Search from over 1000 highly-referenced ELISA kits

5 Steps to Consistent ELISA Results

Enzyme-linked immunosorbent assays (ELISAs) are a gold standard for quantitation of target-specific proteins and provide rapid and consistent results that are easy to analyze. ELISAs are available for many targets across research areas including cytokines, chemokines, growth factors, phospho-specific targets, immunoglobulins, immuno-oncology, and neurobiology. No matter your target of interest or research area, there are 3 format options for ELISA – kits with pre-coated plates, kits with matched antibody pairs to coat-your-own plates, or you can develop your own ELISA by performing your own screening for matched antibody pairs to create a custom assay. With so many variables, it’s sometimes hard to determine how to get consistent results every time.

Here, we provide 5 simple steps to achieve consistent and accurate results when running ELISAs.

Step 1 – Use ELISA kits from the same lot

ELISA kit lot-to-lot variation is important to consider, especially when starting a long-term research project. While many reputable vendors have in-house controls (Figure 1) to avoid drift over time, mitigate any potential lot-to-lot variability by using kits from one lot. Determine how many kits will be needed for the entirety of the project and order accordingly. If space is a concern, reserve enough kits from the same lot from the supplier. Planning in this way prevents any need for a lot change in the middle of the project which can lead to variability.

Special services for any Invitrogen immunoassay product

custom-elisa-proquantum-immunoassay-header

Request lot reservation, bulk packaging or go custom

Contact us and we can help you with a variety of special requests to help you with the start of your next project.

Request a quote

Step 2 – Use pre-coated ELISA kits

When possible, purchase pre-coated versions of the ELISA kits needed. Pre-coated plates are typically manufactured using automated instrumentation and allow less human hand-pipetting variability or error. Once coated, there is validation testing to identify any plate coating variability between wells and between plates to ensure consistency.

Inter-assay precision

Samples were assayed 8 times in 5 different assays to determine precision between assays.

Parameters Sample 1 Sample 2 Sample 3
Mean (pg/mL) 35.3 97.4 236.7
Standard deviation 3.3 6.4 18.5
% Coefficient of variation 9.3 6.5 7.8

Intra-assay precision

Samples of known human IL-6 concentration assayed in replicates of 16 to determine intra-assay precision.

Parameters Sample 1 Sample 2 Sample 3
Mean (pg/mL) 38.8 101.2 242.7
Standard deviation 3.0 5.8 12.5
% Coefficient of variation 7.7 5.7 5.1

Table 1. Inter-assay (between plates) and intra-assay (between wells) CV for Human IL-6 ELISA Kit (Cat. No. KHC0061).

Using Invitrogen pre-coated ELISA kits

View the instructional video showing how to use Invitrogen pre-coated ready-to-use ELISA kits. Please make sure to carefully read and review your ELISA kit’s specific instruction sheet, located inside the package, for detailed information before beginning the procedure.

Step 3 – Use condensed workflows

Where appropriate, use ELISA kits that are designed to have a condensed workflow. Reducing the number of steps will result in less hands-on time and other advantages such as:

  • Fewer instances of introducing variables (human error, buffer preparations, etc.)
  • No need to prepare reagents
  • No need for serial dilutions of standards (i.e.: Instant ELISA)
  • No need to monitor assay every hour for sequential additions

Learn more about condensed workflows: Simplified workflow for Instant ELISA

17 Steps to 7 Steps: An ELISA Condensed Workflow

Fewer steps help to save time and reduce variation. With Instant ELISA kits, all the preparation work has been performed for you. There are only 7 working steps when using an Instant ELISA kit, as opposed to 17 steps when performing a conventional ELISA. Therefore, you can decrease total hands-on time per experiment to about 1 hour by adopting the Instant ELISA technique.

We offer many Instant ELISA options including:

  • Human IFN-γ
  • Human IL-6
  • Human IL-10
  • Human TNF-α

Next generation immunoassay technology

With recent advancements in technology, next-gen ELISA platforms can provide further condensed workflows. ProQuantum immunoassays is an example using matched antibody pairs combined with a qPCR amplification for improved sensitivity, dynamic range, and small sample consumption. The streamlined workflow eliminates all wash steps and requires minimal reagent additions. To learn more, watch the video or visit the informational web page.

ProQuantum Immunoassays: how it works?

Step 4 – Handle samples properly and use controls

Samples, and the way you prepare those samples, may be the most important part of successfully running an ELISA. Proper sample handling and preparation is key to consistent and accurate results. When working with limited or precious samples it is essential to handle them carefully and consistently.

Tips to proper sample prep:

  • After sample collection, use immediately, or aliquot and freeze at -80°C
  • Limit the number of freeze/thaw cycles
  • Compare samples with the same number of freeze/thaw cycles
  • Thaw the samples on ice
  • While handling, keep samples at 2-8°C
  • Use proper control samples in your ELISA assays
    • Run ELISA assays with blank sample and/or negative controls
    • Run ELISA assays with independent positive controls

To learn more about proper sample handling: Proper Sample Handling for Immunoassays
To learn more about pipetting: Good Laboratory Pipetting Guide
To learn more about electronic programmable protocol pipettes: Electronic Pipettes

Step 5 – Boost consistency with reliable equipment

The success of ELISA not only relies on the way the assay is run and the quality of the ELISA kits, but it also relies heavily on the equipment. Use instruments that are guaranteed to give consistent results every time.

The first instrument to consider when running ELISAs is a microplate washer. Plate washers help ensure that wells are washed properly, that wash buffer is allowed to "soak" long enough, and that wash buffer is completely removed from each well. It essential to follow all wash steps as they are stated in an ELISA protocol.

The best results will come from using a reliable automated plate washer with some key features. Using this instrument will wash the ELISA plates effectively while minimizing variability, reducing risk of human error, and saving time.

The advantages of choosing the right automated plate washer include:

  • Reduces risk of human error
  • Convenient and faster than pipetting by hand
  • Get consistent results with identical washes every time
  • Effective washing with minimal residual volumes

It makes running ELISAs easier when using a washer with the following features:

  • Easy-to-clean, removable wash head
  • Automatic priming and rinsing for clog prevention
  • Features designed for lab safety:
    • Liquid level sensors
    • Plate sensors
    • Non-pressurized wash bottles
    • Aerosol covers
  • Easy-to-fill reservoirs and prime lines
  • Optimizable parameters
    • Dispensing/aspiration: height, speed, and location in the well
    • Gentle vs. hard washing to meet specific needs

To learn more about plate washing:
Easy Washing for Reliable ELISA Results
Free poster: Guide to good washing practice

The advantages of choosing the right automated plate washer include:

  • Reduces risk of human error
  • Convenient and faster than pipetting by hand
  • Get consistent results with identical washes every time
  • Effective washing with minimal residual volumes

When it is time to read ELISA plates, use a microplate reader that is reliable to provide consistent results over time. Some key features to seek in an ELISA plate reader include:

  • Integrated incubator and shaker eliminate need for additional instrument for incubated ELISAs
  • Reference calibration system that follows natural variations in the lamp and detector
  • Good plate/well positioning accuracy and precision and methods to verify plate movements
  • Option to use liquid settling delay between plate movement and reading the absorbance
  • Option to use individual plate dimension maps for plates from different manufacturers
  • Fully automatic, continuous diagnostics with reporting about reader status and performance
  • Special functions to ensure data quality and data security
  • Movement verification for every moving component (e.g. plate carrier, optical heads, filter wheels)
  • Option to perform external verification

To learn more about plate reading: Microplate Instruments, Assays and Accessories Guide
To read application notes and learn more, visit thermofisher.com/elisainstrumentsandequipment

Step 1 – Use ELISA kits from the same lot

ELISA kit lot-to-lot variation is important to consider, especially when starting a long-term research project. While many reputable vendors have in-house controls (Figure 1) to avoid drift over time, mitigate any potential lot-to-lot variability by using kits from one lot. Determine how many kits will be needed for the entirety of the project and order accordingly. If space is a concern, reserve enough kits from the same lot from the supplier. Planning in this way prevents any need for a lot change in the middle of the project which can lead to variability.

Special services for any Invitrogen immunoassay product

custom-elisa-proquantum-immunoassay-header

Request lot reservation, bulk packaging or go custom

Contact us and we can help you with a variety of special requests to help you with the start of your next project.

Request a quote

Step 2 – Use pre-coated ELISA kits

When possible, purchase pre-coated versions of the ELISA kits needed. Pre-coated plates are typically manufactured using automated instrumentation and allow less human hand-pipetting variability or error. Once coated, there is validation testing to identify any plate coating variability between wells and between plates to ensure consistency.

Inter-assay precision

Samples were assayed 8 times in 5 different assays to determine precision between assays.

Parameters Sample 1 Sample 2 Sample 3
Mean (pg/mL) 35.3 97.4 236.7
Standard deviation 3.3 6.4 18.5
% Coefficient of variation 9.3 6.5 7.8

Intra-assay precision

Samples of known human IL-6 concentration assayed in replicates of 16 to determine intra-assay precision.

Parameters Sample 1 Sample 2 Sample 3
Mean (pg/mL) 38.8 101.2 242.7
Standard deviation 3.0 5.8 12.5
% Coefficient of variation 7.7 5.7 5.1

Table 1. Inter-assay (between plates) and intra-assay (between wells) CV for Human IL-6 ELISA Kit (Cat. No. KHC0061).

Using Invitrogen pre-coated ELISA kits

View the instructional video showing how to use Invitrogen pre-coated ready-to-use ELISA kits. Please make sure to carefully read and review your ELISA kit’s specific instruction sheet, located inside the package, for detailed information before beginning the procedure.

Step 3 – Use condensed workflows

Where appropriate, use ELISA kits that are designed to have a condensed workflow. Reducing the number of steps will result in less hands-on time and other advantages such as:

  • Fewer instances of introducing variables (human error, buffer preparations, etc.)
  • No need to prepare reagents
  • No need for serial dilutions of standards (i.e.: Instant ELISA)
  • No need to monitor assay every hour for sequential additions

Learn more about condensed workflows: Simplified workflow for Instant ELISA

17 Steps to 7 Steps: An ELISA Condensed Workflow

Fewer steps help to save time and reduce variation. With Instant ELISA kits, all the preparation work has been performed for you. There are only 7 working steps when using an Instant ELISA kit, as opposed to 17 steps when performing a conventional ELISA. Therefore, you can decrease total hands-on time per experiment to about 1 hour by adopting the Instant ELISA technique.

We offer many Instant ELISA options including:

  • Human IFN-γ
  • Human IL-6
  • Human IL-10
  • Human TNF-α

Next generation immunoassay technology

With recent advancements in technology, next-gen ELISA platforms can provide further condensed workflows. ProQuantum immunoassays is an example using matched antibody pairs combined with a qPCR amplification for improved sensitivity, dynamic range, and small sample consumption. The streamlined workflow eliminates all wash steps and requires minimal reagent additions. To learn more, watch the video or visit the informational web page.

ProQuantum Immunoassays: how it works?

Step 4 – Handle samples properly and use controls

Samples, and the way you prepare those samples, may be the most important part of successfully running an ELISA. Proper sample handling and preparation is key to consistent and accurate results. When working with limited or precious samples it is essential to handle them carefully and consistently.

Tips to proper sample prep:

  • After sample collection, use immediately, or aliquot and freeze at -80°C
  • Limit the number of freeze/thaw cycles
  • Compare samples with the same number of freeze/thaw cycles
  • Thaw the samples on ice
  • While handling, keep samples at 2-8°C
  • Use proper control samples in your ELISA assays
    • Run ELISA assays with blank sample and/or negative controls
    • Run ELISA assays with independent positive controls

To learn more about proper sample handling: Proper Sample Handling for Immunoassays
To learn more about pipetting: Good Laboratory Pipetting Guide
To learn more about electronic programmable protocol pipettes: Electronic Pipettes

Step 5 – Boost consistency with reliable equipment

The success of ELISA not only relies on the way the assay is run and the quality of the ELISA kits, but it also relies heavily on the equipment. Use instruments that are guaranteed to give consistent results every time.

The first instrument to consider when running ELISAs is a microplate washer. Plate washers help ensure that wells are washed properly, that wash buffer is allowed to "soak" long enough, and that wash buffer is completely removed from each well. It essential to follow all wash steps as they are stated in an ELISA protocol.

The best results will come from using a reliable automated plate washer with some key features. Using this instrument will wash the ELISA plates effectively while minimizing variability, reducing risk of human error, and saving time.

The advantages of choosing the right automated plate washer include:

  • Reduces risk of human error
  • Convenient and faster than pipetting by hand
  • Get consistent results with identical washes every time
  • Effective washing with minimal residual volumes

It makes running ELISAs easier when using a washer with the following features:

  • Easy-to-clean, removable wash head
  • Automatic priming and rinsing for clog prevention
  • Features designed for lab safety:
    • Liquid level sensors
    • Plate sensors
    • Non-pressurized wash bottles
    • Aerosol covers
  • Easy-to-fill reservoirs and prime lines
  • Optimizable parameters
    • Dispensing/aspiration: height, speed, and location in the well
    • Gentle vs. hard washing to meet specific needs

To learn more about plate washing:
Easy Washing for Reliable ELISA Results
Free poster: Guide to good washing practice

The advantages of choosing the right automated plate washer include:

  • Reduces risk of human error
  • Convenient and faster than pipetting by hand
  • Get consistent results with identical washes every time
  • Effective washing with minimal residual volumes

When it is time to read ELISA plates, use a microplate reader that is reliable to provide consistent results over time. Some key features to seek in an ELISA plate reader include:

  • Integrated incubator and shaker eliminate need for additional instrument for incubated ELISAs
  • Reference calibration system that follows natural variations in the lamp and detector
  • Good plate/well positioning accuracy and precision and methods to verify plate movements
  • Option to use liquid settling delay between plate movement and reading the absorbance
  • Option to use individual plate dimension maps for plates from different manufacturers
  • Fully automatic, continuous diagnostics with reporting about reader status and performance
  • Special functions to ensure data quality and data security
  • Movement verification for every moving component (e.g. plate carrier, optical heads, filter wheels)
  • Option to perform external verification

To learn more about plate reading: Microplate Instruments, Assays and Accessories Guide
To read application notes and learn more, visit thermofisher.com/elisainstrumentsandequipment

Performance guarantee

If an Invitrogen immunoassay does not perform as described on our data sheet, we will replace the product at no cost to you, or if you prefer, we will provide you with a credit for future purchase.*

Learn more about performance guarantee

We're here to help

Our team of field application specialists are trained in immunoassay workflows. We can help you choose an assay or configure a panel, optimize an experiment, troubleshoot and more – face to face or on the phone at no cost.

Contact a field application specialist