To proceed with sample analysis, we optimized ELISA components by cross-titration, using a pool of known positive and negative controls (1
1600 dilutions). The optimal sera and HRP-conjugated secondary antibody dilutions providing maximum signal-to-noise ratio were determined to be 1
50 and 1
5,000, respectively, and used in all further investigations. The variations in reactivity of negative and positive sera among different assays and plates of the same experiment ranged from 3–12%.
We, first, monitored the antigenicity of TcG1, TcG2, and TcG4 using sera samples collected from volunteers enrolled in the study in Argentina in year 2010. Samples were stored at −80°C just after collection, and thawed when utilized. The negative sera samples (n
20) from the endemic area near Argentina-Bolivia border exhibited low reactivity for TcG1, TcG2, and TcG4, similar to that noted for confirmed negative controls (n
42) from non-endemic areas (TcG1: 0.216±0.035 versus 0.211±0.048, TcG2: 0.240±0.04 versus 0.230±0.044, TcG4: 0.225±0.041 versus 0.252±0.038, expressed as mean absorbance ± SD). In comparison, a 4-fold, 2.75-fold, and 2.65-fold increase in sera levels of antibody response to TcG1, TcG2, and TcG4, respectively, was noted in previously characterized seropositive subjects (n
45) from Argentina-Bolivia border (TcG1: 0.81±0.33, TcG2: 0.66±0.20, TcG4: 0.57±0.09, expressed as mean absorbance ± SD, p<0.001 for all, ). The sera levels of antibodies to TcG1, TcG2, and TcG4 were above the meanseronegative
level in 62.2%, 66.6% and 75.5% of the 1st
-phase seropositive subjects. When analyzing plasma samples from the same individuals, we noted a 3.34-fold, 2.4-fold and 2.3-fold increase in plasma levels of antibodies to TcG1, TcG2 and TcG4 in seropositive subjects as compared to seronegative controls (TcG1: 0.77±0.21 versus 0.20±0.05, TcG2: 0.65±0.12 versus 0.21±0.057, TcG4: 0.67±0.09 versus 0.20±0.048, expressed as mean absorbance ± SD, p<0.001 for all, ). The plasma levels of antibodies to TcG1, TcG2, and TcG4 were above the meanseronegative
levels in 71.1%, 77.7% and 80% of the 1st
-phase seropositive subjects. These data suggested that a) TcG1, TcG2 and TcG4 are recognized by antibody responses elicited in human patients infected by T. cruzi
, and b) both plasma and sera samples can be utilized to monitor the antibody response.
Antigenicity of TcG1, TcG2 and TcG4 in inhabitants of Salta Argentina.
We then analyzed the plasma samples that were collected in 2009, and characterized as seropositive (n
65) and seronegative (n
20) by 1st
-phase serology tests. These samples were subjected to two cycles of freezing/thawing during the two-year storage. Our data showed a 6.72-fold, 2.4-fold and 2.9-fold increase in plasma levels of antibodies to TcG1, TcG2 and TcG4 in seropositive subjects as compared to seronegative controls (TcG1: 1.66±0.55 versus 0.204±0.05, TcG2: 0.69±0.13 versus 0.239±0.039, TcG4: 0.75±0.20 versus 0.227±0.05, expressed as mean absorbance ± SD, p<0.001 for all, ). The plasma levels of antibodies to TcG1, TcG2, and TcG4 were above the meanseronegative
level in 61.5%, 64.6% and 81.5% of the seropositive subjects. These results suggest that antibody response to TcG1, TcG2, and TcG4 is stable, and field samples can be utilized to examine antigenicity of the selected candidates in large-scale population studies. Overall, the data presented in also indicate that TcG1, TcG2 and TcG4 are expressed by T. cruzi
isolates circulating in the endemic areas at the Argentina-Bolivia border.
It is important to know if antibody recognition of the three antigens can be expanded for diagnosis of T. cruzi
infection in other countries where different isolates are suggested to be present in domestic and sylvatic cycle of parasite circulation. For example, in Argentina and neighboring countries in South America, TCII isolates are predominantly identified in peripheral blood of seropositive patients, though molecular studies have revealed the presence of TCI parasites also in heart biopsies of chronic chagasic patients 
. In Mexico and Guatemala, TCI is dominantly found in epidemiological evaluation of infected triatomines as well as in blood samples from acute and chronic chagasic cardiomyopathy patients 
. We, therefore, monitored the antigenicity of TcG1, TcG2, and TcG4 in human sera samples from Mexico-Guatemala border area that were characterized as seropositive (n
65) and seronegative (n
34) by 1st
-phase serology tests. Our data showed 6.72-fold, 2.4-fold and 2.9-fold increase in sera levels of antibodies to TcG1, TcG2 and TcG4 in seropositive samples as compared to that noted in seronegative healthy controls (TcG1: 0.7±0.25 versus 0.211±0.047, TcG2: 0.64±0.22 versus 0.25±0.05, TcG4: 0.65±0.20 versus 0.212±0.048, expressed as mean absorbance ± SD, p<0.001 for all, seropositive versus seronegative, ). The sera levels of antibodies to TcG1, TcG2, and TcG4 were above the meanseronegative
level in 69.2%, 76.9% and 72.3% of the seropositive subjects from Mexico. These data demonstrate that TcG1, TcG2 and TcG4 are antigenic, and recognized by antibody responses in chagasic patients from Mexico, and suggest that the three antigens are expressed by T. cruzi
isolates circulating in Mexico-Guatemala border area.
TcG1, TcG2 and TcG4 are recognized by antibody response in human subjects from Mexico.
Next, we determined if the three candidate antigens can be utilized together to improve the diagnosis of exposure to T. cruzi. For this, we coated the 96-well plates with either the mixture of TcG1, TcG2 and TcG4 (0.5 µg/well each) or T. cruzi trypomastigote lysate (TcTL, 2×105 parasite equivalent), and monitored the antibody response by ELISA under similar experimental conditions. When antibody response was captured using the TcGmix, the seronegative controls from the non-endemic areas exhibited a mean absorbance ± SD of 0.225±0.039. Using the controls' mean absorbance+2SD, our data validated 40 of the 45 sera samples (88.8%) from Argentina, characterized as seropositive in 1st-phase screening in the year 2010, were seropositive for TcGmix-specific antibodies (mean absorbance ± SD: 0.73±0.17, maximum OD: 1.2, ). One of the volunteer previously characterized as seronegative exhibited anti-TcGmix antibody response above the meanseronegative level. Similarly, the plasma detection of antibody response to TcGmix identified 102/110 of the seropositive subjects (92.7%) identified in 1st-phase screening in the years 2009 and 2010 (). In comparison, when plates were coated with TcTL to capture anti-T. cruzi antibodies, the seronegative true controls from non-endemic areas, exhibited a mean absorbance ± SD value of 0.233±0.044 (). Using the mean absorbance for controls +2 SD as a cut off, our data validated 97.7–100% sera and plasma (year 2010) and 96.9% plasma (year 2009) samples characterized as seropositive in 1st-phase screening in Argentina were also positive for TcTL-specific antibodies; the mean absorbance ± SD for the positive population was 1.1±0.6 (year 2009) and 0.73±0.08 (year 2010) with the highest value being 2.5 and 0.98, respectively (). One of the volunteer previously characterized seronegative exhibited anti-TcTL antibody response.
TcGmix-based ELISA provides superior efficacy in identifying exposure to T. cruzi infection among inhabitants of Salta Argentina.
To validate that diagnostic potential of the TcGmix based ELISA is not restricted to samples from Argentina, we monitored the antibody response using sera samples collected in Mexico. Of the 65 samples characterized as seropositive in 1st-phase screening, 58 (89.2%) and 63 (96.9%) exhibited reactivity when plates were coated with TcGmix and TcTL antigens, respectively (). The mean absorbance ± SD for the antibody response to TcGmix and TcTL in the positive population positive population was 0.75±0.14 (max: 1.2) and 0.87±0.4 (max: 2.6), respectively, the difference between the two values being observed non-significant. No significant difference was observed when either plasma or sera were used as the source of antibodies.
TcGmix-based ELISA was effective in diagnosing exposure to T. cruzi infection among inhabitants of Chiapas Mexico.
Five of the 110 seropositive/chagasic subjects from Argentina exhibited reactivity for Leishmaina
-specific antibodies and were likely infected with both pathogens. To examine if the antigen-based ELISA is specific for T. cruzi
detection, we performed recombinant antigens-specific ELISA using the sera samples from non-chagasic individuals (total 112), including leishmaniasis patients (n
35), volunteer donors with cardiomyopathy (n
20) and autoimmune diseases (n
15) of other etiologies, and healthy donors from non-endemic areas (n
42), and calculated the specificity of the antigen-based ELISA. Sera samples from leishmaniasis patients (n
35) exhibited very low reactivity for TcG1, TcG2, and TcG4, similar to that noted for sera samples from confirmed negative controls from non-endemic areas (TcG1: 0.18±0.04 versus 0.21±0.048, TcG2: 0.15±0.06 versus 0.230±0.044, TcG4: 0.232±0.05 versus 0.252±0.038, expressed as mean absorbance ± SD). Likewise, sera samples from patients exhibiting symptoms of cardiomyopathy of non-chagasic etiology or autoimmune diseases showed reactivity to TcG1, TcG2, and TcG4 below the cut-off threshold values derived from healthy/seronegative controls. The specificity for TcGmix
was highest (98%), followed by TcG2 (96%), TcG4 (94.6%), TcG1 (93.6%) and TcTL (77.8%), determined by detection of false positive signal for 2, 4, 6, 7 and 25 of the samples, respectively, out of the total 112 samples from non-chagasic individuals that were submitted for antigen- and TcTL-based ELISA. These data indicated that the recombinant antigens were highly specific for the detection of anti-T.cruzi
antibodies than the whole parasite (trypomastigote) lysate, and exhibited no cross-reactivity to Leishmania
Pearson correlation analysis was employed to identify correlation between antigen-specific antibody response and disease state including the data derived from seronegative/healthy controls and seropositive/chagasic subjects. For this, seronegative/healthy subjects were labeled as 0, and patients classified as 0, I, II and III (Materials and Methods
) were labeled as 1, 2, 3, and 4, respectively. Antibody response was titrated using 2-fold sera dilutions (1
1600). We observed no significant correlation between the anti-TcG2, anti-TcG4, anti-TcGmix
and anti-TcTL antibody titration curves and clinical disease category in any of the patient population (data not shown). The representative correlation data from sera levels of anti-TcGmix
and anti-TcTL antibody response (1
50 dilutions) and clinical disease category for the clinically characterized Argentine patients enrolled in the study is shown in . It is worth noting that TcGmix
-specific antibodies exhibited a clear downward trend with patients' disease severity, indicating that presence of antibodies for TcG1, TcG2, and TcG4 is protective during progressive Chagas disease (). No clear trend or correlation was observed for TcTL specific antibody response and disease severity in any of the patient population ().