Biacore 8K – kinetic possibilities in all directions

GE Healthcare

BiacoreTM 8K ? kinetic possibilities in all directions

Veronica Fridh, Linnea Nygren Babol, Anita Larsson, and Olof Karlsson

GE Healthcare Bio-Sciences AB, SE-75184 Uppsala, Sweden

Introduction

Binding kinetic studies with a Biacore system is feasible over a wide dynamic range, making it possible to determine the binding kinetic rate constants for basically all biological interactions. The experimental setup for kinetic analysis in current Biacore systems is based on one sample concentration per cycle, multi-cycle kinetics (MCK), or analysis of all sample concentrations in one cycle, single-cycle kinetics (SCK). The latter has proved to be the preferred choice for many applications where no regeneration is available.

The emergence of Biacore 8K system opens up new possibilities for performing kinetic analysis. This eight needle, high-sensitivity surface plasmon resonance (SPR) system not only shortens time to results by up to eight-fold compared to single-needle systems, it also allows for kinetic analysis in parallel. For instance, the new 2D kinetics functionality offers detailed kinetics from only one sample cycle, thereby significantly reducing assay development time.

This poster presents the high flexibility in assay setup offered by Biacore 8K when determining binding rate constants while still obtaining similar results.

Biacore 8K

Serial setup: concentration series per channel

Channel 12345678

Channel 12345678

Biacore 8K

Parallel setup: concentration series per cycle

Cycle 9 Cycle 8 Cycle 7

Cycle 6 Cycle 5 Cycle 4 Cycle 3 Cycle 2 Cycle 1

Cycle 2 Cycle 1

Biacore 8K: binding kinetics approaches

Depending on the user's preferences and the circumstances of each assay, kinetic analysis can be performed in four different ways.

Multi-cycle kinetics (MCK)

?Suitable for many samples against one ligand ?Suitable when different ligands are to be immobilized

Cycle 9 Cycle 8 Cycle 7 Cycle 6

Cycle 5 Cycle 4 Cycle 3 Cycle 2 Cycle 1

Relative response (RU)

Channel 1

45 40 35 30 25 20 15 10

5 0

-50

0

50 100 150 200 250 300 350 400 450 500

Time (s)

Ex. Cycles 1-9: sample concentrations and blanks are placed per channel

1 ? 107

Anti 2-microglobulin ? 2-microglobulin

MCK Parallel kinetics

SCK 2D kinetics

Single-cycle kinetics (SCK)

?Fast run time ?No regeneration needed

?2?5 concentrations per injection

?Beneficial for long dissociation times and kinetic screen

Cycle 2 Cycle 1

Relative response (RU)

40 35 30 25 20 15 10

5 0

0

Cycle 2

200

400

600

800 1000 1200 1400

Time (s)

Ex. Cycle 2: 5? sample conc. (Cycle 1: 5? blank conc.)

Log ka (M-1 s-1)

Parallel kinetics

?Short run time for few samples ?Kinetic analysis in only two cycles (one blank cycle) ?Beneficial for samples with long dissociation times ?Alternative setup: two samples, four concentrations

Cycle 2 Cycle 1

Relative response (RU)

Cycle 2

40 35 30 25 20 15 10

5 0

-50

0

50 100 150 200 250 300 350 400 450 500

Time (s)

Ex. Cycle 2: sample in 8 concentrations (Cycle 1: blank cycle)

1 ? 106

1 ? 105 1 ? 10-4

1 ? 10-3 Log kd (s-1)

1 ? 10-2

2D kinetics

?In-depth analysis in only one sample cycle ?Sample diluted directly into plate ?Sample diluted in two dimensions to cover a wide range ?No preknowledge of affinity or regeneration needed

Cycle 2 Cycle 1

Relative response (RU)

Cycle 2

100 90 80 70 60 50 40 30 20 10 0

-50 0

50 100 150 200 250 300 350 400 Time (s)

Ex. Cycle 2: sample in 24 concentrations (Cycle 1: blank cycle)

Data evaluation

?Evaluation of kinetic data from all of the above assay setups is supported in Biacore 8K Evaluation Software.

?Curves are grouped either in Serial or Parallel/2D mode prior to curve fitting.

Conclusion

?Biacore 8K supports several different approaches for performing kinetic studies, depending on analysis demand and/or preference.

?Provides flexibility in the assay setup depending on number of ligands, samples, run time, etc.

?New 2D kinetics allows for detailed kinetic analysis of an unknown interaction in a single sample cycle with minimal assay development.

?Biacore 8K allows confident determination of kinetics over an extensive kinetic range; ka 103 to 107 M-1 s-1 (109 for proteins), and kd 10-6 to 0.5 s-1.

GE, GE monogram, and Biacore are trademarks of General Electric Company. ? 2016 General Electric Company. First published Jun. 2016. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare Bio-Sciences Corp., 100 Results Way, Marlborough, MA 01752, USA. For local office contact information, visit contact

29213852 AA 06/2016

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