In medical diagnostics, biomarkers are typically quantified using standard curves derived from calibrators. Results from unknown clinical samples are interpolated against these curves to quantify the targeted biomarker.

However, what if two or more biomarkers need simultaneous quantification in the absence of calibrators? This can be achieved by generating individual titration curves for each biomarker, expressing their titers in arbitrary units (Dilution Factor). The precision of titration methods depends on the number of dilutions tested, with more dilutions yielding greater accuracy.

Standard curves often follow a sigmoidal shape and are best fitted using a 4-parameter equation. The linear portion of the curve provides the most accurate quantification, while the upper and lower plateaus are less suitable due to signal saturation and detection limits, respectively.

MultiCruzi Immunoassay

The MultiCruzi immunoassay is a multiplex, microplate-based diagnostic tool designed for Chagas disease, caused by Trypanosoma cruzi (TC). It simultaneously measures up to 15 distinct antigens derived from TC protein sequences, addressing the unmet clinical need for sensitive and specific diagnostics.

Each immunodominant antigen is represented by a synthetic peptide epitope optimized for sensitivity and specificity based on ROC analysis. Collectively, these antigens capture the diversity of antibodies present in TC-infected individuals, reflecting each patient's unique immune signature. This signature evolves over time, with antibody levels declining at varying rates until complete clearance occurs following parasite elimination.

In a prior study published in Lancet Infectious Diseases (2021), antibody waning in congenital TC infections in children was measured and correlated with parasite clearance. Swift anti-parasitic therapy (e.g., Benznidazole) cured infected children, preventing Chagas disease symptoms.

Extending this approach to chronic adult patients revealed a much slower antibody waning process, potentially requiring decades for full seroreversion (conversion from seropositive to seronegative). MultiCruzi aims to capture dynamic changes in antibody levels over time to predict seroreversion in adults.

DF50: A Novel Approach

To expedite these predictions, the DF50 (Dilution Factor 50) metric was developed. DF50 represents the dilution required to achieve 50% of the maximum signal for a given antigen. This methodology was validated using serial dilutions across the full reactivity range and further optimized to require only three dilutions (1:50, 1:400, 1:3200) within the linear curve segment. While DF50 units are arbitrary, they maintain a linear relationship with antibody concentration.

DF50 values are transformed into log₂ values and analyzed using a nested Linear Mixed Model (LMM). Declines in antibody levels are assessed using two thresholds:

1. A significant decline threshold between treated and untreated patients.
2. A cutoff for the proportion of antigens showing reactivity decline compared to baseline levels.

This dual-threshold approach ensures stringent criteria for detecting clinically relevant antibody waning.

Clinical Applications

The MultiCruzi immunoassay was validated in the BENDITA study, which included an untreated control group alongside six treatment arms. Results were published as open-access research in Nature Communications.

In summary, MultiCruzi provides precise and simultaneous titration of up to 15 antibodies in serum or plasma samples from TC-infected individuals. Its applications include:

1. Confirming seropositive screening, functioning as an immunodot assay.
2. Monitoring antibody dynamics to evaluate therapeutic drug efficacy within short timeframes.
3. Serving as a companion diagnostic to stratify patients likely to respond to anti-parasitic therapy, improving the statistical power of clinical trials.

A mathematical tool for DF50 calculations is available at https://multicruzi.org. Free access is provided to interpret raw data upon request.