• The theoretical background for protein microarray-based ligand binding assays was initially developed by Ekins et al in the late 1980s

• PMAs not only would permit simultaneous screening of an analyte panel, but would also be more sensitive and rapid than conventional screening methods

• 96-well antibody arrays were first created with 144 elements each for standard enzyme-linked immunosorbent assays (ELISAs)

• Various protein microarray platforms have been developed, not only for screening antibodies and antigens but also for discovery– of novel enzymes – enzymatic activities,– protein-ligand,– protein-nucleic acid and – protein-protein interactions

Assay Format

1. Label based– Direct– Indirect

2. Sandwich method

3. Other methods• Whole cell detection• Reverse phase

Direct labeling Indirect detection

Matched pair of AbAnalyte

Biotin/digoxygenin

Secondary AbLabeled with Cy3/Cy5

Cells bind to the array according to their expression of the targeted antigens, and the level of binding to each antibody spot is quantified by dark field microscopy.

Ab

The presence of particular proteins in the lysates are qunatified by incubation of antibodies targeting those proteins.

Protein

Abs

Three types of protein microarrays are currently used to study the biochemical activities of proteins:

• analytical microarrays,

• functional microarrays,

• and reverse phase microarrays.

Analytical MA Analytical microarrays are typically used to• profile a complex mixture of proteins in order

to measure binding affinities, specificities, & protein expression levels of the proteins in the

mixture.

• In this technique, a library of antibodies, aptamers, or affibodies is arrayed on a glass microscope slide.

• The array is then probed with a protein solution.

• Antibody microarrays are the most common analytical microarray

Antibody MA

Antibody Array technology is a powerful chip based technology for profiling hundreds of proteins simultaneously for expression analysis

• Profile hundreds of native proteins simultaneously

• Compare protein abundances in a variety of biological samples

• Obtain results in one day

Definition• An antibody microarray is a specific form of protein

microarrays, a collection of capture antibodies are spotted and fixed on a solid surface, such as glass, plastic and silicon chip for the purpose of detecting antigens.

• Antibody microarray is often used for detecting protein expressions from cell lysates in general research and special biomarkers from serum or urine for diagnostic applications.

Ab Microarrays• Protein profiling tool, • to identify variations in protein levels among

a variety of biological samples, including whole tissue.

Applications • toxicity testing, • disease investigation, and• drug discovery.

• Ab MA is a fluorescence-based analysis

• covalently immobilized antibodies are used to capture fluorescently labelled antigens.

• Ab Microarrays do not measure absolute concentrations.

• They provide a relative measure of protein abundance;

• The protein levels in one sample (Sample A) are compared to those of a second sample (Sample B)

The procedure consists of five main steps:1. extracting protein from 50–200 mg of cells or whole tissue/body fluids

2. labeling protein with Cy5 and Cy3 fluorescent dyes,

3. removing unbound dye by gel filtration,

4. Incubating labeled protein with Ab Microarrays,

5. scanning microarrays to measure bound antigen.

The entire procedure, from protein extraction to array scanning,takes just one day to complete.

Format and layout of Ab Microarrays. The layout shown is that for the Ab Microarray 380. Yellow spots correspond to Cy3/Cy5-labeled BSA spots, which serve as orientation markers.

96-well antibody arrays were first created with 144 elements each for standard enzyme-linked immunosorbent assays (ELISAs)

Similar arrays were used to measure PSA and cytokines

Micro-Array technology in Proteomics

Protein Array

•To Understand the cross-reactivity between allergenic components & to relate clinical significance for allergen avoidance and immunotherapy is important.

•Most studies on cross-reactivity however are limited to a small number of closely related allergens or patient population.

•A large dataset however may provide new insights to relationships between diverse groups of allergenic components.

•An immuno-microarray assay was developed for the rapid quantification of specific IgE to a combination of 190 crude allergens.

• Profiling humoral immune responses to P. falciparum infection with protein microarrays

• The protein MAs were probed with human sera • Information obtained from such analyses will

facilitate:– identifying antigens for vaccine development,– dissecting the molecular basis of immunity,– monitoring the outcome of whole-organism

vaccine trials, – and identifying immune correlates of protection.

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Diagnostic Imaging