**4. Recombinant proteins use: Mixtures vs. fusion proteins**

282 Current Topics in Tropical Medicine

Specific of cardiac disease

Aznar *et al.*, 1995

Diez *et al.*, 2006 Fabbro *et al.*, 2011 Breniere *et al.*, 2002

Engman *et al.*, 1989 Gonzalez *et al.*, 1985 Krautz *et al*., 1995 Fabbro *et al*., 2007 Abate *et al*., 1993 Marcipar *et al*., 2005

Krautz *et al.*, 1998

Chronic infection DaRocha *et al*., 2002

Chronic infection Goto *et al*., 2008

Stages of cardiac disease Stages of cardiac disease

Chronic and acute infection

Cure monitoring patients Cure monitoring patients

Antigen name Characteristics Diagnostic use Described by

All stages

TcE Ribosomal protein Chronic infection Houghton et al., 1999

Chronic infection

Chronic infection Chronic infection

Heat Shock Proteins Chronic infection and cure

monitoring

TcAg48 RNA binding protein Chronic infection DaRocha *et al*., 2002

Tc3 Idem Chronic infection Goto *et al*., 2008 Tc4 Idem Chronic infection Goto *et al*., 2008 Tc9 Idem Chronic infection Goto *et al*., 2008 Tc10 Idem Chronic infection Goto *et al*., 2008 Tc12 Idem Chronic infection Goto *et al*., 2008 Tc15 Idem Chronic infection Goto *et al*., 2008

Table 1C. Other relevant recombinant antigens proposed for diagnostic uses. Abbreviations used: cy-hsp70, cytoplasmic thermal-shock protein; FCaBP, flagellar calcium-binding protein; grp.hsp 78, endoplasmic reticule thermal-shock protein (78 KDa); mt-hsp 70,

Last 13 amino acids from ribosomal

Full length ribosomal

protein.

TcAg29 Alginate regulatory protein

Tc1 Repetitive proteins

obtained by

of the genome

thermal-shock mitochondrial protein (70 KDA).

bioinformatic analysis

P2β protein

Flagellar calcium binding protein

R13

P2β

FcaBP 1F8 Tc-24 F29 Tc-29 Calflagin

cy-hsp70 mt-hsp70 grp-hsp78

The first works dealing with a single recombinant protein for diagnostic purposes reported lack of sensitivity when using only one of those antigens.(Levin *et al.*, 1991;Moncayo & Luquetti, 1990;Peralta *et al.*, 1994) Consequently, most of these proteins have been evaluated not only alone and independently from others, but also together as part of mixtures or as fusion proteins, carrying several recombinant epitopes.(Umezawa *et al.*, 1999;Umezawa *et al.*, 2004;Camussone *et al.*, 2009;Foti *et al.*, 2009) Accordingly, a multicenter study evaluating 6 recombinant proteins separately with a serum panel composed by sera from patients of several countries, described that using the set of results of the 6 proteins together had yield a sensitivity and specificity compatible with the reference assays.(Umezawa *et al.*, 1999) Later, the same group evaluated the mixture of the 6 proteins, supporting the use of the mixture to reach the same sensitivity and specificity.(Umezawa *et al.*, 2003) Soon after, the reactivity of individual antigens vs. antigen mixtures was systematically assessed by ELISA.(Umezawa *et al.*, 2004) This study confirmed that the results obtained with recombinant protein mixtures led to higher media values of optical densities, ODs, than the results produced when using the individual recombinant proteins. Moreover, sera rendering low ODs when examined with individual recombinant proteins produced higher ODs outcomes when using the protein mixtures. Along with this, several commercial ELISA kits with recombinant protein mixtures display equivalent or even higher sensitivities and specificities than those produced by kits with total parasite homogenate.(Gadelha *et al.*, 2003;Pirard *et al.*, 2005;Remesar *et al.*, 2009;Caballero *et al.*, 2007) These works have studied kits using Ag1, Ag2, Ag30, Ag13 together with Ag36 recombinant antigens (Chagatest Rec from Wiener lab, Argentina), and FRA and CRA recombinant antigens (Biomanguinhos, Friocruz, Brazil). However, another study reported that Chagatest Rec v3.0 (Wiener) displayed a rather low 95% sensitivity.(Ramirez *et al.*, 2009)

One of the strategies proposed to enhance reagents production standardization is to obtain multiepitope molecules, designed as a unique construction by fusing several relevant diagnostic antigens.(Houghton *et al.*, 1999;Aguirre *et al.*, 2006;Camussone *et al.*, 2009) It has recently been proved that when using these constructions, the ODs of sera with low reactivity increases, as well as it had been reported for mixtures.(Camussone *et al.*, 2009) Moreover, by this approach the attachment of the antigen turned out to be homogenous and reproducible when using different surfaces such as ELISA plaques, latex particles or bioelectrodes.(Camussone *et al.*, 2009;Gonzalez *et al.*, 2010;Belluzo *et al.*, 2011) It has been proposed that when there is only one molecule exposed to the surface, competition for the active sites is prevented, therefore resulting in a uniform attachment. Furthermore, sensitivity may be increased because a higher number of freely accessible epitopes are available to capture the antibodies present in samples, as depicted in Fig. 1.(Camussone *et al.*, 2009)

A few articles report on the use of this strategy to produce commercial ELISA kits which have demonstrated to be highly satisfying. One of these works, analyzes the performance of the TcF antigen, previously described by Houghton et al in 1999, with which the Biolab Merieux reagent was prepared.(Ferreira *et al.*, 2001) In this case, the recombinant protein used bears the PEP2, TcD, TcE and TcLo1.2 peptides. Recently, Abbot Laboratories have presented a new kit which uses a 4-antigen multiepitope protein containing TcF, FP3 -built up with TcR27 and FcaBP-, FP6 –with TcR39 and FRA- and FP10 -with SAPA and MAP-.(Praast *et al.*, 2011) According to the authors, this kit performed even better than the Biolab Merieux one.

Advances in Serological Diagnosis of Chagas' Disease by Using Recombinant Proteins 285

mothers and their newborns. The recognized IgGs against different *T. cruzi* antigens produced the same signal in sera from newborns and their respective mothers but SAPA antigen was recognized most frequently by antibodies from the infected newborns than it was by antibodies occurring in their mothers serum. Accordingly, the authors proposed it to be used to detect specific anti-*T. cruzi* IgG antibodies in neonates. Other works report that antibodies anti-SAPA allows the discrimination between acute and chronic *T. cruzi* infection because they were not present in the later stage of the infection.(Lorca *et al.*, 1993) Nevertheless, later works described SAPA as reactive when assessed with sera from chronic infected individuals.(Breniere *et al.*, 1997;Camussone *et al.*, 2009) This apparent contradiction could be explained considering the significant differential reactivity of anti-SAPA antibodies generated during the different stages of the infection. Indeed, anti-SAPA antibodies are detected in almost all infected individuals but its reactivity is higher in the acute infection. It has recently been performed a study by following up of 2283 chagasic mothers, from which 209 transmitted the infection to the newborns.(Russomando *et al.*, 2010) This work provides evidence on SAPA utility to serologically diagnose congenital infection before the third month of life, thus turning the protein into a promising inexpensive reagent to reduce the

required time to detect the neonatal infection, and proceed to its early treatment.

described to diagnose the acute phase.

**5.2 Chronic infection diagnosis** 

Stani *et al.,* 2008; Otani *et al*., 2009)

illnesses are co-endemic.(Gil *et al.*, 2011)

Although different reactivity patterns have been described in Western Blot assays which use native *T. cruzi* excretion antigens, to discriminate between acute and chronic infection (Umezawa *et al.*, 1996) no other useful recombinant antigen different from SAPA has been

It has been already mentioned above that, when *T. cruzi* homogenate is used to perform ELISA tests, the assay sensitivity is high leading to a quite reliable result, therefore some authors have suggested that a single assay could be enough to test sera in blood banks. (Sosa

Several multicenter studies carried out on samples from blood-banks, report that ELISA tests which use parasite homogenates perform similarly than those which used recombinant proteins.(Remesar *et al*., 2009; Otani *et al*., 2009) However, cross-reactivity of antibodies towards antigens from *T. cruzi* and *Leishmania sp* has been frequently informed, and can be explained considering the phylogenic proximity between both parasite species.(Chiller *et al.*, 1990;Vexenat *et al.*, 1996;Chiaramonte *et al.*, 1996;Desquesnes *et al.*, 2007;Aguirre *et al.*, 2006) When sera from patients infected with *Leishmania ssp* parasites are included in the evaluations, specificity of recombinant proteins are higher (Umezawa *et al*., 1999; Ferreira *et* 

It has been recently reported that the antigen TSSA2 displays 87.8% sensitivity and 100% specificity to discriminate between chagasic and leishmaniasic patients.(Cimino *et al.*, 2011) TSSA2 is the only reported recombinant antigen, which has displayed specificity to type *T. cruzi* genotypes DTUII, DTUV or DTUVI by specific antibodies from infected patients. (di Noia *et al.*, 2002; Bhattacharyya *et al.*, 2010) As these DTUs are those predominant in South America, the authors proposed using this antigen in confirmatory *T. cruzi* infection diagnostic tests, in regions which are co-endemic for both infections. It was also described that SAPA antigen could be specific and sensitive enough to be used when trying to distinguish between chronic *T. cruzi* and leishmaniasic infections, in regions where both

*al*., 2001; Aguirre *et al*., 2006; Caballero *et al.*, 2007; Camussone *et al*., 2009)

Fig. 1. Illustration of the ELISA plaque sensitizing process and the exposure to the sample: left-hand side, when using a protein mixture of three recombinant peptides RP1+RP2+RP5; right-hand side, when using a multiepitope chimeric protein bearing the same peptides fused in a single protein, CP2. RP: recombinant peptide, CP: chimeric protein obtained by fusion of peptides RP1, RP2 and RP5 together in only one molecule. Reproduction from Camussone et al 2009.
