Competitive ELISA Tutorial 2: How to Use Calbiotech’s Competitive ELISA Kits

Published by Jan Heaney on

Competitive ELISA Tutorial 2: How to Use Calbiotech’s Competitive ELISA Kits

This ELISA tutorial series is kindly sponsored
by Calbiotech. Please click the link below, or in the video description to see what ELISA
kits they have to offer. In this video, we will demonstrate how to
perform a Competitive ELISA using a commercial kit [show picture of Calbiotech’s ELISA kit
& its components]. As most Competitive ELISA kits involve the same basic procedure, this
video will be helpful for anyone wishing to learn this technique. It’s important to note that most Competitive
ELISA kits provide an ELISA plate which has already been coated with antibodies specific
to the protein or metabolite to be measured, and has also been blocked. That means, we
can immediately mix our samples with enzyme conjugate and add the mixture to the ELISA
plate. If this concept is unclear, please see our previous ELISA tutorial to review
how a Competitive ELISA works. Mixing Samples with Enzyme Conjugate Some protocols may allow the user to first
add their samples and standards directly to the ELISA plate, and then add the Enzyme Conjugate
to the plate immediately afterwards. In other words, the mixing of samples with Enzyme Conjugate
is to be done within the ELISA plate itself. This approach, however, can be a little bit
risky, especially if it will take more than a few minutes to add the samples and standards
to the ELISA plate. The reason it’s risky is that the results of a Competitive ELISA
performed this way can potentially be skewed based on which samples were first added to
the ELISA plate. If you think about it, samples added to the plate first will be given a “head-start,”
or have more time to bind to the capture antibodies in the absence of Enzyme Conjugate than samples
added to the plate last. This could result in samples added to the plate earlier having
artificially higher measurements than those added after them. Fortunately, there is an easy way to eliminate
the potential risk of skewing measurements due to time delay between samples. The solution
is to simply pre-mix all of the samples and standards with Enzyme Conjugate prior to adding
them to the ELISA plate. This can be done in Eppendorf tubes or strip tubes, if only
a small number of samples are required, or it can be performed in a non-treated 96-well
plate, which is probably best if you have to handle several samples at once. Remember
to only use a non-treated plate, as the protein in your samples have a higher risk of adsorbing
to, and consequently, being lost if transferred to plates that have been treated for tissue-culture
or other applications. Once all of the samples and standards have
been combined with an appropriate amount of Enzyme Conjugate, the mixture can be conveniently
added to the ELISA plate with a multichannel pipette. Keep in mind that the volume of mixture
in each well should be about 50 µL greater than whatever volume you would like to transfer
to the ELISA plate, otherwise you might have difficulty transferring the full volume of
mixture to the ELISA plate. After the mixture of samples and Enzyme Conjugate
have been added to the ELISA plate, seal and incubate the plate as directed by the manufacturer.
Incubation may take place on a benchtop at room temperature, on a plate shaker, in an
incubator set to 37°C, or in the refrigerator overnight, depending on the specific kit you
are working with. Washing the ELISA Plate When the incubation is over, the next step
is to wash the ELISA plate as directed by the manufacturer. Some kits call for washing
with distilled water, while others provide a bottle of concentrated wash buffer which
can be easily diluted to a working concentration by combining with the appropriate volume of
distilled or deionized water. Wash the ELISA plate generously, as insufficient
washing commonly causes false-positive, or inaccurate measurements. Between each wash,
take care to ensure that the ELISA plate has been completely evacuated by tapping the plate
firmly onto paper towels. The reservoir can also be emptied, rinsed with distilled water,
or replaced between washes in order to prevent contamination of the ELISA plate with solution
left over from any of the previous washes. Adding Substrate & Finishing the Assay Once the ELISA plate has been washed and emptied
for the last time, the next step is to add a clear, colorless substrate, such as TMB,
to the ELISA plate. The substrate will be converted to a colored chemical by any Enzyme
Conjugates bound to the ELISA plate. Incubate the plate as directed by the manufacturer,
and check the plate as needed to ensure that it doesn’t get over-developed. As a rule of thumb, you should immediately
proceed to the next step of adding Stop Solution to the plate if you can see any color change
appearing in the most-concentrated standards present in your standard curve. If this sounds
counter-intuitive, recall that in a Competitive ELISA, the most concentrated samples will
have the least color change, due to there being less Enzyme Conjugate bound to the ELISA
plate. Once the ELISA plate has been incubated long
enough, add the Stop Solution directly to the ELISA plate. Be sure to exercise caution
in this step, as Stop Solution is usually a very strong acid. After the Stop Solution has been added, the
ELISA plate can immediately be assessed in a plate reader for absorbance as directed
by the manufacturer. Most protocols call for measuring absorbance at 450 nm. In the next video, we will demonstrate how
to interpret the data that can be obtained from a Competitive ELISA. If this tutorial was helpful, please click
the “like” button and subscribe to our channel for updates when new videos are released. Thanks for watching!


romina najarro flores · November 17, 2014 at 12:08 am

Thanks a lot for the explanation! 
I want to ask, if a patient is infected with a virus then I should expect to see a yellow color, right?

Reivivus · March 2, 2016 at 6:22 am

Man, this is so useful to see the experiment in real time with those animations!

Namrata Prasad · August 31, 2016 at 4:02 am

Why is it read at 450nm?

Mrs Rekima · July 2, 2017 at 11:21 pm

this is so helpful and useful thank you so much and god bless you

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