ORIENTED IMMOBILIZED ANTIBODIES IN SERS-BASED

IMMUNOASSAY AGAINST BOVINE LEUKEMIA VIRUS

Author: Julija Baniukevic, doctoral candidate of Nanotechnology Research Center,

Chemistry Faculty, Vilnius university, Lithuania

1st International Baku Forum

Outline

What are the immunosensors?

Research object

The ways to achieve the goal

Conclusions

Nanoprospectives

What are the immunosensors?

What are the immunosensors?

Ab + Ag ↔ AbAgSharma SK, Seghal N, Kumar A. Biomolecules for development of biosensors and their applications.

Current Applied Physics. 2003; 3: 307-316. Wang J. Analytical electrochemistry. 2nd ed. New York: Wiley-VCH: 2000; 52-54, 183-188

Antibodies and their structure

Bovine leukemia virus structure

Reverse transcriptase

gp51/gp30

RNA

p15

Integrase

Lipids layer

p24

Nermut MV, Hockley DJ. Comparative morphology and structural classification of retroviruses. Curr Top Microbiol Immunol 1996; 214: 1-22.

Antibody-antigen interaction

Karyakin AA, Presnova GV, Rubtsova MY, Egorov AM. Oriented immobilization of antibodies onto the gold surfaces via their native thiol groups. Anal Chem 2000; 72: 3805-3811

Immunosensor design

Immunosensors devices

Schematic illustration of SERS-based sandwich immunoassay for BLV gp51

detection

Capturing efficiency of antibody on MNP-Au and retained antigen binding

efficiency measured with HPLC.

SAM DTT-1 DTT-2 2-ME

Ca

ptu

ring

effic

ien

cy,%

0

20

40

60

80

100

120

140MNP-Au-Ab immobilization efficiencyMNP-Au-Ab-Ag binding efficiency

88,8 %92,7 % 95,3 %

11,4 %

59,2 %58,7 %

50,3 %

SERS spectra of A) BLV antigen gp51; B) Raman label and anti–gp51 labeled Au-NRs (TNB-labeled anti–gp51-Au-NRs) in

the sandwich immunoassay without antigen; C) Raman Raman label and anti–gp51 labeled Au-NRs (TNB-labeled anti–gp51-

Au-NRs) with 0.1 mg mL-1 of antigen in the sandwich immunoassay.

Raman shift, cm-1

SE

RS

in

ten

sit

y,

a.u

.

Why is it so important?

Bovine leukemia virus consequences

http://www.vetmed.ucdavis.edu/vetext/INF-DA/INF-DA_BovineLeukemia.html

Conclusions

The developed SERS-based immunoassay based on oriented immobilisation of antibodies on MNP-Au and magnetic separation after interaction with analyte presents an indirect, less time consuming and easy-to-use method for antigen detection.

A good linear correlation (R2=0.9983) between the SERS signal and antigen concentration was obtained within the range of 0-0.06 mg mL-

1 of gp51 concentration.

The sensitivity of the developed assay was investigated and the LOD and LOQ of the proposed method for gp51 detection were found to be 0.95 µg mL-1 and 3.14 µg mL-1, respectively.

Conclusions

This method was successfully applied for the detection of bovine leukemia virus antigen gp51 in a milk samples in a rapid, reliable and selective manner. Recoveries of the proposed method in real samples were in the range of 85.5 – 100 %.

It was found that better recovery was achieved by increasing the concentration of analyte. The proposed SERS-based immunoassay using MNP-Au can be applied for the detection of other analytes.

Enhancing public engagement with nanotechnology

NANOPINION, which launched on May 1, 2012, is a €2 million, 30 month project bringing together 17 partners from 11 countries. Investigation of opinion will use an innovative multi-stranded approach, focusing on dialogue. It will include surveys, social media, street labs, and events in public spaces such as science centres and shopping streets. It will provide access to new materials on the risks and benefits of nanotechnologies.

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Thank you for your attention

Thank you for your attention