App note

Checking the Activity of Protein Reference Materials

A concentration measurement tells you how much protein is in the vial. SPR tells you whether it still binds its partner, and how strongly.

SystemP4PRO with AffiPump

ApplicationReagent and reference-material QC

SamplePurified recombinant proteins

In short: The National Research Council Canada produced two reference proteins for SARS-CoV-2 serology tests: human ACE2 and the Omicron BA.4/5 spike. They used Affinité SPR as the functional check. The spike bound ACE2 with the affinity expected from the literature, which confirmed that both materials are active.

The need

Serology tests from different makers use different antigens and antibodies, so their results are hard to compare. Shared reference materials fix that, but only if each vial holds a protein that is pure, at a known concentration, stable in storage, and still able to bind. The first three can be measured by chromatography, mass spectrometry and UV absorbance. The last one needs a binding measurement.

The same question comes up in every lab that buys or makes proteins: a new lot of antigen, an antibody from a new supplier, a protein after six months in the freezer. Is it still active?

How it works

The ACE2 protein carries one biotin at a single site. On a biotin sensor with a streptavidin layer, every ACE2 molecule is captured the same way, with its binding site facing out. The spike protein then flows over the surface at increasing concentrations, and the sensorgrams give the rates of binding and release, and from them the affinity (KD) (Figure 1).

Biotin sensor, streptavidin layer, oriented biotinylated ACE2, and spike protein binding — Figure 1. Oriented capture on a biotin sensor: streptavidin layer, biotinylated ACE2, then the spike protein binding and releasing as it flows past.

What the study showed

	Result
Omicron BA.4/5 spike reference material on ACE2	KD ≈ 4.4 nM, consistent with published values: the material is active
Compared with the original (ancestral) spike reference material	About 4 times tighter binding
Where the difference comes from	Mostly a slower release from ACE2, not a faster binding

That last point is why kinetics matter for QC. Two proteins can look similar in an end-point assay and still behave differently: here the Omicron spike stays bound more than twice as long (Figure 2).

Simulated sensorgrams of the Omicron and original spike proteins on ACE2 at the same concentration — Figure 2. The two spike proteins at the same concentration, simulated from the rate constants reported in the paper. Both bind at a similar rate; the Omicron spike releases more slowly, so more of it stays bound.

Full method and data: Stocks et al., Analytical and Bioanalytical Chemistry 2024 (reference below).

Why Affinité SPR fits protein QC

What a QC lab needs	What Affinité SPR brings
Proof of activity, not just quantity	Direct binding to the protein's real partner, label-free
A number to compare lots	Rates and KD, so a change in binding or release shows up even when an end-point signal does not
The same surface every time	Biotin Sensor and Streptavidin Kit for oriented, single-point capture of biotinylated proteins
Measurements in your own lab	Benchtop flow system: the P4PRO with the AffiPump
A reference in every run	Several sensing channels, so a reference channel runs alongside the sample

Beyond reference materials

The same check works for any protein with a known partner: antigen lots for an immunoassay, antibodies from a new supplier, in-house recombinant proteins after purification or storage. For antibodies, see also our note on rapid quality control of antibodies. For planning the experiment itself, see TN-07, designing a binding experiment.

What you need

Item	Source
P4PRO with AffiPump	Affinité Instruments
Biotin Sensor (SAMBT-01) and Streptavidin Kit (STRP-KIT)	Affinité Instruments
Biotinylated ligand (here: ACE2, biotinylated at one site)	Your protein, or a commercial biotinylated version
Analyte in a dilution series	The protein lot you want to check
Running buffer with surfactant; BSA to block the surface	Standard reagents

Reference

B. B. Stocks, M.-P. Thibeault, D. L'Abbé, M. Umer, Y. Liu, M. Stuible, Y. Durocher, J. E. Melanson, "Characterization of biotinylated human ACE2 and SARS-CoV-2 Omicron BA.4/5 spike protein reference materials," Analytical and Bioanalytical Chemistry, vol. 416, pp. 4861–4872, 2024. doi:10.1007/s00216-024-05413-7 (open access, CC BY 4.0).

Illustrations by Affinité Instruments. Figure 2 is simulated from the rate constants in the reference above, not measured data.