Ambergen Technology

Production of protein antigens is a critical step in a variety of diagnostic applications including autoantigen discovery and detection in autoimmune disease, tumor associated autoantigen (TAA) discovery and detection in cancers, as well as production allergens and pathogens in the diagnosis of allergy and infectious diseases.

The necessary antigens are normally produced recombinantly or isolated from various tissue sources, but these methods are slow, difficult, subject to variability and expensive, particularly when multi-antigen assays are necessary (e.g. autoantigen signatures).

Ambergen utilizes a better and innovative means of producing protein antigens, that is, cell-free (in vitro) protein production. General advantages of cell-free protein synthesis include:

• Simple: Eliminates the need for tedious cell transfection, cell culture and protein extraction.
• Fast: Can express specific proteins in less than 1 hr, even in eukaryotic and mammalian systems.
• Flexible: Can express proteins that cellular systems cannot due to cytotoxicity or interference with host cell physiology.
• Consistent: A uniform antigen source which can be coupled with a rapid, simple, and unified method of protein purification from the cell-free reaction.

Ambergen has integrated cell-free antigen production into a patented epitope tag based ELISA platform (T²-ELISA). Figure 1 compares the front-end antigen production using conventional recombinant methods to Ambergen's cell-free based approach.

T²-ELISA

Similar to Ambergen's patented ELISA-PTT technology, T²-ELISA (Figure 2) uses cell-free expressed and epitope tagged proteins. Following cell-free antigen production, purification is integrated into the ELISA assay by direct capture onto the wells of the microtiter plate, in 30 min, without additional manipulations (e.g. no protein extraction). Capture is mediated by a common C-terminal epitope tag in the expressed antigens (C-Tag) and the corresponding antibody coated onto the ELISA plate, ensuring only full-length antigens are bound. Patient test sera are then added to the ELISA plate to allow binding of specific serum antibodies, which are then detected with a reporter labeled secondary antibody. Detection of a common N-terminal epitope tag (N-Tag) in the antigens provides a normalization signal for quantification. Ambergen's innovative dual-reporter technology allows detection of both the test serum antibody and N-Tag in every well of every ELISA plate, thus providing an internal normalization signal for maximum quantitative accuracy.

T²-ELISA In Practice:

T²-ELISA can be used both in screening mode, for instance to validate the results of biomarker discovery assays, or as the final commercial diagnostic assay itself. In screening mode, an additional advantage is the ability of cell-free protein synthesis systems to express from PCR products (in addition to plasmid DNAs), in order to more rapidly produce libraries of putative antigens for screening, without the need for cloning. The overall advantages of T²-ELISA are summarized as follows:

• Industry standard, automatable, microtiter plate format.
• Rapidly express antigens from PCR products or plasmid DNA and without cell cultures.
• Facile production of properly folded antigens, even in mammalian cell-free expression systems, including expression of membrane proteins.
• Uniform antigen source and tighter control over background.
• Ultra-high sensitivity chemiluminescent readout.
• Dual-reporter and internal normalization strategies for quantitative accuracy.
• Significantly sorter assay development times and reduced costs per antigen.
• Proven concordance with existing conventional antigen and autoantigen ELISAs.