Automatisierung des Bio-Plex Pro Analyseverfahrens

Für den simultanen Nachweis mehrerer Analyten innerhalb einer Probe ist die Bead-basierte Multiplexanalytik ein häufig verwendetes Verfahren und wird beispielsweise zur Quantifizierung von Proteinen genutzt. Die aufwendige Durchführung der Assays soll durch Automatisierung einerseits dem Anwender abgenommen werden, während andererseits die Genauigkeit und Reproduzierbarkeit der Analyse gesteigert wird. Die Automatisierung des Bio-Plex Pro™ Assays ist mit dem Pipettierroboter Tecan Freedom EVO 200 umgesetzt worden. Es ist ein Skript mit der Software Freedom EVOware® entwickelt worden, welches die Probenvorbereitung des Assays vollständig übernimmt. Für einen Vergleich der manuellen und automatisierten Methode sind die humanen Zytokine IL-2, IL-4, IL-10, GM-CSF, IFN-γ und TNF-α in einer achtstufigen Standardverdünnungsreihe und in unterschiedlich konzentrierten Proben gemessen worden. Die Berechnung der Streuungen (Standardabweichung und Variationskoeffizient) der einzelnen Standardverdünnungsreihen sowie der Vergleich von gemessenen und erwarteten Konzentrationen der automatisierten und manuellen Methode zeigen, dass die Automatisierung neben der zeitlichen Optimierung auch die Genauigkeit und Reproduzierbarkeit der Analyse verbessert.Bead-based multpiplex analysis is frequently used for the simultaneous detection of multiple analytes within a sample. Such assays are commonly used to quantify proteins. The automation of the process relieves the user from the complex assay conductance and on the other hand increases the accuracy and reproducibility of the analysis. The automation of the Bio-Plex Pro™ assay has been successfully implemented with the pipetting-robot Tecan Freedom EVO 200. A script has been developed, using the Freedom EVOware® which has the ability to perform the complete assay procedure. For a comparison between manual and automated methods, the human cytokines IL-2, IL-4, IL-10, GM-CSF, IFN-γ and TNF-α were analysed by an eight-point standard dilution series and with samples of different concentrations. The calculation of the variances (standard deviation and coefficient of variation) of the single standard dilution series as well as the comparison of the observed and expected concentrations of the manual or automated method show the higher precision and reproducibility of the automated process in addition to its time-saving nature

In Vitro Evaluation of Glycoengineered RSV-F in the Human Artificial Lymph Node Reactor

Subunit vaccines often require adjuvants to elicit sustained immune activity. Here, a method is described to evaluate the efficacy of single vaccine candidates in the preclinical stage based on cytokine and gene expression analysis. As a model, the recombinant human respiratory syncytial virus (RSV) fusion protein (RSV-F) was produced in CHO cells. For comparison, wild-type and glycoengineered, afucosylated RSV-F were established. Both glycoprotein vaccines were tested in a commercial Human Artificial Lymph Node in vitro model (HuALN®). The analysis of six key cytokines in cell culture supernatants showed well-balanced immune responses for the afucosylated RSV-F, while immune response of wild-type RSV-F was more Th1 accentuated. In particular, stronger and specific secretion of interleukin-4 after each round of re-stimulation underlined higher potency and efficacy of the afucosylated vaccine candidate. Comprehensive gene expression analysis by nCounter gene expression assay confirmed the stronger onset of the immunologic reaction in stimulation experiments with the afucosylated vaccine in comparison to wild-type RSV-F and particularly revealed prominent activation of Th17 related genes, innate immunity, and comprehensive activation of humoral immunity. We, therefore, show that our method is suited to distinguish the potency of two vaccine candidates with minor structural differences