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OWLS APPLICATION NOTES NO-007

LABEL-FREE IMMUNOSENSOR FOR OCHRATOXIN - A
using OPTICAL WAVEGUIDE LIGHTMODE SPECTROSCOPY (OWLS) detection

Abstract
Ochratoxins are toxic metabolites produced by Penicllium verrucosum and several Aspergillus species, and can be present in a wide range of food and feed commodities. Because of the persistence of aflatoxins in the food chain, exposure to the compound is a potential human health hazard. This has prompted adoption of regulatory limits in several countries which, in turn, implies the development of suitable validated and official anal ytical m ethods and rapi d screeni ng tests for cost-effecti ve food control on a l arge sc al e. OW LS offered a hi ghl y sens iti v e label -free method to develop imm obili zed antigenBSA conjugate based competitive immunosensor.

Application of OWLS sensors

Standard inhibition curve
of the immobilized antigen conjugate based competitive aflatoxin immunosensor

as competitive immunosensor for the detection of Ochratoxin A

Surface Chemistry of OWLS sensors
Amino functionalisation of waveguide surface by 3ami nopropyl tri ethoxysil ane Imm obi lis ati on of Oc hratoxin-BSA c onjugate (10µg/ml ) on the OW LS s ens or s urfac e b y gl utaral deh yde (2,5% ).

Competitive assay format
S t a n d a r d s a n d s a m p l e s w e r e m i xe d wi t h a n t i b o d y , i n c u b a t e d f o r a c e r t a i n p e r i o d a n d t h e m i xt u r e w a s injected into the O W LS system. During d e t e r m i n a t i o n , t o xi n p r e s e n t i n t h e s a m p l e c o m p e t e s for binding of the antibody to the toxin conjugate immobilised on the sensor surface. Upon incubation, only antibodies remaining in free form in the sample m i xt u r e b i n d t o t h e a n t i g e n s i m m o b i l i z e d o n t h e sensor surface. Thus, the amount of antibodies bound to the surface of the chip was inversely proportional to the Ochratoxin A content in the samples.

The sensitive detection range of the competitive d e t e c t i o n m e t h o d w a s b e t w e e n 0 . 5 - 1 0 n g m l - 1 wh e n m e a s u r i n g O c h r a t o xi n A .

Sample measurement
E xt r a c t i o n p r o c e d u r e
W eight 1 g of sample and add 10 mL of acetoni tril e/water (6:4; v/v) m i xture. (C ompl ete cereal grains should be grinded)Stirred for 5 min Decante and filtrate using a UF mem brane with 1 00 . 00 0 N MW L. (5 min centrifugation at 5000 rpm) Dilute the filtrate at 100 fold dilution with 100 fold dil uti on of acetoni tril e/water mi xture i n TR IS buffer.

Optimization of antiserum dilution
The antibody concentration employed is one of the parameters of key importance, because increasing t o xi n c o n c e n t r a t i o n s i n t h e s a m p l e r e s u l t i n l a r g e r decreases in the assay signal.

Com pari ng the res ults from OW LS m easurem ent to that obtained by ELISA method, the calculated regression coefficient (R2) for Ochratoxin A was 0.97. It can be stated that the competitive imm unosensor based on O W LS detection coul d be suitable for the quick determination of Ochratoxin level in grain samples.

References
1. Measuri ng c ycl es of di fferent dil uti ons of m onoclonal anti ochratoxin A antibody (1 - 0.01µg/ml, 2 - 0.1µg/ml, 3 0.25µg/ml, 4 - 0.5µg/ml, 5 - 1.0µg/ml, 6 – 2.5µg/ml, 7 – 5.0µg/ml, 8 - 10µg/ml, 9 –25µg/ml, 10 – 1.0µg/ml) Vörös, J. J. Ramsden, G. Csucs, I. Szendrõ, S.M. De Paul, M. Textor, N. D. Spencer (2002): Optical Grating Coupler Biosensors. Biomaterials 23 3699-3710. Adányi N ., Levkovets I.A., Rodri guez-Gi l S., Ronal d A., Váradi M. Szendrı I. (2006): Development of immunos ens or bas ed on OW LS t echni que f or determining Aflatoxin B1 and Ochratoxin A • Biosensors and Bioelectronics,22(6) 797-802. www.owls-sensors.com

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