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Volume: 11 Issue: 6 December 2013 - Supplement - 2

FULL TEXT

ORAL PRESENTATION
Rapid Biosensor For Testing Acute Rejection After Liver Transplantation

Introduction: Neutrophil activation is very important response to organ transplantation. Activation process may cause acute organ rejection. Rapid monitoring of inflammatory cells with membrane markers is an indicator for activation. Flow cytometry is current laboratory tool for testing activation. Sample preparation and measurement is a long, expensive and labor intensive method. Objective of this study is to design and manufacture alternative assays for testing cellular response to organ transplantation.

Materials and Methods: Twenty QCM crystals that were 12 MHz used for analysis. The electrode surface was pretreated sequentially with pure acetone, pure methanol and 0.5 M NaOH (30 minutes for each step) to obtain a clean, silver surface. After the pretreatment, the crystals were rinsed with deionised, distilled water in an ultrasonic washer and air-dried. cystamine molecule has two functional groups: the SH group is called a thiol, and the NH group is called an amine. With this property, the molecule was attached to the crystal surface by the thiol group. Glutaraldehyde was used as a spacer arm and has two functional groups. The amine groups on the cystamine molecules and aldehyde groups on the glutaraldehyde molecules were reacted and covalently bonded. During the immobilisation of ligands, the active regions of the ligand molecules participate in specific and rapid interactions with the analyte molecules. The spacer arm prevents steric interference during the interaction between the analyte and ligand molecules. A blood sample from a healthy donor was collected, and leucocytes were prepared at four different concentrations for testing the biosensor in vitro. In total, four different samples were prepared to test the biosensor in vitro. To prepare diluted samples of leucocytes, blood was centrifuged at 3000 rpm, and leucocytes were collected from the buffy coat and diluted to concentrations of 50%, 35%, or 10% in PBS.

Results: At the highest concentration of neutrophils the frequency value was much lower than that at the lowest concentration of neutrophils. This trend may occur because the activation of leukocytes is increased in samples with higher concentrations of neutrophils. AFM measurements of these samples showed that the antibodies were immobilised on the surface.

Conclusions: Neutrophils play an essential role in the inflammatory response. A rapid test for neutrophil activation would be a useful tool for acute rejection screening. The testing of the inflammatory response should be rapid, inexpensive and easy. In this study, surface modified QCM crystals were investigated for their use in measuring the inflammatory response. The results of this study suggested the use of frequency changes in surface-modified QCM crystals to measure the inflammatory response. The results presented here were supported by AFM surface topography measurements and SEM images.

References

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Volume : 11
Issue : 6
Pages : 45


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