Technology base: hybcell
Anagnostics has developed a novel microarray technology in recent years. The microarray is based on a rotating cylinder, which comes along with a special cartridge. The rotation of the microarray offers significant advantages in comparison to „flat" microarrays, explains Dr. Bernhard Ronacher, inventor of hybcell technology and founder and CSO of Anagnostics:
Previously my work with conventional microarrays was quite frustrating: complex handling was rewarded with uncertain results, as these results were too dependent on environmental conditions and variations due to manual processing. Technological limits like the static end point added to that frustration. Therefore the aim of our developments was from the very beginning to offer a highly productive system producing reproducible high quality data".
Paralleled results and high number of samples
Patented hybcell technology combines the „parallelism" of microarrays with a automated sample manipulation - up to 96 samples can be processed without any user interference. All processing steps (handling, hybridisation, cycling, scanning and analysis) are fully automated. An intuitive software interface facilitates the design of „new" analytical tasks without limiting the high flexibility of process design. The strict separation of scientific and routine users with help of different user levels guarantees an efficient application of the technology by routine users.
Affinity measurement of proteins with help of hybcell
The usage of fluorescence scanning enables highly sensitive and selective tests. The cylindrical design of hybcell offers kinetic scans and analysis. Kinetics is relevant for describing binding characteristics of proteins. A „strong" binding, for example between a vaccine antibody and its antigen, results in a low „off-rate", which manifests in a low (or not noticeable) reduction of signal intensity. A strong binding of a vaccine antibody is associated with long-lasting effectiveness within the bodies of patients.
Hybcell technology helps to identify „successful" drug candidates in an early stage of a project. In contrast to conventional SPR technology hybcell technology analyzes highly paralleled (more than 100 candidates) and selective, what increases lab productivity (fig. 1).
Stratification of patients by hybcell sequencing of up to 2,000 DNA sequences with help of OnSpot Primer Extension
„Targeted therapy" and „companion diagnostics" are buzzwords describing a trend in drug discovery. Only a few new drugs work for all patients in the same manner. In fact more and more drugs work differently for different groups of patients. One differentiator between such groups is the presence of certain genetic variations, for example point mutations.
Prominent example is the mutation of the human KRAS2 gene. The approval authorities both in the US and the EU have ruled that the prescription of the two therapeutic antibodies, Erbitux? and Vectibix? which effect the growth receptor EGFR during colon cancer treatment, is dependent on a molecular diagnostic test. The studies have shown that a treatment is only likely to be successful if the KRAS2 gene is not mutated.
There are several other approved (oncological) drugs (IRESSA?, GLEEVEC?) or drugs in development, whose effectiveness is dependent on the (non-)presence of genetic mutations (e.g. PI3K Gen). These new developments and expected approvals are basis of further growth of companion diagnostics.
Mutation Diagnostics and „Mini-Sequencing" with help of the hybcell
Integration of thermocycling, washing, hybridisation and detection in one device - the hyborg - and the flexible choice of process sequences offer the possibility of highly multiplexed and simultaneous OnSpot primer extension and the identification of a wide range of DNA sequences of interest.
Primers (DNA-oligomers) complementary to DNA sequences of genes of interest are spotted onto the hybcell. Up to 2,000 different sequences can be spotted. Sample (template) DNA dissolved within 300?l extension solution is pipetted into the sample container.
All further steps are processed by hyborg fully automated. Primer extension starts with a short denaturing step at 94°C to melt sample (target) DNA and to activate polymerase. Even temperature distribution and constant sample agitation is assured by rotation of the hybcell within the sample container.
Sample DNA anneals at 55°C to the immobilized (spotted) primers. Immobilized primers are extended along sample (template) DNA at 72°C. This temperature cycle (94°C - 55°C - 72°C) is repeated for another 3 times. Finally DNA is denatured once again (94°C) and washed away with the help of hybcell's „flow-through-cell-functionality".
Highly specific results in 45 minutes
Successful primer extension is made visible through hybridisation of labeled probes (22nt CY5 labeled) complementary to the extended sequence. The integrated analysis software derives results automatically, e.g. indicating if certain mutations are present in the sample.
Advantages of hybcell sequencing in comparison to conventional sequencing are its high specificity (distinct differentiation of DNA sequences) and its easy and fast handling. The 45 minutes sequencing test saves time, money and sample.
