Ways to check your set up is correct

by | Sep 9, 2019

Already have a project in mind? – Contact out team to get started

You’ve spent all of your lab time making the perfect SPR assay. Your data looks great, the Sensorgrams overlay like they are a single curve and then the inevitable happens; someone asks:

“How do you know it’s reliable?”

Assessing the fit

First thing to do is look at the sensorgram and the fit to it:

Ways to check

In the above example the data is in blue and the fit is the solid black line. So this data fit overlays the actual assay data very well so it’s expected that we’d observe confidence values that reflect this. Parameters that are commonly used to help define precision in an assay include:


Ways to check


Chi2 is a measure of the average deviation of the experimental data to form the fitted curve. Lower chi2 values indicate a better fit, but assessment of chi2 must be taken in the context of the binding level of the system. Basically the further away the fit is from your original data, the higher the Chi2 value will be and vice versa. As the Chi2 value is derived from the experimental data it’s hard to advise an exact number, but as a general rule of thumb ≤10% Rmax.

So using the data in the table above we have an average Rmax of 23.5 RU and therefore, we should ‘accept’ a Chi2 of less than 2.35. Out actual Chi2 is 0.00389 so we are much lower than that, so that gives us confidence in the fit.


One of my favourite values for confidence in data is T-values. These provide nice and simple feedback on the individual fit parameters. To determine these, you need the standard error (SE), which is generated for you by the BiacoreTM software. For SE you’re looking for a small a number as possible because a small SE indicates that changes in the parameter’s value would have a significant effect on the fitting (i.e., the confidence value is high).
To calculate T-values you simply divide the parameter’s value by the SE. The larger the number the better for T-values. My rule of thumb is:

  • 10 = good
  • 100 = great
  • 1,000 = happy days

So the T-values of 251.3, 705.0 and 1,023.1 for T(ka), T(kd) and T(Rmax) give me increased confidence in the data.

Percentage coefficient of variation (%CV)

Much maligned but still a standard value that people expect to see, the coefficient of variation (CV) is defined as the ratio of the standard deviation to the mean. There are multiple guidelines for what an acceptable %CV is but a good rule of thumb is that ≤15% is good for intra-assay triplicate repeatability and ≤20% for inter-assay repeatability. Therefore, the %CV values in the table above are good for the kinetic parameters and for the T-values.

It’s worth mentioning here that %CV can be misleading, in the above table we can see that KD has a %CV of 8.44% as the numbers are close together. But as we’re working in the pM range a small change in value can have a large impact on the %CV.

For example, if I change the kd of Antibody 1.2 to 6.38E-5 from 7.38E-5, we can see that the %CV for Kd and KD is massively affected:


Ways to check


In real terms, the KD of Antibody 1.2 has changed from 19.63 pM to 16.97 pM but the %CV increases from 8.44% to 16.15%. I’d argue that a affinity shift of 2.66 pM is hardly biologically relevant but the mathematics behind it can play tricks on you.

As the saying goes – keep your mind open but not so open your brain drops out…


There you have it, all the information you need to defend your beautiful assay (should you ever need to) and the reason that sometimes the %CV can play tricks upon you. I really enjoy writing posts like these, so please keep asking questions and feedback on what you would like covered next.

<a href="https://www.antibodyanalytics.com/author/stuknowling/" target="_self">Chris McHenry</a>

Chris McHenry

A Multidisciplinary Creative; producing and deploying engaging content that converts. Creative duties include Animated Video Production, Medical Visualisation, Augmented Reality Design, Social Media Content Curation and Marketing Strategy optimisation.

Get In Touch

[contact-form-7 404 "Not Found"]

Popular Posts

Ways to check your set up is correct

How can you and your team build and have confidence in the data generated? This section covers a few ways to give confidence that your data is the best it can be, and if you’ve followed everything above, it certainly should be!

Control Your Variables

This section delves into the nitty gritty of ensuring that your assay setup is optimised before you start, by looking at the role of start-ups, chip conditioning and effects certain buffers can have on your assays.

What’s your Question?

To avoid wasting time and money, it is critical to start with the end in mind, and focus on clearly deciding what question you’re looking to answer. Do you want to know the kinetics of a single interaction or against a large panel of antibodies? Do you just want a yes / no screen of a panel of antibodies or targets? Do you want to know the affinity of an interaction but don’t mind if you don’t know the kinetics? Do you want to toggle-switch epitope select antibodies?… the list is endless!

Effector cell considerations

Redirection of cytotoxic effector T cells is the primary mechanism of action for CD3-engaging bispecific molecules. So choosing the right effector cell preparation is pretty important! Our last TMC blog goes through what preparations you can use, choosing the right cell source and how you can monitor your cells through your assay.

Key Materials And Reagents

One of the easiest ways to make beautiful assays is to ensure that you have effectively considered what sensor chips, buffers and other reagents are right for the job. we hope that in this post, you will find some helpful advice which help you to accelerate your assay preparation and overall project timelines.

Know Your Protein

As SPR is generally used for assessing binding to proteins in one form or another it’s a good idea to ensure that you have as much information about the proteins you’re assessing as possible.

Already have a project in mind? – Contact out team to get started

Relevant Posts

No Results Found

The page you requested could not be found. Try refining your search, or use the navigation above to locate the post.

No Results Found

The page you requested could not be found. Try refining your search, or use the navigation above to locate the post.

No Results Found

The page you requested could not be found. Try refining your search, or use the navigation above to locate the post.

Already have a project in mind? – Contact out team to get started

Contact Us
close slider

Product of Interest