Detection of multiple proteins in an antibody-based protein microarray system

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Abstract

A new antibody-based protein array assay is described. This assay combines the advantages of the specificity of enzyme-linked immunosorbent assays (ELISA), sensitivity of enhanced chemiluminescence (ECL) and high-throughput of microspot. In this system, the capture proteins, either antibodies or antigens are spotted onto membranes in an array format. Biological samples are then incubated with membranes. After antigens or antibodies in the samples bind to their corresponding targets and unbound proteins are washed away, the membranes are exposed to Horseradish Peroxidase (HRP)-conjugated antibody(ies). The signals are finally visualized with ECL system. Experiments demonstrate that multiple cytokines and antibodies can be simultaneously detected using this new approach. The procedure is so simple that no sophisticated equipment is required. The concept should be able to be extended to develop a high-throughput protein array system. Future applications of this new approach include direct protein expression profiling, immunological disease diagnostics and discovery of new biomarkers.

Introduction

With the complete sequence of human genome, we now should be able to analyze whole genomic expression. This will provide vital information on the coordinate regulation among many genes since almost all cell activities or phenotypes are the sum of a series of molecular and biochemical events interacting with each other in a complex and multifaceted fashion. However, a complete understanding of normal and disease states can never be obtained from genomic research alone. Although DNA is an information archive, proteins do almost all the work of the cell. Experimental evidence clearly shows a disparity between the relative expression levels of mRNA and their corresponding proteins (Gygi et al., 1999). More importantly, the protein-based analysis is able to study post-transcriptional control, post-translational modifications and protein–protein interaction. Therefore, the field of proteomics with an ultimate goal to assemble a complete library of all the proteins is becoming increasingly important Anderson et al., 2000, Legrain et al., 2000. Unlike the cDNA microarray technology, the methodology that allows detecting entire pool of proteins does not exist yet. Two systems, two-dimensional polyacylamide gel electrophoresis (2-D gel) coupled with mass spectrometry Emmert-Buck et al., 2000, Page et al., 1999, Haynes and Yates, 2000, Celis et al., 1999 and surface-enhanced laser desorption and ionization (SELDI) Kuwata et al., 1998, Bruenner et al., 1996 are currently being used in analysis of multiple protein expression. However, the requirement of sophisticated devices greatly limits their accessibility. In this study, I demonstrated that numerous proteins could be detected simultaneously and specifically using an ELISA-based protein array system. This method can be used to detect multiple secreted proteins and antibodies.

Section snippets

Materials and methods

Immunoglobulins (IgGs) and corresponding Horseradish Peroxidase (HRP)-conjugated monoclonal antibodies were purchased from several different companies as shown in Table 1. IgGs were prepared as stock solutions at a concentration of 4 mg/ml and diluted with TBS to 100 μg/ml as working solutions prior to experiments.

Pairs of antibodies against cytokines were obtained from BD PharMingen (San Diego, CA). Cytokines were purchased from Peprotech (Rochy Hill, NJ). Cytokines were prepared as stock

A simplified model: detection of HRP-conjugated antibodies

To simultaneously detect multiple proteins and antibodies, a microspot approach was developed. In this approach, capture proteins, either antibodies or antigens, are spotted onto a membrane. The membrane is then exposed to a sample containing protein of interest. The corresponding antigen binds to its cognate antibody spotted onto membrane and detected by a developing antibody.

As a first step, a simplified system was applied to test the feasibility of this assay. Various known specific

Discussion

The array format for global analysis of gene expression, mainly cDNA microarrays and DNA chip technology has revolutionized biological and medical research. Recently, the array format has been used in systematical analysis of protein–protein interaction (Emili and Cagney, 2000). Arrays have the advantage of being scalable, flexible and easy to perform. The nature of arrays allows a high-throughput screening using robotic, imaging, or analytical methods. However, no protein array method in the

Acknowledgements

This work was supported by NIH/NCI grant CA89273 (RPH) and ACS grant RPG-99-164-01-CNE (RPH). We would like to express our thanks for the support by the Helen Dyar King Fund at the Arizona Community Foundation for Cancer Research. We are grateful to Dr. Sampath Parthasarathy for providing sera in this study.

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