What is Antibody-Array

Antibody array (also known as antibody microarrays) is one of the most versatile approaches within multiplexed immunoassay technologies. Antibody array presents significant advantages compared with other traditional methods of protein analysis, which is widely used ina variety of research or disease diagnosis such as disease marker discovery for diagnosis, prognosis, and drug response, characterization of signaling and protein pathways, etc.

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Introduction of Antibody Array

Antibody array is a method by immobilizing antibodies for a parallel analysis of multiple targets in a given sample. Besides common antibodies, various antibody formats have been used in arrays, such as nanobodies, single-chain variable fragments (scFvs) and fragment antigen-binding (Fab)-fragments. In addition, other technologies such as phage display and ribosome display, combined with advanced materials and bioinformatics development also facilitate the development of antibody array in recent years.

The typical workflow of an antibody array includes several steps. Simply, antibodies are primarily immobilized onto a chemically functionalized or otherwise modified surface. Once blocking the reactive groups of the surface, a sample containing targeted proteins is incubated on the array, and the proteins are captured by the antibodies. The binding results are reported directly by fluorescent labelling of the sample or by the addition of a secondary detection reagent.

Antibody arrays can be used to study a diverse number of biological processes such as protein-protein interactions, signal pathway analysis, studies of post-translation modifications, and detection of toxins. During clinical context, arrays have been used to identify novel disease biomarkers as well as generating unique proteome signature by comparing healthy and disease states. This information provides better disease management through improved diagnostics and the ability to track disease status and therapeutic efficacy.

Antibody arrays have significant advantages compared to traditional, single analyte methods of protein analysis (such as ELISA and WB). The method has become more standardized and user-friendly experimental procedures since it is high throughput, high sensitivity, and requires small sample volume. Besides, the process of antibody microarray assays is fast and takes less than 24 h from sample preparation to data interpretation compared with mainstream proteomics strategies, especially mass spectrometry (MS).

Theoretically, antibody microarrays can be designed to host a few to thousands, or even ten-thousands, of features. The array can be constructed to host multiple features for each sample, or it can be designed to divide the array into arrays that allow multiple samples to be studied at the same time. Importantly, antibody arrays can produce high dimensional data using the well-established DNA microarray software, since the images scanned from planar antibody microarrays are similar to DNA microarrays.

Antibody arrays are most often used during the biomarker discovery process. However, the cross-reactivity to non-specific proteins is still a challenge for the single binder version of the assay. Thus, uncontrolled reactions with other target proteins display one of the main obstacles in establishing high performance and high specificity assays.

Fig.1 Review of planar antibody microarray technologies and their applications in the field of proteomics. (Chen, 2018)

Applications of Antibody Array

· Signalling Pathway Related Research

Change of signaling pathways represents a hallmark of many disease conditions, including cancer, diabetes and neurodegenerative disorders. Using antibody arrays has helped researchers to study changes in protein profiles and modifications in signaling pathways in normal biological processes and disease states through capture reagents targeting proteins in the signaling cascade of interest. A range of biological sample materials can be used in antibody array, including cell lysates, tissue extracts, and plasma.

· Drug Mechanism Research

Antibody array assays are also used to study drug mechanisms in a systematic and efficient manner. Among many possible applications, tumor-induced angiogenesis has been studied since it plays a critical role in cancer progression. For example, P11 as a novel peptide ligand with high affinity to integrin αvβ3 has been identified from a hexapeptide library (PS-SPCL) using an antibody array. The pharmacological mechanism of P11 was confirmed using a specifically designed antibody array assay containing 48 cancer-related antibodies. Results showed that P11 inhibits bFGF-induced human umbilical vein endothelial cell proliferation via mitogen-activated protein kinase and extracellular-signal regulated kinase inhibition. Additionally, the antibody array also helps to reveal that P11 can cause the upregulation of apoptotic marker p53, leading to apoptosis induction.

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