Performing an ELISA with the VIAFLO 96/384 handheld electronic pipette

  • Performing an ELISA with the VIAFLO 96/384 handheld electronic pipette

    Enzyme-linked immunosorbent assays with VIAFLO 96/384 handheld electronic pipette

    ELISA (enzyme-linked immunosorbent assay) is a plate-based assay that is commonly used to quantify and detect peptides, proteins, antibodies or hormones. Well to well and plate to plate consistency and reproducibility are key success factors for ELISAs, which consist of multiple pipetting steps that are time-consuming and tedious to perform manually. Using the VIAFLO 96/384 handheld electronic pipette can vastly improve ELISAs. The 96 channel pipetting head makes the whole process faster, less error prone and much more reproducible compared to traditional single or multichannel pipettes by transferring the samples and reagents into all 96 wells of the plate at the same time. The VIAFLO 96/384 electronic pipette offers clear gains in productivity, consistency and reproducibility while remaining as easy to use as a traditional handheld pipette.

  • Table of contents

      ELISA (enzyme-linked immunosorbent assay) is a plate-based assay that is commonly used to quantify and detect peptides, proteins, antibodies or hormones. Well to well and plate to plate consistency and reproducibility are key success factors for ELISAs, which consist of multiple pipetting steps that are time-consuming and tedious to perform manually. Using the VIAFLO 96/384 handheld electronic pipette can vastly improve ELISAs. The 96 channel pipetting head makes the whole process faster, less error prone and much more reproducible compared to traditional single or multichannel pipettes by transferring the samples and reagents into all 96 wells of the plate at the same time. The VIAFLO 96/384 electronic pipette offers clear gains in productivity, consistency and reproducibility while remaining as easy to use as a traditional handheld pipette.

    Key benefits

    • The VIAFLO 96/384 allows easy and rapid simultaneous transfer of all samples for optimal productivity, reproducibility, and massive time reduction.
    • All the steps needed to run the ELISA protocol can be saved on the pipette as a custom program containing all the necessary settings to guide the user through the process via easy to follow prompts.
    • The pipetting heights are saved to avoid any crashes and scratching of the coated surface.
    • For higher throughput assays, multiple plates can be quickly filled using the Repeat Dispense mode of the VIAFLO 96/384.

    Overview of the sandwich ELISA steps and corresponding programs:

    The following protocol shows an example set-up for performing a sandwich ELISA using the VIAFLO 96/384 handheld electronic pipette together with a three position stage. In this protocol, position A is dedicated to the GripTip box and the liquid waste reservoir, position AB to reagent reservoirs containing the various reagents and washing solutions required, and position B to the ELISA plate. A 96 channel pipetting head (10-300 μl) is used together with 300 μl Sterile, Filter, Low Retention GripTips. Customized VIALINK programs are provided for performing a sandwich ELISA with the VIAFLO 96/384.

    Note: The VIALINK programs provided can be easily adapted to any other ELISA type, e.g. direct, indirect or competitive ELISAs, by just adding or removing steps from the protocols supplied.

    Downloads: Protocols for automated ELISA with VIAFLO 96/384

    Here you can download the complete protocols used in this application and use them on your VIAFLO 96/384. The protocol can be customized with the VIALINK software.

    Step-by-step procedure

    1. Coat the ELISA plate

    Adding the capture antibody to coat the ELISA plate.

    Place the capture antibody, prediluted in the coating buffer, into a 150 ml automation friendly reagent reservoir. Select and run the VIALINK E_REAGENTS program, then simply follow the instructions on the pipette. Pipette 100 μl of the capture antibody into the ELISA plate and then incubate it.

    Tips: A z-height limit is defined to ensure an optimal tip immersion depth, preventing both air entering into the tips during the aspiration step and the pipette tips from touching the bottom of the plate. We also recommend setting the Tip Align support strength to 3 for this application. To increase your throughput, the Repeat Dispense mode of the VIAFLO 96/384 electronic pipette can be used to rapidly prepare several plates at once. The VIALINK program E_REAGENTS_3 PL is an example of a multiple plate filling protocol.

    Adding the capture antibody to coat the ELISA plate.

    2. Block the plate

    Blocking the ELISA plate’s non-specific binding sites.

    Select the VIALINK program E_REAGENTS to remove the coating buffer from the plate, then use the E_WASH program to wash the plate three times with 200 μl of washing buffer from a 300 ml automation friendly reagent reservoir. Firmly blot the plate against clean paper towels. To block the ELISA plate, select the corresponding E_BLOCK program and transfer 300 μl of the blocking buffer from a 150 ml automation friendly reservoir into the plate before incubation.

    Tips: All wells are treated simultaneously and in the same way. This ensures not only the well to well consistency of the assay, but also the reproducibility from plate to plate. The z-height is set 1 mm above the bottom of the plate, preventing the tips from scratching the coated surface, which can lead to inaccurate or inconsistent assay results.

    Blocking the ELISA plate’s non-specific binding sites.

    3. Add the controls and samples

    Adding the diluted samples and controls to the ELISA plate.

    Select and run the E_BLOCK program to remove the blocking buffer, then wash the plate using the E_WASH program. In the current example, the controls and samples are dispensed into a 96 well plate. The E_REAGENTS program allows rapid, simultaneous transfer of all samples and controls into the ELISA plate before incubation.

    Tips: Adding all the samples at once ensures optimal productivity and reproducibility of the ELISA. If your samples are stored in microcentrifuge tubes, using a VOYAGER 8 channel 300 μl adjustable tip spacing pipette allows you to quickly reformat them from the tubes to the plate at the simple touch of a button, while reducing transcription errors when compared to a single channel pipette. ELISA buffers often contain surfactants, such as Tween 20. Using Low Retention GripTips reduces the residual volume in the tips for maximum sample recovery.

    Adding the diluted samples and controls to the ELISA plate.
    Figure 3: The image highlights the advantages of using Low Retention GripTips (left) versus standard GripTips (right) when pipetting buffers containing surfactants.
    The image highlights the advantages of using Low Retention GripTips (left) versus standard GripTips (right) when pipetting buffers containing surfactants.

    4. Add the detection antibody

    Adding the diluted detection antibody to the ELISA plate.

    After incubation, select the E_REAGENTS program to remove the sample buffer, then use E_WASH to wash the plate three times. Place the detection antibody into a 150 ml automation friendly reagent reservoir on position AB. Run the E_REAGENTS program to rapidly transfer the detection antibody into the ELISA plate before incubation.

    Adding the diluted detection antibody to the ELISA plate.

    5. Add the enzyme conjugate

    Adding the enzyme conjugate to the ELISA plate. 

    Repeat step 4 to add the enzyme conjugate. Incubate the plate.

    Adding the enzyme conjugate to the ELISA plate. 

    6. Add the substrate

    Adding the TMB substrate and incubating the plate until the color develops sufficiently.

    Remove the buffer then thoroughly wash the plate six times before adding 100 µl of TMB substrate to the ELISA plate. Incubate the plate at room temperature: the color of the solution changes from transparent to blue in wells where the samples have reacted with the antibodies. The color intensity depends on the sample concentration.

    Adding the TMB substrate and incubating the plate until the color develops sufficiently.
    Example of a sandwich ELISA plate after incubation  with TMB substrate;
    Example of a sandwich ELISA plate after incubation with TMB substrate; positive (blue) and negative (clear) reaction of the samples with the antibodies. The color intensity directly correlates with the sample concentration.

    7. Stop the reaction

    Adding the stop solution to the plate before detection.

    Use the VIALINK E_REAGENTS program to add the stop solution to the plate; the color changes from blue to yellow in wells where the samples have reacted with the antibodies. The plate is now ready for detection.

    Adding the stop solution to the plate before detection.
    Example of a sandwich ELISA plate after incubation  with TMB substrate and addition of the stop solution;
    Example of a sandwich ELISA plate after incubation with TMB substrate and addition of the stop solution; positive (yellow) and negative (clear) reaction of the samples with the antibodies. The color intensity directly depends on the sample concentration.

    Remarks

    Partial plate

    If your particular ELISA doesn't require processing of 96 samples, the VIAFLO 96/384 is able to work with any number of tips loaded, giving you the benefit of simultaneous and precise dispensing of a smaller number of samples.

    Automation

    The instrument can also operate on its own, reducing user interaction, which in turn improves ergonomics and reproducibility. This also makes the VIAFLO 96/384 ideal for use in tight spaces, such as under a laminar flow cabinet

    VIAFLO 96/384 is able to work with any number of tips loaded and automation.

    Conclusion

    • The VIAFLO 96/384 allows all wells of a 96 well plate to be treated at the same time, offering optimal consistency and reproducibility of the results from well to well, and plate to plate.
    • Thanks to its unique operating concept, the VIAFLO 96/384 electronic pipette is as easy to use as any traditional handheld pipette.
    • Optimized pipetting settings ensure easy and fast sample and reagent transfers, while avoiding the risk of damaging the coated surface of the plate.
    • The VIAFLO 96/384 electronic pipette is adaptable to your needs, either by working with a reduced number of tips for partial plate processing or by working on its own for improved ergonomics, especially when used in tight spaces, such as a laminar flow cabinet.

    April 2020, by

    • Lydiane Saucède, PhD

      Application Scientist

      Email
    • Jan-Peter Baldin, PhD

      Product Manager

      Email

    Any questions? I'm happy to help!

    • Tom Bentivegna

      Product Manager

      Email

    Used instruments and accessories

    VIAFLO 96/384, Handheld Electronic Pipette Three position stage for 96 and 384 well plates 96 Channel Pipetting Head, 300 µl VOYAGER, 8 Channel, 300 µl Automation Friendly Reagent Reservoir, 300ml Automation Friendly Reagent Reservoirs, 150 ml GripTip, 300 µl, Sterile, Filter Greiner Bio-One: 96 Well Polypropylene Microplates

    VIAFLO 96/384, Handheld Electronic Pipette

    With this 24, 96 and 384 channel pipette, INTEGRA meets the need for an affordable, compact and easy to use pipette that provides increased productivity. The unique operating concept makes VIAFLO 96/384 as easy to use as any traditional handheld pipette.

    Part No. 6001 / 6031

    See how it work
    The VIAFLO 96/384 is handheld, electronic pipette

    Three position stage for 96 and 384 well plates

    This accessory expands available stage positions for microplates, reservoirs and tips on a VIAFLO 96 or VIAFLO 384 from 2 to 3 thereby enhancing workflow in a wide range of applications. Beneficially the Three Position Stage also features an indexing function to allow access to 384-well plates using a 96-channel pipetting head.

    Part No. 6230

    See all plate holder options
    Three position stage for 96 and 384 channel pipetting

    96 Channel Pipetting Head, 300 µl

    Users can simply change the pipetting head of their VIAFLO 96 and VIAFLO 384 to guarantee optimal matching of the available volume range to the application performed. Changing a pipetting head of VIAFLO 96 and VIAFLO 384 is easy and takes less than one minute.

    Part No. 6103

    Find out more about VIAFLO 96/384 Pipetting Heads Specifications
    VIAFLO 96/384 96 channel pipetting head

    VOYAGER, 8 Channel, 300 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. 4743

    See it work
    VOYAGER, 8 channel series

    Automation Friendly Reagent Reservoir, 300ml

    INTEGRA offers two types of automation friendly reagent reservoirs:

    • 150 ml and 300 ml Clear Advantage™ reagent reservoirs which are made of both crystal clear virgin polystyrene and polypropylene. Both options fit into a reusable automation friendly base with bold, crisp, clearly visible graduation markings.
       
    • The 300 ml open well, 12 column and 8 row polypropylene reservoir blocks offer improved chemical compatibility.
    Find out more about Automation Friendly Reagent Reservoirs
    300 ml Reservoir filled

    Automation Friendly Reagent Reservoirs, 150 ml

    150 ml Automation Friendly Clear Advantage™ Reservoirs (Polystyrene) which are made of crystal clear 100 % virgin polystyrene and fit into a reusable automation friendly base with bold, crisp, clearly visible graduation markings.

    The SureFlo™ Array located on the flat bottom reservoir prevents pipette tip seal off. This allows users to rest the pipette tips directly on the bottom of the reservoir offering an incredibly low dead volume of less than 3 ml!

    Find out more about Automation Friendly Reagent Reservoirs
    Reservoir for automation with SureFlo™ anti-sealing array

    GripTip, 300 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. 6435

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

    Greiner Bio-One: 96 Well Polypropylene Microplates

    SAPPHIRE MICROPLATE, 96 WELL, PP, FOR RT PCR, WHITE, HALF-SKIRT, FOR LIGHT CYCLER, 10 PCS./BOX

    Source: Website Greiner Bio One

    Visit supplier website

    Customer’s voice

    We just received our second ViaFlo 96 pipettor with a 1250 µL head and unexpectedly, and immediately, put it to use washing ELISA plates with minimum wash buffer dead volume. We were changing pipetting heads three and four times a day on one instrument between our 12.5 µL, 50 µL and 300 µL 96-well heads. We now spend less time changing heads as well with the second pipettor. The simple things that make for a happy lab.

    Steve Miller, Aerie Pharma