Surface expression of amastigote epitopes is variable among different T. cruzi strains and clones
We established that our ELISA could reliably identify differences in the surface expression of epitopes defined by our mAbs. The nature of the epitopes recognized by mAbs 1D9, 4B5 and 4B9 was determined previously (35). Briefly, 10 mM sodium m-periodate treatment of blots containing protein amastigote (G strain) extracts abolished immunoblot reactivity of mAbs 1D9 and 4B9 but not 4B5, suggesting that only the last mAb may recognize a protein moiety, while the other two recognize carbohydrate epitopes. The observations that L-fucose inhibits the binding of 2C2 to glutaraldehyde-fixed amastigotes in ELISA indicate that the corresponding epitope may also be a carbohydrate in nature (Verbisck NV, Da-Silva S and Mortara RA, unpublished data).
We next determined the relative expression of the epitopes recognized by mAbs 1D9, 2C2 and 4B5 in IA and EA isolated from either culture supernatants (TCA) or LIT medium (Extra). Figure 2 shows that, unlike mAb 2C2 that is expressed abundantly in all forms, the epitopes recognized by mAbs 1D9, 4B9 and 4B5 were considerably more abundant in intracellular parasite form.
We subsequently examined the reactivities of the mAbs in ELISA, using extracellular amastigotes of different strains. The results are summarized in Figure 3 and indicate that the mAbs can detect substantial inter-strain variation in their relative expressions. Several interesting features emerge from the analysis of these results: i) the epitope identified by 1D9 (Figure 3A) was scarce in the Y and CL strains, and present in intermediate amounts in MD, Tulahuen, and F amastigotes; ii) the epitope recognized by mAb 2C2 was more evenly represented in all strains (Figure 3B); iii) the mAb 4B5 target was more abundant in Tulahuen, G, and CL parasites and was moderately expressed in Y and MD isolates (Figure 3C); iv) the mAb 4B9 epitope was poorly expressed in Tulahuen and CL parasites but was more abundant in MD, G and Y parasites (Figure 3D). We also observed with some mAbs marked differences in epitope expression between clones and parental strains. For instance, whereas 2C2 expression by CL.B was higher than CL expression, the opposite pattern was observed with the other epitopes which were more abundant in the parental strain (Figure 3). Similarly, epitopes recognized by mAbs 4B9 and 2C2 were equally abundant in clone D11 and in the G strain but mAbs 1D9 and 4B5 had a higher expression in the parental strain (Figure 3).
Expression of amastigote antigens was polymorphic among T. cruzi isolates
Immunoblotting analysis using mAbs 1D9, 2C2, 4B5, and 4B9 revealed a polymorphic pattern of antigen expression among the different strains and clones. As shown in Figure 4, several bands present in some isolates were clearly not detected in others (e.g., 1D9 in CL, Y strains), while the relative molecular masses may also vary to a substantial extent (e.g., 4B5 in G, Sylvio X-10/4, and F strains). However, the absence of immunoblot reaction in some strains that give positive reaction by ELISA (e.g., 1D9 reaction with Y, CL and CL.B) can be explained by the behavior of this mAb under different experimental conditions, since this antibody is still capable of immunoprecipitating an 84-kDa component or to surface label Y and CL (but not CL.B) extracellular amastigotes by immunofluorescence (35). In a similar way, mAb 2C2 immunoprecipitated an 84-kDa major component of G, D11, Sylvio X-10/4 and CL.B extracellular amastigotes despite its poor or absent reaction with these parasites in immunoblot (data not shown; Figure 4). Conversely, immunoblot reaction of mAb 4B5 was not observed in ELISA, e.g., the poor reaction with D11, CL.B and Sylvio X-10/4. This apparent lack of correlation could be explained if we assume that there is a significant reaction of this mAb with D11 cytoplasmic components of amastigotes as shown by immunofluorescence and immunoelectronmicroscopy (35).