Proteins are essential biological macromolecules that can be found in every single cell. They take over many important tasks in the cell to provide a correct function.One of those proteins is the so called Heatshock protein 70 (Hsp70). As a chaperone, his purpose is to help other proteins to fold and get in the correct shape, so they can do their work properly. Furthermore, they also protect other proteins when they are exposed to extreme situations, so they don’t unfold and denaturate.
In such extreme situations, cells increase the production of Hsp70 highly, to protect their proteins.
Cancer cells, however, change their metabolism after the “Warburg effect” and multiply in an extremely high and disproportionate manner. This enormous growth leads to a constant stress the cancer cells are exposed to. Therefore, they also produce a huge amount of Hsp70. This phenomenon has been observed at any tested cancer type.
A unique behaviour of cancer cells is that they transport the Hsp70 to their membrane and also release it bounded in vesicles to their environment.
If a cell dies, a large amount of Hsp70 is released. It is very important to distinguish between the active release of the living cell, in which the Hsp70 is located inside an exosome and the release as a result of the lysis in which the Hsp70 is free in an unbound condition.
When the Hsp70 concentration in the blood is tracked during a therapy (for example chemotherapy), different phases can be observed: From the first few days to 1-2 weeks a high increase of Hsp70 in the blood will be determined because many cells are dying, and a strong reaction of the immune system is caused. After about a month finally, a drastic decrease of the concentration will be measured because the Hsp70 that was released through apoptosis is decomposed. The reduction of this concentration in comparison to the beginning indicates a succesfull therapy.
Enzyme linked immunosorbent essay (ELISA) is a commonly used analytical biochemistry assay.
We are using this technology in form of a Sandwich-ELISA to immobilize @@ LABEL verbesserung @@ and label the Hsp70 and therefore are able to detect it.
@@Vereinfachen@@As shown in the animation, capture-antibodies are immobilized at a substrate. These special antibodies bind both to free and exosomal bound Hsp70. If a blood sample of a patina is flushed over the substrate, only Hsp70 binds to those capture-antibodies. Afterwards, biotin-labelled detection-antibodies are added, which also bind specific to the captured Hsp70. After a certain incubation time, any unbound fractions are removed with a wash-solution. Then, streptavidin, bound to Horseradish peroxidase is added. The streptavidin binds to the biotin of the detection-antibody. In the last step, a chemiluminescence-solution (Dioxetane) is added which then is transposed by HRP into photons (light).