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 enzymelinked 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

Secondary Ab Labeled with Cy3/Cy5 Biotin/digoxygenin

Matched pair of Ab

Analyte

Abs Protein

Ab

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.

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

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 50200 mg of cells or whole tissue/body fluids 2. 3. 4. 5. labeling protein with Cy5 and Cy3 fluorescent dyes, removing unbound dye by gel filtration, Incubating labeled protein with Ab Microarrays, 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 enzymelinked 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