from nitrocellulose strips. Sequencing of this clone provided a 682 nucleotide sequence and the translation of the cDNA resulted in only 1 open reading frame (ORF), corresponding to 57 amino acids (Valli et al, 2007, Oliveira et al (2006 synthesized 7 peptides, of which 3 were based on the amino acid sequence of cathepsin B and 2 on the ORF sequence of clone ET03. When used in an enzyme-linked immunosorbent assay (ELISA), these peptides showed high immunoreactivity against a serum pool from individuals with intestinal schistosomiasis.…”

“…In experiments with clones of an adult worm cDNA library, the clone designated as ET03 was the most reactive during screening with anti-Sm31/Sm32 antibodies eluted from nitrocellulose strips. Sequencing of this clone provided a 682 nucleotide sequence and the translation of the cDNA resulted in only 1 open reading frame (ORF), corresponding to 57 amino acids (Valli et al ., 2007 ). In 2006, Oliveira et al .…”