ProteOn XPR36, User Manual

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ProteOn XPR36 System | Analysis
128
When you select Langmuir, you can choose whether to analyze both the
on-rate (k
a
) and off-rate (k
d
) or to analyze just the off-rate:
• Langmuir mass transfer — simple 1:1 biomolecular interaction,
but where analyte diffusion to the surface is slower than the
interaction. Mass transfer (or transport) is the process of an analyte
diffusing from the bulk solution to the biosensor chip surface.
Because binding of the analyte by the ligand involves both diffusion
of the molecules from the bulk solution and attraction between the
two molecules, it is necessary to measure the binding kinetics and
not the rate of diffusion. Mass transport limited interactions occur
when the analyte binds to the ligand on the chip surface faster than
it can diffuse from the bulk solution. Therefore, the flow of the
analyte should be sufficient to eliminate mass transfer from the
equation. However, if it is not, this model can be used to correct the
data. This model is helpful when diffusion is the rate limiting step.
This can be detected by injecting a certain analyte concentration at
different flow rates. If the binding curves are dependent on the flow
rate, then mass transfer is limiting, and this model should be
applied. In contrast, if the binding curve is independent of the flow
rate (all binding curves overlay), then the diffusion is not limiting,
and the simple Langmuir model can be applied
• Bivalent analyte — interaction where the analyte has two binding
sites for the ligand. When an analyte, like an antibody, has two
binding sites, this yields two separate binding events. The first
event will yield traditional 1:1 kinetic fits where the second binding
event will cause the ligand-analyte complex to stabilize, thus
changing the kinetics of the reaction. Therefore, the bivalent
analyte sensorgram is the sum of two different kinetic fits. This
model is used when the analyte has two binding sites
• Heterogeneous analyte — interaction where two analytes
compete for a single ligand site. When an analyte is heterogeneous
it may physically bind to the ligand in two different locations, or it
may have two different affinities for the same epitope. This can
occur naturally or through modifications of the analyte. Whichever
happens, the sensorgram data associated with it changes. In the
former, the sensorgram comprises the sum of the two interactions.