Overview: Performing an endotoxin test using a rFC assay

The ASSIST PLUS is used to perform an ENDOZYME II GO endotoxin test from bioMérieux, using a pipetting program generated by the VIALAB software.
Note: Prepare the assay reagent manually in advance by combining enzyme, substrate and assay buffer, according to the manufacturer’s protocol (ENDOZYME II GO, bioMérieux).

Downloads: Protocols for automated endotoxin testing with ASSIST PLUS

Here you can download the complete protocols and reports used in this application and use them on your ASSIST PLUS. The protocol can be customized with the VIALAB software.

Experimental set-up

Deck position A: 25 ml multichannel reservoir – filled with 12 ml assay reagent (Figure 1, in blue).
Deck position B: 96 well flat bottom (GOPLATE) placed on a microplate heater (HeatPAC, INHECO).
Deck position C: tube rack for 15 ml centrifuge tubes – with 5000 μl of sample (Figure 1, in green). Alternatively, a custom rack for ENDOGRADE® Glass Test Tubes can be used with 1000 μl of sample.

Step-by-step procedure

1. Add water and samples to the dedicated wells

Distribute samples into the 96 well GOPLATE.

Use the Repeat Dispense mode of the ASSIST PLUS to equally distribute samples from the tube rack (position C) into the dedicated wells of the GOPLATE (position B).

Tip: Insert a pre- and post-dispense step to improve accuracy and precision while pipetting. The recommended pre- and post-dispense volume is 3-5 % of the nominal volume of the pipette. Insert an air gap to eliminate any risk of contamination. Perform a tip touch to the wall of the well to remove droplets that may cling to the end of the pipette tips. The pipetting height is set to 65 mm so that the tip does not touch the solution.

2. Heat the GOPLATE containing the samples

Incubate your samples on the microplate heater (INHECO).

Start the incubation of the plate on the INHECO microplate heater at the appropriate temperature and incubate the plate for five minutes (ENDOZYME II GO, bioMérieux). If there is no microplate heater available on the deck, the plate can be manually removed and incubated on an external microplate heater or in the microplate fluorescence reader.

3. Add the assay reagent

Add the assay reagent to the dedicated wells of the 96 well GOPLATE.

Use the Repeat Dispense mode to transfer assay reagent from position A into the GOPLATE (position B).

Tip: Adjusting the pipette settings (tip travel, tip touch and pipetting height) increases the precision and accuracy of pipetting. The tip touch automatically touches the tip to the side of the well after each dispense to remove droplets clinging to the tip end. In this protocol, the tip touch of the samples and assay reagent is set to the opposite well sides to avoid the risk of cross-contamination between samples.

4. Run the assay in a fluorescence reader

Determine the endotoxin content with a microplate fluorescence reader.

After adding the assay reagent, place the GOPLATE immediately in a fluorescence microplate reader and start your readings according to the manufacturer’s protocol ENDOZYME II GO, bioMérieux).