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Abstract. Ligand binding assays (LBAs) have been the method of choice for protein analyte measurements for more than four decades. Over the years, LBA methods have improved in sensitivity and achieved larger dynamic ranges by using alternative detection systems and new technologies. As a consequence, the landscape and application of immunoassay platforms has changed dramatically.

The combination of flexible and robust immunoassay technology with the sensitivity of DNA as a signal amplification template is realized in Immuno-PCR ("IPCR"). Classical ELISA is converted to IPCR by exchange of antibodyenzyme detection conjugates with antibody-DNA conjugates. The value of ultra-sensitive analytics deriving from this for pharmaceutical R&D is threefold: (I) Deeper understanding of biomolecular interactions for the development of new compounds. (II) Monitoring of compounds at very low concentrations in toxicokinetic and pharmacokinetic clinical studies. (III.) Control of compound functionality and therapeutic effects by surveillance of characteristic biomarkers and/or immunogenicity reactions. This review summarizes background information about general selection of IPCR targets, actual standard assay procedures and work with biological matrices. Quantitative real-time detection as well as optimized reagents and protocols revealed a typically 100-10,000-fold increase in sensitivity and a broad dynamic range compared to ELISA. Case studies are discussed for (I) the analysis of biomolecular interaction with proximity ligation technologies and IPCR, (II) pharmacokinetic studies of novel drugs with validation data for assay precision and recovery and (III) biomarker profiling, including cytokine multiplex assays and polyplex trace analysis in extremely small sample volumes. In a survey of results from recent innovations, the potential of this emerging field of applications is evaluated for novel pathways in study design and analytics.

The main objective of the MACIVIVA European consortium was to develop new Good Manufacturing Practice pilot lines for manufacturing thermostable vaccines with stabilized antigens on influenza virosomes as enveloped virus-like particles. The HIV-1 gp41-derived antigens anchored in the virosome membrane, along with the adjuvant 3M-052 (TLR7/8 agonist) on the same particle, served as a candidate vaccine for the proof of concept for establishing manufacturing processes, which can be directly applied or adapted to other virosomal vaccines or lipid-based particles. Heat spray-dried powders suitable for nasal or oral delivery, and freeze-dried sublingual tablets were successfully developed as solid dosage forms for mucosal vaccination. The antigenic properties of vaccinal antigens with key gp41 epitopes were maintained, preserving the original immunogenicity of the starting liquid form, and also when solid forms were exposed to high temperature (40°C) for up to 3 months, with minimal antigen and adjuvant content variation. Virosomes reconstituted from the powder forms remained as free particles with similar size, virosome uptake by antigen-presenting cells in vitro was comparable to virosomes from the liquid form, and the presence of excipients specific to each solid form did not prevent virosome transport to the draining lymph nodes of immunized mice. Virosome integrity was also preserved during exposure to <−15°C, mimicking accidental freezing conditions. These "ready to use and all-in-one" thermostable needle-free virosomal HIV-1 mucosal vaccines offer the advantage of simplified logistics with a lower dependence on the cold chain during shipments and distribution.

Semisynthetic DNA-protein conjugates are versatile tools for many applications in bioanalytics and nanobiotechnology. We here report a method based on expressed protein ligation (EPL) for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins. The latter contain a C-terminal thioester, enabling the mild and highly specific reaction with N-terminal cysteine compounds. To conveniently couple commercially available DNA oligomers with cysteine groups a universal chemical modifier was developed, containing a protected cysteine and an amino-reactive N-hydroxysuccinimide group connected by a hexaethyleneglycol moiety. Using maltose-binding protein (MBP) and green fluorescent protein mutant EYFP as a model systems, we demonstrate the feasibility of this approach, as well as the integrity and functionality of the DNA-protein conjugates synthesized. We anticipate that our concept will enable many applications, such as the generation of large arrays of surface-bound, recombinant proteins assembled by means of DNA-directed immobilization.