Screen drugs for specific inhibition of anaerobes/aerobes (in cell lines/tumor tissue/blood culture).

Filter anaerobes. Screen cells (PBMCs, tissue) for anaerobic growth capacitiy  in disorders where switch to anaerobic metabolism has significant impact on the outcome of the disease.

Implement into AI classifiers. Advantage: standardized binary data set (anaerobic vs aerobic growth) with profound tumorbiological impact for improved evaluation of clinical data with artificial intelligence based classifiers (e.g. machine learning, deep learning, broad learning).  

Apply to your automated platforms. Measurement of enzyme activities using NADH decrease at 340nm is easy to transfer to automated analyzers (e.g. Cobas® from Roche, ARCHITECT cSystems™ from Abbott) using open channel cartridges.

German State Research Award winning technology introduced at iwCLL 2017 in New York City!

Preanalytic: Half of the sample is incubated at 37°C over night in cell culture medium under normoxic condition. In parallel the other half is incubated under anoxic condition by means of an O2-impermeable sachet (e.g. GasPak™ from BD). The next day, the cytosolic proteins are extracted (e.g. by ultra sound) with EnFin®-Buffer Solution.

Test Kit: Activities of the three enzymes PK low affinity, PK high affinity and LDH are compared between the normoxic and the anoxic sample. 

After the preparation of the Kit Reagents, samples’ protein concentration is determined (e.g. Bradford Assay). The desired protein concentration of 0.1 µg/µl is adjusted with Buffer Solution. Now, 1 µg protein of the normoxic and anoxic sample is pipetted into a 96-Well microtiter plate. Then the negative and positive control is added. Finally, the addition of the enzyme-specific Start Reagent initiates the enzymatic decomposition of NADH to NAD+. The pipetting steps follow the scheme in the figure. The declining NADH content is recorded every 5 min up to 30 min with a microplate reader at 340 nm and 37°C. The measured raw data are used to calculate the EnFin Score. This can be done manually or with the help of our specifically developed software package.

Figure 1: Kit components for detection of aerotolerant anaerobic cells preferably growing under anoxia.

Starting reagents are supplied as lyophilisates. Pipetting steps: 1. Loading sample homogenate (1 µg protein), 2. Loading negative control (EnFin®-Buffer Solution), 3. Loading provided positive controls for each enzyme and 4. Loading the appropriate starting reagent (ad 200µl reaction volume using a 8-channel pipette). Enzymatic activity is measured in a micro-plate-reader detecting O.D. decrease at 340 nm. 

Required Instruments 
Incubator (37°C, 5% CO2, humidity 95%, pH 7.2-7.5), homogenisator (e.g. Bioruptor), micro plate reader (kinetic mode).