IMMOBILIZATIONS OF ANTI JEV ANTIBODIES IN HUMAN SERUM FOR DIRECT IMMUNOSENSORS

IMMOBILIZATIONS OF ANTI-JEV ANTIBODIES IN HUMAN SERUM FOR DIRECT IMMUNOSENSORS Tran Quang Huy1,2, Phan Thi Nga1, Nguyen Thi Hong Hanh1, Mai Anh Tuan2 1 National Institute of Hygiene and

IMMOBILIZATIONS OF ANTI-JEV ANTIBODIES

IN HUMAN SERUM FOR DIRECT

IMMUNOSENSORS

Tran Quang Huy1,2, Phan Thi Nga1, Nguyen Thi Hong Hanh1, Mai Anh Tuan2

1) National Institute of Hygiene and Epidemiology (NIHE), N°1 – Yersin Str., Hanoi

2) International Training Institute for Materials Science (ITIMS), Hanoi University of

Technology (HUT), N°1 Dai Co Viet Road, Hanoi

INTRODUCTION

Mechanism: based on the

specific bio-chemical reactions:

A perspective of traditional methods

and biosensors

Based on the specific reaction of

antigen - antibody

Virus: envelop (poteins or

antigens) and capsid

(containing genetic

material: DNA or RNA)

Antibody: immunoglubulin molecule to recorgnize and block strange agents

(antigens of viruses, bacteria, etc)

1- Transducer (fabrication technology)

2 – Immobilization of sensitive biological elements (antibody)

IMMOBILIZATIONS

- Molecules may be immobilized either passively through:

Hydrophobic

Ionic interactions

Covalently by attachment to activated surface groups.

- The immobilization process should occur selectively in the presence of common functional groups, including amines, thiols, carboxylic acids, and alcohols.

Preparation of Surface for Antibody Immobilization:

• Modification of the electrodes surface to create functional groups.

• Modification of antibodies for covalent attachment to the surface.

• Cleaning the surface of electrode

• Modification of the surface to create fuctional groups by:

PREPARED MICRO-ELECTRODE SENSORS

Up-down sensors

CLEANING PROCEDURES

sensors

3-aminopropyl-triethoxy-silance (APTES)

Experimetal immobilizations of anti JEV serum

1 APTES – Antibody

- Immobilization with 1 mg/ml anti-JEV antibodies contained human serum for 1 hour

- Blocked with 2% BSA solution in PBS for 30 minutes.

- Washing with phosphate buffered saline (PBS) at pH 7.0 and air dried

2 APTES – Glutaraldehyde – Antibody

- The silanized sensor with 10% (v/v) aqueous glutaraldehyde solution for 30 min

- Immobilization with 1 mg/ml anti-JEV antibodies contained human serum for 1 hour

-Blocked with 2% BSA solution in PBS for 30 minutes.

- Washing with phosphate buffered saline (PBS) at pH 7.0 and air dried.

3 APTES – Glutaraldehyde – Protein A - Antibody

- The silanized sensor with 10% (v/v) aqueous glutaraldehyde solution for 30 min.

- 5µl protein A solution (1mg protein A/1ml PBS, pH 7.0) was added to the working

electrodes area for 30 min

- Immobilization with 1 mg/ml anti-JEV antibodies contained human serum for 1 hour.

- Blocked with 2% BSA solution in PBS for 30 minutes Washing with phosphate buffered saline (PBS) at pH 7.0 and air dried.

Covalent immobilisation of antibody using

glutaraldehyde 10%

RESULTS AND DISCUSSION

Biosensors after anti - JEV serum immobilization

Sensors are immobilized with antibodies Immunosensors are kept in tubes at 40C

(A) - The cleaned sensor surface (B) - The sensor surface coated with APTES only and directly immobilized with anti-JEV antibodies contained serum (1mg/ml in PBS pH 7.0) (C) - The sensor coated with APTES, added 10% glutaraldehyde (G.A) and then immobilized with anti-JEV antibodies contained serum (1mg/ml in PBS pH 7.0) (D) - The sensor coated with APTES, added 10% glutaraldehyde (G.A), then protein A (1mg/ml in PBS) and immobilized with anti-JEV antibodies contained serum (1mg/ml in PBS pH 7.0)

Morphology of the sensor surfaces treated with

different immobilization methods

[FE-SEM S-4800 (NIHE)]

FTIR spectra

FTIR spectra showing the stretching modes of chemical bonds on the sensor surface modified with different chemical

methods for antibody immobilization The region of 3600–3200 cm−1 is characteristic of free amino functional groups of

the surface treated with APTES only (Fig A) The vibration band for asymmetric Si-O-Si stretching mode can be seen in the range of 1100 – 1050 cm-1, and C- N stretching mode in 1300 – 1000 cm-1 However, the stretching modes are shifted slightly by different treatments on the sensor surface [Fig B, C, D]

[Nicolet 6700 FT-IR machine (HUT)]

JEV detection using immunosensors

The detection signal of the sensor using APTES – 10% glutaraldehyde for immobilization of anti-JEV antibodies contained human serum is 2- to 3- fold higher than other methods The response time of JEV antigen detection was about 5 minutes and got stable condition within 20 minutes after immersion of the immunosensor in JEV antigen solution, even it was 60-times diluted in comparison with the initial solution used for MAC ELISA test

[SR 830 Lock-In Amplifier (ITIMS)]

CONCLUSION

 Potential development of direct immunosensors using anti JEV serum

as probes for rapid JEV detection in outbreaks

 The signal obtained by using 3-aminopropyl-triethoxy-silance (APTES) and 10% glutaraldehyde to immobilize the antibodies was 2 to 3-fold

as higher as those obtained using different attachment methods

 The response time of JEV antigen detection occurred was 5 minutes and stable within 20 minutes after immersion of the immunosensor in the diluted solution of JEV antigen, 60 times in comparison with the initial solution used for MAC ELISA test

Four biosensors can be integrated on the chip

Development of Biosensors/Biochips at ITIMS

BIOSENSOR GROUP AT ITIMS

THANK YOU FOR YOUR

ATTENTION!