GE HEALTHCARE Biacore T100, Handbook

Page: 101 / 238

Biacore T100 Software Handbook BR-1006-48 Edition AE
101
Methods 5
Calibration-free
• At least one assay step is required with purpose Sample , connected to a
cycle type that includes one Sample injection with flow rate set to
Variable .
• Each sample must be injected at least twice using different flow rates.
• Evaluation variables D(20°C) , MW and Blank must be included for the
Sample injection.
5.10.2 Kinetics/Affinity
See Chapter 9 for a description of kinetics and affinity evaluation.
• At least one assay step is required with purpose Sample , connected to a
cycle type that includes one Sample command.
• Sample concentration must be specified in the variable Conc . If weight-
based units are used, a molecular weight for the analyte must be specified
in the variable
MW .
• As a recommendation for multi-cycle kinetics and affinity, there should be
a concentration series with at least four non-zero analyte concentrations
and one zero concentration. At least one of the non-zero concentrations
should be measured in duplicate. Although kinetic and affinity evaluation
can be applied to runs with fewer sensorgrams, the results will generally
be less reliable if these recommendations are not followed.
• Five sample concentrations are recommended for single-cycle kinetics
and affinity, injected in order of increasing concentration. Duplicate cycles
are recommended to ensure robust evaluation. For each determination,
there should be a blank cycle corresponding to the sample cycle, with
buffer replacing the sample for each injection.
5.10.3 Thermodynamics
See Chapter 10 for a description of thermodynamics evaluation.
Thermodynamics evaluation requires that kinetics or affinity (see Section 5.10.2)
is determined at two or more (recommended 5) temperatures.
5.10.4 Affinity in solution
See Chapter 11 for a description of affinity in solution evaluation.
• At least one assay step is required with purpose Calibration and one with
purpose Sample . Both assay steps must be connected to a cycle type that
includes one Sample command. The two assay steps will normally be
connected to the same cycle type.