DD7

Define Immunodetection?

A general term to describe the detection of a specific antigen using antibody binding

Based on the affinity of an antibody raised against a particular antigen

What techniques can be used in Immunodetection?

• Immunohistochemistry / immunocytochemistry

• [Western blotting]

• ELISA

• Flow cytometry

What does ELISA stand for?

Enzyme-Linked Immunosorbent Assay

What is ELISA used for?

Used to detect a specific antigen or antibody in a sample (qualitative or quantitative)

What enzymes commonly used for ELISA?

Alkaline phosphatase (AP), horseradish peroxidase (HRP)

What is the difference between ELISA and Sandwich ELISA

SELISA starts with a well incubated with a capture antibody

Same gets caught on antibody rest continues the same

Why use a sandwich ELISA?

No clean, low concentration samples

What can be detected by ELISA for cancers?

high levels of proteases can result in degradation of fibrin and these breakdown products can be detected by ELISA

Why is PSA measurable in early stage cancer?

early stage cancer get disorganisation of the vasculature and PSA can get into blood stream

What level of PSA may indicate cancer?

High (>4 ng/mL)

What does Immunocytochemistry work on?

Cultured cells

Describe Flow Cytometry?

• A method for detecting specific molecules on and within cells

• Using individual cells

• Also be used for sorting/isolating specific sub-populations of cells

What is Flow Cytometry important in?

immunophenotyping, diagnosis, cell sorting

How can Flow Cytometry used to identify antigens?

Incubating a cell population with antibodies fluorescently tagged to antigens of interest

What are the positives of Flow Cytometry?

Multiple antigens can be quantified simultaneously

Extra- and intracellular

Multiple wavelengths of light

Can be used for DNA and RNA as well

How can Flow Cytometry? be used in diagnosis?

flow cytometry on cells to determine the expression patterns of particular antigens and determine whether those are important in the disease states or not

What features do modern screening machines need

• High-throughput – hundreds of samples per hour

• Multiplex – multiple analytes quantified per tube/well

• Low volume

• Sensitive and accurate

Describe the method for Electrochemiluminescence

• Using antibodies to capture an analyte

• Biotin and streptavidin bind extremely strongly

• Binds to the magnet

• Ruthenium emits a photon and the luminescence is detected by a photomultiplier

• Brighter the more analyte you have

Describe the method for Luminex?

A multiplex immunoassay system

• Each type of bead is tagged with a different coloured fluorescent marker and captures a specific protein

• Detection antibody that has biotin bound

• Secondary antibody has a PE (phycoerythrin)

• Beads are read in a flow-based system or, if magnetic, in a layer at the bottom of a well

• One laser classifies the type of bead, the second quantifies the PE signal

What could a liquid biopsy test for?

• Circulating tumour cells

• Extracellular vesicles

• Cell free DNA

• Micro RNAs

What were limitations of cancer SEEK?

• Patients had known disease – should ideally be able to detect in general population at earlier stage

• Controls were healthy individuals – some disease states may generate false positives

Describe methylation as a cancer biomarker?

Generally, cfDNA is unmethylated

Methylation patterns change in tumours and are characteristic of tumour location

Cancer patients will have a mixture of methylation patterns from healthy and tumour cells

Can be used to identify cancer and it’s origin