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TECHNIQUES

The project utilises a range of analytical techniques to bring new light to diagnosing GvHD. Genomics will be utilised to develop the prognostic index; proteomics to identify the potential early biomarkers and differentiate between GvHD and RA. The biochip development will utilise proprietary technology developed by Orla Technologies.

Genomics
SNP Analysis (Single Nucleotide Polymorphism)

Single nucleotide polymorphisms (SNPs) are DNA sequence variations that occur when a single nucleotide (A,T,C,or G) in the genome sequence is changed, which occur approximately once every 100 to 300 bases. There are many techniques for SNP detection and genotyping, such as restriction fragment length polymorphism PCR (RFLP-PCR), SSCP, allele specific hybridization, primer extension, allele specific oligonucleotide ligation, sequencing.

ELISA ( Enzyme-Linked Immunosorbent Assay)

ELISA is a simple and highly sensitive method of analysis that allows for simultaneous and rapid quantification of a large number of samples. The assay is based on the specific recognition of the target compound (analyte / antigen) by antibodies which bind to the compound. The antigen-antibody complex is detected and measured with the aid of an enzyme-labelled antibody or antigen. Upon addition of a non-coloured reagent the enzyme produces a colour reaction where the colour intensity is directly or inversely proportional to the concentration of the analyte in the sample.

Proteomics

Protein separation can be performed using two-dimensional gel electrophoresis, which usually separates proteins first by isoelectric point and then by molecular weight. Protein spots in a gel can be visualized using a variety of chemical stains or fluorescent markers. Proteins can often be quantified by the intensity of their stain. Once proteins are separated and quantified, they can be identified. Individual spots are cut out of the gel and cleaved into peptides with proteolytic enzymes. These peptides can then be identified by mass spectrometry, specifically matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. In this procedure, a peptide is placed on a matrix, which causes the peptide to form crystals. Then the peptide on the matrix is ionized with a laser beam and an increase in voltage at the matrix is used to shoot the ions toward a detector in which the time it takes an ion to reach the detector depends on its mass. The higher the mass, the longer the time of flight of the ion. In a MALDI-TOF mass spectrometer, the ions can also be deflected with an electrostatic reflector that also focuses the ion beam. Thus, the masses of the ions reaching the second detector can be determined with high precision and these masses can reveal the exact chemical compositions of the peptides, and therefore their identities.

Protein Biochip

“Protein biochip” usually refers to the protein microarray, which has an orderly arrangement of many different proteins, such as antibodies, whose coordinates on the chip are known. Those proteins serve as capture agents for selected proteins in a sample. The captured proteins are made detectable either by labelling them directly with a fluorophore or, in the case of antibody chips, by adding a layer of labelled antibodies to make a sandwich. The position at which a protein binds reveals the type of antibody to which it binds and therefore its functional identity. The strength of the signal from that location indicates the protein’s concentration. Antibody microarrays have the advantage of high throughput—in theory, they could simultaneously analyze thousands of proteins in a drop of sample, such as serum.

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