Performing an endotoxin test (ENDOZYME® II GO) using a recombinant factor C (rFC) assay and an automated pipetting robot

  • Performing an endotoxin test (ENDOZYME® II GO) using a recombinant factor C (rFC) assay and an automated pipetting robot

    Automated endotoxin test with ASSIST PLUS

    The ENDOZYME® II GO kit was developed by bioMérieux. It includes a novel 96 well microplate (GOPLATE™) pre-loaded with the required Control Standard Endotoxin (CSE) concentrations for a standard curve, as well as Positive Product Controls (PPC). The ASSIST PLUS pipetting robot provides a solution to minimize hands-on time and further optimize the assay workflow, without the need for dedicated personnel. Automation of the protocol with the ASSIST PLUS helps to prevent pipetting errors and ensures outstanding data reproducibility.

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      The ENDOZYME® II GO kit was developed by bioMérieux. It includes a novel 96 well microplate (GOPLATE™) pre-loaded with the required Control Standard Endotoxin (CSE) concentrations for a standard curve, as well as Positive Product Controls (PPC). The ASSIST PLUS pipetting robot provides a solution to minimize hands-on time and further optimize the assay workflow, without the need for dedicated personnel. Automation of the protocol with the ASSIST PLUS helps to prevent pipetting errors and ensures outstanding data reproducibility.

    Key benefits

    • The ASSIST PLUS reduces hands-on time for preparing a full 96 well GOPLATE from 10 minutes to less than two minutes.
    • Optimal pipette settings, such as fixed pipetting heights, angles and speeds, lead to increased reliability, precision and accuracy.
    • An on-deck heating solution – an INHECO microplate heater – can be placed on the ASSIST PLUS deck in order to further streamline the workflow and increase the throughput by 33 %.
    • The ASSIST PLUS ensures process security to minimize pipetting errors.

    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.

    VIALAB deck setup for ENDOZYME GO
    Figure 1: Deck set-up.

    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).

    Conclusion

    • The ENDOZYME II GO kit can be easily automated with the ASSIST PLUS.
    • Using an on-deck heating solution reduces manual handling and increases the throughput by 33 %.
    • Accuracy and precision of the semi-automated workflow is equal to or higher than that of manual pipetting.
    • Using the semi-automated process approach described in this application note offers higher efficiency and throughput of recombinant factor C (rFC) endotoxin testing.

    April 2020, by

    • Lydiane Saucède, PhD

      Application Scientist

      Email
    • Jan-Peter Baldin, PhD

      Product Manager

      Email

    Any questions? I'm happy to help!

    • Stephanie Stursberg

      Product Manager

      Email

    Used instruments and accessories

    ASSIST PLUS, Pipetting Robot VOYAGER, 6 Channel, 1250 µl GripTip, 1250 µl, Sterile, Filter Multichannel Reagent Reservoir, 25 ml Rack for 15 ml centrifuge tubes, 4x6 tubes bioMérieux: ENDOZYME® II GO bioMérieux: ENDOGRADE Glass Test Tubes INHECO: HeatPac with flat bottom adapter and Single TEC Control

    ASSIST PLUS, Pipetting Robot

    INTEGRA has developed the ASSIST PLUS pipetting robot to streamline routine pipetting tasks at an affordable price. Using INTEGRA electronic multichannel pipettes, the system:

    • automates pipetting tasks,
    • eliminates physical strain and
    • ensures superior reproducibility and
    • error free pipetting.

    Part No. 4505

    Visit product page
    ASSIST PLUS Pipetting Robot

    VOYAGER, 6 Channel, 1250 µl

    VOYAGER pipettes allow the tip spacing to expand anywhere between 4.5 mm and 33 mm at the push of a button.

    • Single handed operation leaves the other hand free to handle labware.
    • On the fly access to up to 3 user-defined tip spacings.
    • These user defined tip spacings are saved. No need to memorize spacings of different labware formats.

    Part No. 4764

    See it work
    VOYAGER, 6 channels, 1250μL

    GripTip, 1250 µl, Sterile, Filter

    GripTip pipette tips perfectly match the multi-lobe tip fitting, snapping firmly on during loading. This guarantees a perfect seal on every tip, preventing them from loosening, leaking or completely falling off. All tips are precisely aligned horizontally, enabling accurate touch-offs, even when pipetting with 384 tips.

    Part No. 6445

    Learn more about GripTips pipette tips for Benchtop Pipetting Systems
    Integra-Relaunch_Website-Output-Produktebilder-Product_Overview-GripTips.png

    Multichannel Reagent Reservoir, 25 ml

    • SureFlo™ anti-sealing array prevents pipette tip seal off which can cause liquid to pop into the tip, filter or even the pipette itself
    • Unique surface treatment spreads liquid evenly offering even lower dead volumes
    • Reusable base with bold, crisp, clearly visible graduation markings
    • Nest inside each other, making it possible to get twice as many reservoirs in half the space of other products on the market
    • Reducing inventory space requirements and shipping costs
    • Available as crystal clear polystyrene reservoirs or polypropylene reservoirs for improved chemical compatibility
    Find out more about Multichannel Reagent Reservoirs
    SureFlo™ anti-sealing array

    Rack for 15 ml centrifuge tubes, 4x6 tubes

    This tube rack is designed to hold 15 ml centrifuge tubes in a 4x6 ANSI/SLAS format.

    Part No. 4542

    Show all labware accessories
    Rack for 1.5/2 ml microcentrifuge tubes

    bioMérieux: ENDOZYME® II GO

    ENDOZYME® II GO is intended for quantitative determination of endotoxin (chemically lipopolysaccharide, LPS) in liquid samples such as water. It can also be used for quantitative determination of endotoxin in pharmaceutical final products, in-process control and medical device testing.  Assay range 0.005 to 50 EU/mL.  Assay time 60 minutes. The kit contains reagents for 192 tests.

    Source: Website bioMérieux

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    bioMérieux: ENDOGRADE Glass Test Tubes

    Endotoxin-free borosilicate glass test tubes with aluminum screw cap. Ideal vessels for samples and the dilution of endotoxin standards contained in the ENDOZYME® II and ENDOLISA® kits. Manufactured according to a dry heat depyrogenation procedure.

    Source : Website bioMérieux

    Visit the website of the supplier

    INHECO: HeatPac with flat bottom adapter and Single TEC Control

    The INHECO HeatPAC is especially made for integration on deck of robotic workstations. It is designed for stationary heating up to 135°C of almost any ANSI/SLAS format disposable. Due to the very low profile of only 40mm, deepwell plates or other high disposables will fit on most liquid handling instruments, even when limited space is available between the pipetting head and the deck.

    Source: Website INHECO

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    INHECO Cooling & Heating for ASSIST PLUS

    Customer’s voice

    We have an Assist Plus to prepare samples for enzymatic assays and protein-ligand interaction studies. It's perfect for making dilution series of small molecule compunds to be assayed, and to add reaction components and enzymes before screening using various methods such as fluorescence, absorbance or microscale themophoresis.

    Bjorn Dalhus, Oslo University Hospital