Regarding protein homogeneity, the preparations of rK9 and rK26 showed at least one significant protein impurity as verified by SDS-PAGE, and such recombinant antigens were assayed by immunoblotting against a Leishmania infected human panel. The proteins K9 + K39 were analysed by ELISA using a canine serum panel (20 positive and 20 negative sera), and the values of SP (100%) and SE (95%) learn more obtained were identical

to those found for rLci2B. ELISA performed with rLci2B employed a higher number of canine serum samples (138 positive and 119 negative sera) than that used in K9 + K39 immunological assay. The comparison between chimera K9-K39-K26 and rLci2B, in respect to ELISA values, shows that for rLci2B, the SE values were superior (100% vs. 95%), while the SP values were inferior (95% vs. 100%). However, it should be noted that the construction of the chimera K9-K39-K26 with two tags

is a difficult task, and the chimera recovery was low and estimated at approximately 10 mg/L bacterial culture buy BGJ398 (34). Considering the number of serum samples tested using rLci2B and the chimera K9-K39-K26 as being statistically consistent, the values obtained in this study are significant especially those related to the SE parameter (100%) that eliminates the false negative cases. On the other hand, the value of SP equal to 100% obtained for the chimera protein minimizes the false positive Uroporphyrinogen III synthase cases. Therefore, the ELISA results obtained for both proteins, mainly rLci2B and the chimera K9-K39-K26, can be considered excellent as commented by Chappuis et al. (20). The recombinant proteins rLci2B and rLci1A did not show cross-reactivity with serum samples of dogs infected with T. caninum, B. canis and E. canis, although cross-reactivity has been observed in serum samples obtained from dogs infected with L. brasiliensis, a parasite responsible for American Cutaneous

Leishmaniasis (ACL) (Table 1). The cross-reactivity for rLci2B (11·7%) and rLci1A (2·9%) observed with L. brasiliensis (n = 34) infected sera is probably due to the fact that this parasite belongs to the same genus of L. chagasi. For canine VL, the sacrifice of dogs positive for ACL is also recommended because there is no effective treatment and the animal also constitutes an important reservoir of this disease (35). In conclusion, based on data obtained from protein recovery (rLci2B: 105 mg/L and rLci1A: 225 mg/L bacteria cultures), protein purity and sensibility/specificity values, both proteins can be proposed as alternative antigens for Leishmania serological assay. We thank the researchers of Centro de Pesquisa Aggeu Magalhães, Pernambuco and Centro Gonçalo Muniz, Bahia, Brazil, especially to Dr. Geraldo G. Oliveira, for the donation of the modified E. coli plasmids containing the genes concerning the recombinant proteins rLci2B and rLci1A. We would also want to thank Dr.