Antibody microarray

Samples of antibody microarray creations and detections.

An antibody microarray (also known as antibody array) is a specific form of protein microarray, a collection of capture antibodies spotted and fixed on a solid surface such as glass, plastic or silicon chip, for the purpose of detecting antigens. Antibody microarrays are often used for detecting protein expressions from cell lysates in general research and special biomarkers from serum or urine for diagnostic applications.^[1][2]

Background

The concept and methodology of antibody microarrays were first introduced by Tse Wen Chang in 1983 in a scientific publication^[3] and a series of patents,^[4][5][6] when he was working in Centocor, Inc. in Malvern, Pennsylvania. Chang coined the term “antibody matrix” and discussed “array” arrangement of minute antibody spots on small glass or plastic surfaces. He demonstrated that a 10×10 (100 in total) and 20×20 (400 in total) grid of antibody spots could be placed on a 1×1 cm surface. He also estimated that if an antibody is coated at a 10 μg/mL concentration, which is optimal for most antibodies, 1 mg of antibody can make 2,000,000 dots of 0.25 mm diameter. Chang's invention focused on the employment of antibody microarrays for the detection and quantification of cells bearing certain surface antigens, such as CD antigens and HLA allotypic antigens, particulate antigens, such as viruses and bacteria, and soluble antigens. The principle of "one sample application, multiple determinations", assay configuration, and mechanics for placing absorbent dots described in the paper and patents should be generally applicable to different kinds of microarrays. When Tse Wen Chang and Nancy T. Chang were setting up Tanox, Inc. in Houston, Texas in 1986, they purchased the rights on the antibody matrix patents from Centocor as part of the technology base to build their new startup. Their first product in development was an assay, termed “immunosorbent cytometry”,^[7] which could be employed to monitor the immune status, i.e., the concentrations and ratios of CD3⁺, CD4⁺, and CD8⁺ T cells, in the blood of HIV-infected individuals.

The theoretical background for protein microarray-based ligand binding assays was further developed by Roger Ekins and colleagues in the late 1980s.^[8][9][10] 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 Project.

The first antibody array used for protein-protein interaction and protein post-translational modification analysis in mammalian cells was reported in 2000 by Eugene Chin and colleagues.^[11][12] 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.^[13] Nowadays, the sensitivity of antibody microarrays is in the range of ELISA.^[13][14] 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.^[15] For cancer-related research the development and application of an antibody microarray comprising 810 different cancer-related antibodies was reported in 2010.^[16]

See also

• ELISA
• protein microarray
• DNA microarray
• tissue microarray
• chemical compound microarray

References