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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. Choosing your sensor chip An important part of setting up an assay is to ensure that the correct chip is chosen before you start. Beginning with […]

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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.

Choosing your sensor chip

An important part of setting up an assay is to ensure that the correct chip is chosen before you start. Beginning with the wrong chip can lead to optimising an assay format that isn’t suitable. For most general antibody / protein assays, the CM5 sensor chip is commonly thought of as the industry standard, but it’s always important to assess your individual needs.

No matter which chip you choose to start with, if issues are found with non-specific binding, avidity or other artefacts then there a large variety of sensor chips are available to try.

Sensor chips and insights

C1

Carboxymethylated, matrix-free surface. Low binding capacity. Flat surface allows interactions to take place closer to surface.

CM3

Carboxymethylated, short dextran matrix surface with similar properties to sensor chip CM5. Allows the interaction to take place closer to the surface.

CM4

Carboxymethylated, dextran matrix surface like CM5 but with a reduced net negative charge. Well suited for use with negatively charged molecules.

CM5
Carboxymethylated dextran covalently attached to a gold surface. The ‘industry standard’ starting point.

PEG
PEG surface for analytes that demonstrate unwanted binding to dextran-based surfaces.

L1

Lipophilic groups covalently attached to carboxymethylated dextran. Suitable for direct attachment of lipid membrane vesicles such as liposomes.

Protein A

Carboxymethylated dextran matrix with a recombinant Protein A (PrismA) variant covalently attached. Binds only to the heavy chain within the Fc region, ensuring binding to the surface in a specific orientation. Does not bind Fab fragments.

Protein G

Carboxymethylated dextran matrix with a recombinant Protein G covalently attached. Binds only to the heavy chain within the Fc region,ensuring binding to the surface in a specific orientation.

Protein L

Carboxymethylated dextran matrix with a recombinant Protein L covalently attached. Binds to kappa light chain subtypes, without interfering with its antigen-binding site.

SA

Carboxymethylated dextran matrix with streptavidin covalently attached. Binds to biotinylated interaction partners. Controlled biotinylation enables orientated capture.

CAP

Carboxymethylated dextran matrix with DNA oligo covalently attached. Complementary DNA strand labelled with streptavidin allows reversible capture of biotinylated molecules in biomolecular interaction studies.

Buffer

The running buffer is of critical importance in SPR assays as it’s the carrier of your analyte and therefore, any composition, pH or other excipient issues can affect the interaction between the analyte and ligand. For me, a basic starting point would be to use buffer HBS-EP+. Typically I use the ‘+’ versions of buffers, as they prevent non-specific sticking to the Biacore’s micro-fluidics and tubing which can lead to inaccuracies in results.

From there it’s possible to change the buffer choice or add components to enhance the ligand binding or reduce non-specific binding for example with the addition of 0.1% BSA, which helps stop proteins getting stuck in all the wrong places, extra NaCl or carboxymethyl-dextran, which can also help decrease the likelihood of non-specific binding (as well as assessing the PEG sensor chip).

No matter what buffer is chosen, it’s vital that the buffer is filtered and degassed prior to use in the machine, as introducing micro bubbles of gas and dirt into the system leads to the system quickly degrading. As the running buffer is also to be used to dilute your analyte, I would advise you use high antibody concentrations to minimise any carry over of buffer excipient into your assay. This can ultimately lead to poor quality data.

Capture molecules

The beauty of SPR is that in addition to a multitude of sensor chip chemistries, you have access to an almost unlimited library of capture molecules. This freedom allows you to make assays that are orientation specific, leading to higher quality assays and better data that you can have confidence in.
Aside from the previously discussed affinity tags and their corresponding capture antibodies (anti-Fc, anti-Fab and anti His) there are also Fc-silenced versions, which can help when using Fc receptors as analytes. With a vast array of FAb and F(Ab’)2 fragments that can be used in your assay setup, it’s always best to have a look at what’s out there and maybe test a few different molecules prior to deciding on a specific one.

As an example of testing a variety of antibody capture methods, we recently tested 3 different ways of capturing an antibody:

  • Light chain specific
  • Heavy chain specific
  • Mixture

As you can see, the mixture and heavy chain specific show stable binding of the antibody whilst the light chain specific does not. It’s clear from the Sensorgrams that the heavy chain specific is the preferred choice for general use, as the mixture also contains the light chain specifc. It is always worth reassessing each capture molecule when swapping between antibodies and especially antibody classes in order to really understand what’s happening.

Temperature

SPR is a great tool for assessing interactions at different temperatures, with current machines allowing temperatures ranges of 4 – 40 °C. As with CM5 being the first port of call for sensor chips, you can pretty much guarantee that the standard ambient temperature of 25 °C will be used for the assay. It’s important to consider what information you want from the assay, and build this into your set up e.g. If you want biologically relevant data, then perhaps 37 °C is a better choice. Just be sure to stick to one temperature once you’ve made your choice as the kinetic rate constants change with temperature as do the absolute responses that you will observe.

Summary

Armed with the best choice of chip, the best capture molecule, optimised buffer and optimal temperature conditions, you’re setting your assay up for the best chance of success. With this, and all of the information I’m sure you have gleaned from the first article, you are ready to get started…Or are we? What question are you trying to answer with your assay?

Antibody Analytics Ltd.

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