imagination at work

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30 35 40 45 50Temperature (°C)

kd

(s-1)

45°C

Response(RU)

Response(RU)

Time (s)

6

50

2

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14

10 30

Time (s)

Response(RU)

Time (s)

Response(RU)

Response(RU)

Response(RU)

Time (s)

5010 30

0

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14

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Response(RU)

Time (s)

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0 5e-6 1,5e-5 2,5e-5 3,5e-5

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18

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Time (s)

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5 25 45

2

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18

0 1e-5 2e-5 3e-5 4e-5Concentration (M)

Concentration (M)

Analysis Kinetic model Steady state modeltemperature (°C) KD (M) KD (M)

40 2.9 2.8

45 4.9 5.1

From

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Concentration (µg/ml)

Measurement 1Measurement 2

Response(RU)

Response(RU)

Response(RU)

20 25 30 35 40 45Temperature (°C)

ka

(M-1 s-1)

Response(RU)

Time (s)

From

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0

1×107

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3×107

4×107

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6×107

1

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0

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20°C

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3

0 200 400 600

Response(RU)

Time (s)

Biacore T100

Response(RU) Biacore 3000 Biacore T100 Biacore T200

-0.4

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-0.1

0

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1998 2005 2010

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Biacore T200

Response(RU)

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0 10 20 30 40 50 60 Time (s)

S NH2

O

O

CH3

0

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0 5e-4 1e-3 1.5e-3 2e-3

Response(RU)

Concentration (M)

KD = 4.9×10-4 M

Biacore™ T200: Sensitivity for confident analysis of the most challenging interactions

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Biacore is a trademark of GE Healthcare companies. All third party trademarks are the property of their respective owners.

© 2010 General Electric Company – All rights reserved.

All goods and services are sold subjects to the terms and conditions of sale of the company within GE Healthcare wich 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 AB, Björkgatan 30, 751 84 Uppsala, Sweden.

Ewa Pol, Åsa Frostell-Karlsson, Jos Buijs, GE Healthcare Bio-Sciences AB, 751 84 Uppsala, Sweden

IntroductionAdvances in label-free protein interaction technology by Biacore result in continuously improving sensitivity. Considerably lower responses are at present reliably evaluated, which brings numerous benefits discussed here.

Pushing detection limit to the edgeReliable interaction analysis of the smallest compoundsBinding of Methanesulfonamide, 95 Da, to Carbonic Anhydrase (7000 RU)

ConclusionsThe implications of confident analysis at extremely low response levels are:

• Extended kinetic range: ka ≤ 5×107 M-1 s-1 (for proteins: 3×109 M-1 s-1), kd ≤ 1 s-1

• No molecular size limit of detection

• Limit of quantification for concentration assays < 1 RU, corresponding to about 1 ng/ml antibody

• Higher throughput, as the injection time can be reduced

• Increased assay flexibility – consistently accurate characterization of

antibodies – more stable capture on NTA chip

The same kinetic analysis performed in Biacore T100 and in Biacore T200.

Baseline noise reduction throughout the instrument development.

Confident detection of low protein concentrationsConcentration assay of antibody Xolair® using Human Antibody Capture Kit (from GE Healthcare)• Quantification range: 2 ng/ml to 1 mg/ml (3 min injections)• Limit of detection calculated as a mean of 10 blanks + 3×SD• 2 ng/ml (~13 pM) of human antibody is detected

Extending kinetic space

References1. Karlsson, R. J. Mol. Recognit. 1999;12:285–292

2. Markova, N. et al. To be published

To examine the limits of association and dissociation rate constants in the high sensitivity instrument we analysed the interaction of the fast binding (Melagatran) and fast dissociating (Dansylamine) molecules at increasing temperatures.

Increased ka limitAccording to a relationship between the maximal binding response, Rmax, the mass transport constant, kt , and the association rate constant, ka (Ref. 1), low response levels are required to overcome mass transport limitation for very fast binding rates, especially for LMW compounds.

Rmax ≤

Binding of Melagatran to Thrombin at 20°C, 30°C, and 40°C (n=4). The ka values are plotted against temperature and error bars represent SD.

Higher concentrations, marked by arrows, have to be added to approach equilibrium when using the steady state model.

The affinities obtained from kinetic and steady state evaluations for the interactions at 40°C and 45°C are in excellent agreement. The benefit from fitting to the kinetic model is (in addition to resolving kinetic rate constants) that the use of higher concentrations can be avoided.

Expanding assay flexibilityAntibody characterization without avidity effectsA careful characterization of an antigen-antibody interaction is of great importance in the assessment of antibodies as therapeutic, analytical and diagnostic tools.

To avoid avidity, the antibody is usually immobilized on the sensor surface. However, in some situations, e.g., to save precious target, the immobilization of antigen in required instead. In this case, by using low antigen density surface, avidity effects can be prevented.

In our experiment, several surfaces with decreasing IgE densities were prepared and a concentration series of anti-IgE antibody was analysed at each surface. As the amount of target attached to dextran matrix (visualized as the maximal binding response, Rmax) is reduced, avidity effects decrease, and below Rmax ~5 RU, disappear.

4.0×10–9

6.0×10–9

8.0×10–9

1.0×10–8

1.2×10–8

1.00×105

1.25×105

1.50×105

1.75×105

1 10 Rmax (RU) 1 10 Rmax (RU)

Rmax (RU)1 10

5.0×10–4

1.0×10–3

1.5×10–3

2.0×10–3

0

1

100

Respons(RU)

Respons(RU)

Respons(RU)

Respons(RU)

200 300 400 500Time (s)

100

Respons(RU)

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100

Respons(RU)

Respons(RU)

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Respons(RU)

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AbAg

Ab(Ag)2Rmax= 80 RU

Rmax = 1 RU

ka (M-1 s-1)

kd (s-1) KD (M)

The changes of kd, ka, and KD accompanying a decrease in maximal binding response (Rmax) together with the corresponding sensorgram data sets. All sensorgram data is fitted to a 1:1 binding model.

Superimposed, normalized sensorgrams of a single antibody concentration at two surfaces of different antigen density. The sensorgram shape is clearly connected to the degree of avidity.

Stable His-capture to NTA chipHis-tag is the most widely used tag today and can be used to easily attach a His-tagged protein to NTA chip. A drift that may occur after capture injection may be avoided by using low capture levels.

His-tagged protein is titrated to stable binding level by reducing the injection time from 180 s to 30 s. At about 1000 RU the capture level becomes stable.

Kinetic profile of the binding of compound XAV939 (310 Da) to Tankyrase 1, captured on NTA chip to 760 RU (Ref. 2).

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XAV939Response

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8F

N

NO

S

FF

Increased kd limit

Binding of Dansylamine to Carbonic Anhydrase at 30°C, 35°C, 40°C, and 45°C (n=2). The kd values are plotted against temperature and error bars represent the SD values.

5 × kt

ka

Advantages:

• Homogeneous orientation of the proteins on the surface

• Immobilization under neutral conditions

• Increased sensitivity enabling stable capture and reliable kinetics on the NTA chip

• Low target consumption: 0.2 µg/compound

NTA

NTA chip

N

COO- CH2COO-

CH2COO- His

Ligand

HisHis

HisHis

Ni2+

Biacore