Performing an ELISA with the ASSIST PLUS pipetting robot

  • Performing an ELISA with the ASSIST PLUS pipetting robot

    Enzyme-linked immunosorbent assays with ASSIST PLUS pipetting robot

    The enzyme-linked immunosorbent assay (ELISA) is a standard method used to detect and quantify peptides, proteins, antibodies or hormones in a sample. It consists of multiple repetitive steps that are time consuming and tedious to perform manually. The ASSIST PLUS pipetting robot allows this process to be automated, which not only increases the reproducibility of your results, but also gives you more time to focus on your science. Any VIAFLO or VOYAGER electronic pipette can be automated using the ASSIST PLUS; the VOYAGER adjustable tip spacing pipette enables reformatting of samples from one labware type to another in the blink of an eye. All the steps needed to run an ELISA are saved on the pipette as a VIALAB program – the smart and easy-to-use pipetting automation software of the ASSIST PLUS. Simply place the labware on the deck, choose the program corresponding to the ELISA step and let the ASSIST PLUS do the work.

  • Table of contents

      The enzyme-linked immunosorbent assay (ELISA) is a standard method used to detect and quantify peptides, proteins, antibodies or hormones in a sample. It consists of multiple repetitive steps that are time consuming and tedious to perform manually. The ASSIST PLUS pipetting robot allows this process to be automated, which not only increases the reproducibility of your results, but also gives you more time to focus on your science. Any VIAFLO or VOYAGER electronic pipette can be automated using the ASSIST PLUS; the VOYAGER adjustable tip spacing pipette enables reformatting of samples from one labware type to another in the blink of an eye. All the steps needed to run an ELISA are saved on the pipette as a VIALAB program – the smart and easy-to-use pipetting automation software of the ASSIST PLUS. Simply place the labware on the deck, choose the program corresponding to the ELISA step and let the ASSIST PLUS do the work.

    Key benefits

    • Optimal pipette settings – including tip immersion depth, pipetting speeds and angles – maximize the consistency and reproducibility of the ELISA.
    • The full automation capability of the ASSIST PLUS frees highly valuable time that you can use for more important tasks.
    • VOYAGER and VIAFLO electronic pipettes, in combination with the ASSIST PLUS, provide unmatched pipetting ergonomics.
    • Repeat Dispense and Multi Aspirate steps can be used for fast dispense and removal of reagents to speed up the process. The automatic Tip Change ensures assay contamination is avoided.
    • The ASSIST PLUS pipetting robot is perfectly adapted to handle different plate layouts, increasing the flexibility of your work depending on your needs.
    • Various sample input tubes can be used. The samples are easily transferred to the assay plate using multichannel and adjustable tip spacing pipettes, increasing the assay productivity while avoiding reformatting errors.

    Overview of the sandwich ELISA steps and corresponding programs:

    The ASSIST PLUS is used to perform a sandwich ELISA. The pipetting robot operates a VOYAGER 8 channel 1250 μl electronic pipette with 1250 μl Sterile, Filter, Low Retention GripTips. The use of Low Retention GripTips guarantees optimal liquid recovery when pipetting ELISA buffers that contain surfactants, such as Tween 20. 

    Below is an example set-up for a sandwich ELISA with a standard curve and 24 samples in triplicate. The pipetting programs are prepared with the VIALAB software. The protocol is divided into eight programs that guide the user through the eight steps of the ELISA.

    Downloads: Protocols and reports for automated ELISA 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.

    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, in a 10 ml polypropylene multichannel reagent reservoir. Select and run the first VIALAB program, 1_E_Coating. The pipette automatically transfers 100 μl of the capture antibody into the ELISA plate using the Repeat Dispense mode. The plate is ready to be incubated.

    Adding the capture antibody to coat the ELISA plate.

    2. Block the plate

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

    Select the VIALAB program 2_E_Blocking and set up the deck with the required labware.

    The program incorporates all the necessary pipetting steps, including removing of the coating buffer from the plate followed by washing three times with 200 μl of the washing buffer. In our example, we included a 15 second incubation time, which can be easily adjusted to your protocol using the VIALAB software. The use of the Repeat Dispense and Multi Aspirate modes speeds up this fully automated process. At the end of the washing steps, the user is prompted to blot the plate against clean paper towels – the only manual step of this ELISA protocol. After confirming that this step has been completed, the ASSIST PLUS pipetting robot continues by adding the blocking buffer into the ELISA plate using the Repeat Dispense mode. Finally, the pipette informs the user that the plate is ready for incubation.

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

    3. Prepare your samples

    Diluting your samples 1:10.

    In this example, centrifuged blood samples are stored in EDTA collection tubes placed in an INTEGRA rack. The plasma is diluted 1:10 with the dilution buffer. 

    Select and run the 3_E_Sample_Preparation program. The pipette automatically fills the microcentrifuge tubes with 900 μl of the dilution buffer. This is followed by transfer of the plasma samples from the EDTA tubes to the microcentrifuge tubes, and careful and thorough mixing. The samples are then ready to be used.

    Tips: Using a VOYAGER adjustable tip spacing pipette together with the ASSIST PLUS allows automatic and error-free sample reformatting. The ASSIST PLUS pipetting robot ejects and loads the tips automatically, eliminating any risk of sample cross-contamination.

    Diluting your samples 1:10.

    4. Add your controls and samples

    Adding the controls and diluted samples to the ELISA plate.

    Prepare the deck of the ASSIST PLUS.

    Select and run the 4_E_Sample_Addition program to remove the blocking buffer and subsequently wash the plate. In our example, each sample is added to the ELISA plate in triplicate. The ASSIST PLUS pipetting robot uses the Repeat Dispense mode to transfer the triplicate samples into the plate, replacing the pipette tips before aspirating the next series of samples.

    Tip: Each pipetting step is done in exactly the same way, ensuring the reproducibility of the assay from row to row, and plate to plate.

    Adding the controls and diluted samples to the ELISA plate.

    5. Add the detection antibody

    Adding the diluted detection antibody to the ELISA plate.

    After incubation, prepare the deck and select program 5_E_Detection_Antibody. The addition of the detection antibody is performed automatically by the ASSIST PLUS. Incubate the plate again.

    Adding the diluted detection antibody to the ELISA plate.

    6. Add the enzyme conjugate

    Adding the enzyme conjugate to the ELISA plate.

    Set up the ASSIST PLUS deck. Select program 6_E_Enzyme_Conjugate, which includes the removal of the previous solution, the three washing steps, and the addition of the enzyme conjugate to the ELISA plate. The plate is ready for incubation.

    Adding the enzyme conjugate to the ELISA plate.

    7. Add the substrate

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

    Select and run program 7_E_Substrate. The ASSIST PLUS removes the previous buffer then washes the plate six times before adding the TMB substrate. Incubate the plate at room temperature until the color is sufficiently developed. The color of the solution changes from transparent to blue in wells where the samples have reacted with the antibodies. The color intensity is dependent on the sample concentration.

    Tips: The pipetting robot automatically processes the plate, regardless of the multiple and repetitive pipetting steps, freeing up time for you to concentrate on other tasks. The ASSIST PLUS tells you when to add the TMB substrate into the corresponding row of the reservoir, preventing the photosensitive substrate from being exposed to light for too long a period of time.

    Adding the TMB substrate and incubating the plate until the color develops sufficiently.
    Example of a sandwich ELISA plate
    Example of a sandwich ELISA plate after incubation with TMB-substrate, showing positive (blue) and negative (clear) reactions of the triplicate samples with the antibodies. The color intensity directly depends on the sample concentration.

    8. Stop the reaction

    Adding the stop solution to the plate before detection.

    Select and run the final program, 8_E_Stop_Solution.

    The ASSIST PLUS adds the stop solution to the sample triplicates in the plate; the color changes from blue to yellow in the wells where the samples 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 using a TMB-substrate after addition of the stop solution
    Example of a sandwich ELISA plate using a TMB-substrate after addition of the stop solution, showing positive (yellow) and negative (clear) reactions of the triplicate samples with the antibodies.

    Remarks

    Partial plate

    If your particular ELISA doesn’t require processing of 96 samples, the ASSIST PLUS is able to work with any number of columns. Simply adapt the VIALAB program to fit your need.

    VIALAB software

    The VIALAB programs can be easily adapted to your specific labware and protocols.

     

    Partial plate and VIALAB software.

    Conclusion

    • ELISAs can be fully automated using the ASSIST PLUS pipetting robot, offering users increased walk-away time.
    • Optimized pipetting settings and tip immersion, together with the use of Low Retention GripTips, guarantee the consistency and reproducibility of the ELISAs.
    • Using the ASSIST PLUS pipetting robot allows various sample tube types and multiple reagents to be accommodated on the deck, for improved productivity and unrivaled flexibility.
    • Automatic Tip Change avoids any assay contamination while using the Repeat Dispense and Multi Aspirate modes whenever possible speeds up the process.
    • Thanks to the VIALAB software, the pipetting programs can be easily adapted to specific protocols and labware.

    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, 8 Channel, 1250 µl GripTip, 1250 µl, Sterile, Filter, Low Retention Multichannel Reagent Reservoir, 10 ml, PP Automation Friendly Reagent Reservoirs, 150 ml Automation Friendly Reagent Reservoir, 300ml 8 Row Polypropylene Reservoirs

    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, 8 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. 4724

    See it work
    VOYAGER, 8 channel series

    GripTip, 1250 µl, Sterile, Filter, Low Retention

    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. 6545

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

    Multichannel Reagent Reservoir, 10 ml, PP

    • 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
    Reagent reservoirs 10ml, 25ml, 100ml

    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

    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

    8 Row Polypropylene Reservoirs

    The polypropylene design improves the reservoirs chemical compatibility and also allows the reservoirs to be autoclaved.

    • Reagent reservoirs for 96 well plates
    • Pyramid bottom
    • Each row holds 32 ml

    Part No. 6371

    Show all automation-friendly reagent reservoirs
    8 Row Reagent Reservoirs Blocks, Non-Sterile, Sterile

    Download

    Download this App Note as PDF file

    Download PDFPerforming an ELISA with the ASSIST PLUS pipetting robot

    Customer’s voice

    This product has so many features, whatever your pipetting needs are there is either a built in program that can save you time or you can create a program to suit your needs. I also love that you can customize the menu if you want to eliminate things you will never do. The ability to add text prompts and sounds to protocols facilitates training of new members. I have received top notch customer service from my rep Courtney, who explained the product in detail, confirmed understanding of myself and my undergraduate assistants, as well as helped us incorporate the pipettes into our workflow. By taking the time to understand our laboratory methods, Courtney was able to help us write new protocols to save time and reduce error. (Review published on Select Science)

    Nicole Osier, UT Austin