Background: Suspension array technology has surpassed ELISA for automated, simultaneous detection and quantification of soluble biomarkers such as virusspecific antibodies. We describe assays in which antigens are attached to a lipid bilayer surrounding colorcoded particles. Methods: We used layer-by-layer technology to establish a multiplex suspension array with distinguishable microbeads coated with authentic viral surfaces to catch and quantify virus-specific antibodies in a flow cytometric analysis. Antigenic surfaces were generated by chimeric and wild-type baculoviruses plus 2 different influenza A virus subtypes fused to a lipid bilayer surrounding distinctly colored particles. Specificity of binding of chosen antibodies and sera was detected by immunofluorescence. Results of multiplex analysis were compared with results of ELISA. Results: Titrations of virus-specific antibodies in the multiplex suspension array demonstrated specific binding to the viral surface proteins. The multiplex suspension array gave positive results for up to log 5-diluted primary antibodies with an ϳ5-to 10-fold reduced dynamic range compared with the respective ELISA. Conclusions: The bead-based multiplex suspension array is customizable and easy to establish. By displaying native influenza A virus surfaces and recombinant

Colloidal particles have become valuable tools in biotechnology and medicine. Even a small sample provides a huge surface which can be engineered in many ways. One of the most versatile ways to engineer the surface of colloidal particles is to coat them with a polyelectrolyte multilayer by means of the layer-by-layer (lbl) technology. [1] Multifunctional composite colloidal devices can be fabricated in this way with nanometer precision in a radial direction. Since most of the important biomolecules-nucleic acids, proteins, and many carbohydrates-are essentially polyelectrolytes, the lbl technology has the potential to generate complex colloidal multilayer composites consisting of biological and artificial building blocks. This technology can thus be seen as a promising platform for ** This research was supported by a grant from the Volkswagenstiftung within the framework of the program "Complex Materials"(VW foundation I/78 168). We thank Wolfgang Ernst for advice and discussions, Alexandra Spenger for the construction of AcCOIN-3D6, Nicole Borth for help with FACS analysis, and Harald Berger for support with CLSM imaging. Many thanks to Julia Romanova and Boris Ferko for influenza A viruses and Bernhard Benke for designing Figure 1.

Die Kombination macht's: Werden kolloidale Partikel, deren Oberfläche mithilfe der Layer‐by‐Layer(LbL)‐Technik mit einer Polyelektrolytmultischicht belegt wurde, mit Virusoberflächen‐Displaysystemen kombiniert, können die Partikel mit unterschiedlichen biologischen Funktionen versehen werden (siehe Bild). Die auf der kolloidalen Oberfläche präsentierten Proteine lassen sich mit modernen Sortier‐ und Analysetechniken bestimmen.

To create a tool for eukaryotic surface display, this approach is aimed at demonstrating a direct modification of the native envelope protein gp64 of Autographa californica NPV without disturbing viral infectivity. Short affinity-tag peptides, the biotin mimic streptagII, and the gp41 amino-acid motif ELDKWA of HIV-1, specific for the human monoclonal antibody 2F5, were engineered into the baculovirus major coat protein gp64 and presented on the viral surface. Two different streptag peptides were inserted at the naturally occurring Not I site at aminoacid 278 of gp64. Additionally, the ten-amino-acid peptide GG-ELDKWA-GG, containing the epitope of mAb 2F5, was introduced into gp64 envelope protein at the same position. In all cases we were able to propagate viable virus-achieving infectious titers in the range of wild-type AcMNPV. Streptag and ELDKWA-epitope surface localization on purified virus particles was demonstrated by flow cytometry and Western blot analysis. We could also show selective retention of mutant viruses by specific interaction between chimeric virions and their target counterparts, recognizing the epitope or the streptag peptide in the viral envelope. These data provide evidence that altering the surface properties of the baculovirus virion could be of value in improving baculovirus display technology and developing new applications.Keywords: baculovirus; Autographica californica nuclear polyhedrosis virus; insect cells; gp64 envelope protein; streptag peptide.To generate baculovirus-budded virions as a tool for eukaryotic surface display, different strategies have been investigated. When a foreign gene is fused to a second copy of the entire coat protein of AcMNPV, gp64 [1], or alternatively to the membrane anchor domain of gp64, simultaneous expression of the fusion construct and the native gp64 envelope protein results in packaging of both proteins into the viral coat and its presence on the surface of baculovirus-infected insect cells and budded virions [2±4]. Boublik and colleagues demonstrated the display of glutathione-S-transferase/gp64 fusion protein on the baculoviral surface, forming a hetero-oligomer with the wild-type gp64 coat protein [2]. Also, they targeted the gp120 surface glycoprotein of HIV-1 to the viral surface and showed that gp120 was still active, using a CD4 binding assay. Similar proteins such as the gp41 ectodomain of HIV-1 were directed to the baculoviral envelope thereby investigating the influence of different viral promoters on the efficiency of the process of viral packaging [3], indicating that only a minor portion of the target protein was incorporated and could be detected on budded virus.The native gp64 is abundantly expressed during the infection cycle and transported to the cell surface to be incorporated into budded virions. Two functional domains have been characterized, an oligomerization domain within a predicted a helix, necessary for oligomerization and transport, and a fusion domain as part of a hydrophobic cluster [5]. The essential role of gp64 ...

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