Antibody microarray
Encyclopedia
An antibody microarray is a specific form of protein microarray
s, 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 expression
s from cell lysates in general research and special biomarkers from serum
or urine
for diagnostic applications.
.
The first antibody array used for protein-protein interaction and protein post-translational modification analysis in mammalian cells was reported in 2000 by Chin and colleagues. The first array approaches attempted to miniaturize biochemical and immunobiological assays usually performed in 96-well microtiter plates. 96-well antibody arrays were first created with 144 elements each for "standard enzyme-linked immunosorbent assays" (ELISA
). Similar arrays were used to measure prostate-specific antigen (PSA) and cytokines.
Filter membranes were also initially used because of their superior protein binding capacity. They were mostly probed with antibodies using ELISA techniques. A low density array of 48 purified proteins involved in transcription was developed for the investigation of specific interactions of proteins with radiolabeled DNA, RNA, ligands, and other small chemicals. A membrane-based high density array was developed for the purpose of screening a human fetal brain cDNA expression library consisting of 37830 clones. Purified proteins were spotted onto PVDF membranes at a density of 300 samples/cm2. Other filter based arrays were constructed but the limitations were the low resolution and considerable background making it difficult to use them in applications with limiting sample quantities such as protein expression profiling of tumor biopsies. In the last ten years the sensitivity of the method was improved by an optimsation of the surface chemistry as well as dedicated protocols for their chemical labeling. Nowadays, the sensitivity of antibody microarrays is in the range of ELISA. Small array sizes often make use of a sandwich approach with a second set of analyte specific antibodies. For more complex arrays, usually only one set of highly specific antibodies is used and the protein samples are labelled directly by fluorescent dyes or haptens.
Nowadays, antibody microarrays are used for profiling experiments on tissue samples, plasma or serum samples and many other sample types. One main focus in antibody microarray based profiling studies is cancer . For cancer-related research the development and application of an antibody microarray comprising 810 different cancer-related antibodies was reported in 2010 .
Protein microarray
A protein microarray, sometimes referred to as a protein binding microarray,provides a multiplex approach to identify protein–protein interactions, to identify the substrates of protein kinases, to identify transcription factor protein-activation, or to identify the targets of biologically active...
s, 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 expression
Protein expression
Protein expression is a subcomponent of gene expression. It consists of the stages after DNA has been translated into polypeptide chains, which are ultimately folded into proteins...
s from cell lysates in general research and special biomarkers from serum
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...
or urine
Urine
Urine is a typically sterile liquid by-product of the body that is secreted by the kidneys through a process called urination and excreted through the urethra. Cellular metabolism generates numerous by-products, many rich in nitrogen, that require elimination from the bloodstream...
for diagnostic applications.
Background
The theoretical background for protein microarray-based ligand binding assays was initially developed by Ekins et al. in the late 1980s. According to the model, antibody microarrays not only would permit simultaneous screening of an analyte panel, but would also be more sensitive and rapid than conventional screening methods. Interest in screening large protein sets only arose as a result of the achievements in genomics by DNA microarrays and the Human Genome ProjectHuman Genome Project
The Human Genome Project is an international scientific research project with a primary goal of determining the sequence of chemical base pairs which make up DNA, and of identifying and mapping the approximately 20,000–25,000 genes of the human genome from both a physical and functional...
.
The first antibody array used for protein-protein interaction and protein post-translational modification analysis in mammalian cells was reported in 2000 by Chin and colleagues. The first array approaches attempted to miniaturize biochemical and immunobiological assays usually performed in 96-well microtiter plates. 96-well antibody arrays were first created with 144 elements each for "standard enzyme-linked immunosorbent assays" (ELISA
ELISA
Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
). Similar arrays were used to measure prostate-specific antigen (PSA) and cytokines.
Filter membranes were also initially used because of their superior protein binding capacity. They were mostly probed with antibodies using ELISA techniques. A low density array of 48 purified proteins involved in transcription was developed for the investigation of specific interactions of proteins with radiolabeled DNA, RNA, ligands, and other small chemicals. A membrane-based high density array was developed for the purpose of screening a human fetal brain cDNA expression library consisting of 37830 clones. Purified proteins were spotted onto PVDF membranes at a density of 300 samples/cm2. Other filter based arrays were constructed but the limitations were the low resolution and considerable background making it difficult to use them in applications with limiting sample quantities such as protein expression profiling of tumor biopsies. In the last ten years the sensitivity of the method was improved by an optimsation of the surface chemistry as well as dedicated protocols for their chemical labeling. Nowadays, the sensitivity of antibody microarrays is in the range of ELISA. Small array sizes often make use of a sandwich approach with a second set of analyte specific antibodies. For more complex arrays, usually only one set of highly specific antibodies is used and the protein samples are labelled directly by fluorescent dyes or haptens.
Nowadays, antibody microarrays are used for profiling experiments on tissue samples, plasma or serum samples and many other sample types. One main focus in antibody microarray based profiling studies is cancer . For cancer-related research the development and application of an antibody microarray comprising 810 different cancer-related antibodies was reported in 2010 .
See also
- ELISAELISAEnzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
- protein microarrayProtein microarrayA protein microarray, sometimes referred to as a protein binding microarray,provides a multiplex approach to identify protein–protein interactions, to identify the substrates of protein kinases, to identify transcription factor protein-activation, or to identify the targets of biologically active...
- DNA microarrayDNA microarrayA DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome...
- tissue microarrayTissue microarrayTissue microarrays consist of paraffin blocks in which up to 1000 separate tissue cores are assembled in array fashion to allow multiplex histological analysis.-History:...
- chemical compound microarrayChemical Compound MicroarrayA chemical compound microarray is a collection of organic chemical compounds spotted on a solid surface, such as glass and plastic. This microarray format is very similar to DNA microarray, protein microarray and antibody microarray...