OWLS APPLICATION NOTES NO-001

LABEL-FREE IMMUNOSENSOR FOR HSP70 DETECTION
Abstract

using OPTICAL WAVEGUIDE LIGHTMODE SPECTROSCOPY (OWLS) detection OWLS immunosensor offers a real time label-free detection of heat shock protein, HSP70 (molecular weight of 70 kDA). For biosensing monoclonal antibodies raised against HSP70 protein under investigation were used. To form regenerable sensor surface, the waveguide sensor surface was modified with amino group and sensitized by immobilizing antibody/antigen molecules covalently to the surface.

Application of OWLS sensors

as immunosensor for the detection of the heatshock protein HSP70

Calibration curve for HSP-70 sensor
12 Se nsor re sponse (a.u.) 10 8 6 4 2 0.0001 0.001 0.01 0.1 1 10 100

sample

HSP70 monoclonal antibody 100 - 150 nm

laser light EM field distribution

waveguide glass support

HSP70 conce ntration (ng/ml)

Surface Chemistry
• • Amino functionalisation of waveguide surface by (γ-aminopropyl)triethoxysilane Immobilisation of biomolecules on the amino surface of the OW LS sensors by glutaraldehyde.

Measurement of anti-HSP70 antibody

HSP70 molecules are covalently attached to the surface and different dilutions of anti-HSP70 a n t i b o d y we r e m e a s u r e d .
400 10 µ g/ml Sensor response (a.u.) 350 5 µ g/ml 300 250 200 150 1 µ g/ml 20 µ g/ml

Sensitization of the sensor surface
with monochlonal anti-HSP70 IgG

600
HCl HCl

500 Sensor response (a.u.) 400
Blocking

300 200
GA MoAb

100
TRIS

70

90

110 Time (min)

130

150

0 0 20 40 60 80 100 120 140 Time (min)

Conclusion Regeneration cycles of the measurement
Antibodies are covalently attached to the surface a n d l i f e t i m e o f s e n s o r s we r e e x a m i n e d a n d optimized. The OW LS Sensor is a very sensiti ve and costeffective tool for HSP70 and anti-HSP70 antibody detection. The lowest detectable amount of HSP70 is 1 pg/ml.

500
5 pg/ml 1 pg/ml 10 pg/ml 20 pg/ml

References
1.
Vörös, J. J. Ramsden, G. Csucs, I. Szendrõ, S . M . D e P a u l , M . T e xt o r , N . D . S p e n c e r “Optical Grating Coupler Biosensors” Biomaterials 23 (2002) 3699-3710 Levkovets I., Adányi N., Trummer N., Váradi M., Szendrı I., Starodub N.F., Székács A., “Developm ent of optical (O W LS) immunosensors for macromolecules and s m a l l a n a l y t e s ” , B I O K É M I A X XV I I I / 1 , 2 0 0 4 w w w . o wl s - s e n s o r s . c o m

Sensor response (a.u.) xx

450

400

350

2.

300

250 100 150 200 Time (min) 250 300

3.

MicroVacuum Ltd. H-1147, Kerékgyártó u. 10., Budapest, Hungary
Web: http://www.owls-sensors.com/; E-mail: info@owls-sensors.com

Phone: + 36 1 252 1991; +36 1 467 0108

Fax: +36 1 221 7996

Copyright @ Microvacuum, 2006

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