Molecular approaches to diagnosis of Chagas' disease: use of defined antigens and a target ribosomal RNA sequence.
We evaluated the performance of two defined antigens in the serological diagnosis of Chagas' disease. One of them is a recombinant protein named B13 isolated from a genomic library of Trypanosoma cruzi in the expression vector lambda gtll. We show that the gene corresponding to B13 is conserved in the evolutive stages of the two "polar" strains of T. cruzi. The protein epitopes cloned in B13 are represented in 140 kDa, 116 kDa and 35 kDa polypeptides of trypomastigotes. The other antigen chosen for serodiagnosis is a lipopeptidophosphoglycan (LPPG). This glycoconjugate is also widely distributed in T. cruzi strains. The use of a rabbit serum to LPPG allowed the demonstration that this molecule bears epitopes in common to LPPG-like components and to 80-90 kDa glycoproteins of trypomastigotes. Both B13 and LPPG were evaluated in serodiagnosis by ELISA and RIA using a panel of normal human, Chagasic and Leishmaniasis sera. It was observed that B13 presents high sensitivity and specificity for Chagasic sera. For LPPG it was also concluded that this reagent discriminates between individuals infected and non-infected with T. cruzi. A heterogeneity in the level of antibodies to LPPG in Chagasic patients was detected. No correlation was found between the clinical form of Chagas' disease and the preferential reactivity to B13 or LPPG. We also report preliminary studies towards the characterization of a 100 bp sequence of the 24S alpha rRNA as a target for DNA-based detection systems for diagnosis. We show that polymerase chain reaction of total DNA of different trypanosomatids lead to the specific amplification of a 100 bp fragment only for T. cruzi. Northern blots confirmed the presence of the target region in the mature 24S alpha rRNA. Titration experiments based on the direct amplification of RNA with Taq DNA polymerase allowed the detection of 50 parasites. Studies are in progress to increase the sensitivity of the proposed system.
Zingales
B
B
Departamento de Bioquímica, Universidade de São Paulo, Brazil.
Golgher
D B
DB
Marmorato
P G
PG
Souto
R P
RP
Gruber
A
A
D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood / Q000276:immunology; D000953:Antigens, Protozoan / Q000235:genetics* / Q000276:immunology; D014355:Chagas Disease / Q000175:diagnosis*; D003001:Cloning, Molecular; D018503:Genome, Protozoan; D006801:Humans; D010457:Peptidoglycan / Q000276:immunology; D010743:Phospholipids / Q000276:immunology; D016053:RNA, Protozoan / Q000235:genetics*; D012335:RNA, Ribosomal / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014349:Trypanosoma cruzi / Q000235:genetics* / Q000276:immunology*
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D010457:Peptidoglycan; D010743:Phospholipids; D016053:RNA, Protozoan; D012335:RNA, Ribosomal; D011993:Recombinant Fusion Proteins; C014643:lipopeptidophosphoglycan