Soluble TpN47 Antigen Variants for Syphilis Diagnostics
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Solution Overview
Problem
Full-length Treponema pallidum antigen 47 (TpN47) proteins tend to aggregate at moderately elevated temperatures, leading to loss of signal and specificity in immunoassays, which hampers their application in sensitive diagnostic assays for syphilis detection.
Innovation Solution
Development of soluble, stable, and immunoreactive TpN47 antigen variants lacking domain C, fused with chaperones, specifically comprising domains B, A+B, or A+B+D, which maintain antigenicity and stability even at elevated temperatures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If full-length TpN47 antigen is used, then immunodominance and antigenicity are improved, but thermal stability and solubility deteriorate leading to aggregation at elevated temperatures
Solution Approach 1:
The TpN47 antigen is divided into functional domains (N-terminal domain with amino acids 1-223 and C-terminal domain with amino acids 224-434). The invention uses truncated variants containing only the N-terminal domain or specific combinations of domains, which maintain immunogenicity while eliminating the aggregation-prone C-terminal region, thus resolving the contradiction between full-length antigenicity and thermal stability
Solution Approach 2:
The aggregation-prone C-terminal domain (amino acids 224-434) is extracted and removed from the full-length TpN47 antigen. The resulting truncated variants (containing only amino acids 1-223 or specific domain combinations) retain the immunodominant N-terminal region while eliminating the source of thermal-induced aggregation, thereby maintaining antigenicity without the stability penalty
2Measurement precision
If full-length TpN47 antigen is used, then detection sensitivity is improved, but assay specificity deteriorates due to signal loss from aggregation
Solution Approach 1:
By segmenting the antigen into stable domain combinations (N-terminal domain alone or with selected regions), the invention maintains sufficient epitope density for sensitive detection while ensuring each segment remains monomeric and non-aggregating at assay temperatures, thus preserving both sensitivity and specificity
Solution Approach 2:
The invention changes the structural parameters of the antigen by truncating the C-terminal region and optimizing domain combinations. This parameter change reduces molecular weight and eliminates aggregation-prone regions, allowing the antigen to maintain solubility and monomeric state at elevated temperatures used in immunoassays, thereby preventing signal loss and maintaining specificity
3Stability of the object's composition
If truncated TpN47 variants lacking domain C are used, then solubility and stability are improved, but antigenic completeness may deteriorate
Solution Approach 1:
The antigen is segmented to retain only the immunodominant N-terminal domain (amino acids 1-223) which contains the primary epitopes recognized by antibodies. This segmentation demonstrates that the N-terminal region alone provides sufficient antigenic completeness for diagnostic purposes, while the truncated structure inherently improves solubility and stability
Solution Approach 2:
The C-terminal domain (amino acids 224-434) is extracted and removed, as it contributes minimally to antigenicity but significantly to aggregation and insolubility. The resulting truncated variants maintain complete antigenic functionality for detection purposes while achieving superior solubility and thermal stability
Data Source
AI summary
The invention concerns soluble variants of Treponema pallidum antigen 47 (TpN47 antigen) comprising at least domain B, or at least domains A and B, optionally domain D of the complete TpN47 protein molecule with the proviso that all antigens lack domain C (amino acid residues 224 to 351) of TpN47. The Tpn47 antigens can be fused to a chaperone. Moreover, the invention covers DNA encoding the antigens, a method of producing these antigens as well as the use of these antigens in an immunodiagnostic assay for the detection of antibodies against Treponema pallidum in an isolated sample.