Immunoassays: Protein Arrays vs. ELISA and Westerns

IN Discussion Topics, ONCYTE nitrocellulose film slides, Product Applications, Reagents, Uncategorized

Studies of biological systems have expanded beyond the ‘one gene, one protein’ paradigm to the field of proteomics, or studying large numbers of proteins that act in a concert of complex biological systems. Thus in the post-genome era, technology has been driven to deliver multiplex assays that allow us to monitor multiple proteins in parallel, resulting in more biologically relevant information. Multiplex assays are used for basic research and drug discovery to look at thousands of proteins, or a ‘proteome’ in a sample. For clinical diagnostics, monitoring only a few proteins along with controls is sufficient. In both cases, however, specificity, sensitivity, sample availability, reagent costs, and convenience of use are major criteria for selecting a protocol.

Immunoassays are still the preferred platform for most protein studies, particularly clinical diagnostics and drug development,  where specificity is critical . Antibody-based assays including immune-capture in lateral flow devices, immunoblot (Western blot) and ELISA (Enzyme-Linked Immunosorbent Assay) have been around for decades, and are widely adapted into clinical diagnostics.  Most often the first choice is convenience- using the technology that is at hand, and that which has already been set up in the lab or neighboring community. This article describes the relative features and benefits of antibody-based assays, comparing Western, ELISA and protein microarrays.

Western Blots are typically done to determine the presence and integrity of a specific protein. In this assay, a mixture of proteins is first separated based on molecular weight and/or charge by electrophoresis in a gel matrix, then transferred to a membrane and probed with antibody specific to the protein of interest. The relative amount of protein in different samples may be compared and approximated. Because the proteins in a mixture are first separated according to physical properties, the specificity of the Western can be very high, and any cross-reactivity of the detecting antibody with other proteins in the mixture can be distinguished by the known molecular weight of the protein of interest. Western blots are limited to detection of denatured protein because all proteins in the sample are denatured prior to the electrophoresis step. On the downside, Western blots are relatively technically complex, requiring many steps and relatively large sample volume and thus are not easily automated and are relatively time consuming and expensive.

ELISA assays are often performed in 96-well plates and are adaptable to higher throughput than Western blots, but like Western blots offer only monoplex data, or results of a single protein per assay.  Unlike the Western assay, an ELISA can be used to detect native proteins, and protein interactions that require intact three-dimensional structure.  ELISA assays can be highly quantitative when run with a standard curve of the known protein, and well-characterized antibodies.  However, these assays are not highly specific and can give false positives due to cross-reactivity of the detecting antibody with other proteins in the sample. ELISA assays are very useful for looking at protein interactions that require native conformation, and for studying binding competition. In addition, ELISA assays are technically less difficult than Western blots, and can be adapted to higher throughput with automated plate handling and detection systems.

Protein microarrays are more similar to ELISA than Western blot because generally the proteins in a sample are not fractionated prior to the assay.  In constructing microarrays, the proteins are deposited in small (100-300 um) spots on a specially coated microscope slide. The slide is often coated with a polymer like nitrocellulose, or gel that increases the binding capacity of the protein. Microarrays offer advantages of higher throughput, multiplex analysis, low reagent consumption, high sensitivity and lower sample requirement compared to either the Western or ELISA assays (see Tables I and II).

Commercially available protein arrays are now available that offer pre-printed arrays of hundreds to thousands of proteins, typically antibodies used to capture proteins much like an ELISA.  The manufacture of microarrays is in fact a time-consuming and costly process that is not generally feasible for the individual researcher. More importantly, the antibody content spotted onto microarrays is the most valuable and costly component. However, the amount of  antibody required for a microarray as well as the sample requirement is much less than for ELISA. The protein microarray conserves reagents as well as precious samples. Thus for high-throughput, or highly repeated assays the reduced reagent costs, time-savings and sample conservation for microarray can outweigh the relative expense of setup (i.e. printing or purchasing the arrays, see Table II). In addition, the amount of protein or antibody may be limited in some cases (for example, limited amounts of polyclonal antibody or patient sample for diagnostics).

Grace Bio-Labs has developed leading technology for sensitive and high-quality protein microarrays. Our nitrocellulose film-slides provide the highest protein binding capacity,  leading to  excellent sensitivity (down to XX pg/ml). We have developed a suite of reagents designed to optimize results with these arrays in terms of sensitivity (signal/noise) and reproducibility. If you are interested in developing immunoassays in a protein microarray format, we invite you to partner with us with the content of your choice.

Table I: Relative properties of Western Blot, ELISA and Microarray Assays.

Pros

Cons

Best Applications

Western Blot

High specificity

Labor Intensive/time consuming

Denatured protein

Medium quantitation

Low-medium throughput

High sample use

Confirmation of other screening method

Protein-protein interactions

Protein degradation or turnover

ELISA

Medium specificity

High sensitivity

High quantitation

Low variability

Automation potential

Good reproducibility

False positives

High sample use

High reagent use

Time consuming

Labor intensive

Costly setup for automation or high throughput

High throughput screening, automation

Receptor inhibition

Relative quantitation

Ample sample availability

Microarray

Medium specificity

High sensitivity

Highest throughput

Low reagent use

Low variability

Low sample use

Good reproducibility

Multiplex capability

Costly setup for low throughput, mainly costs of scanner and high density arrays.

False positive/negative

High throughput screening

Limited sample availability

Relative quantitation

Multiplex analysis

Proteome assessment

Screening antibody cross-reactivity§

Pathology/diagnosticsŦ

 § Ma et al, 2012 Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology. BMC Biotechnology 2012, 12:88

 Ŧ Duburcq et al., 2004, Peptide−Protein Microarrays for the Simultaneous Detection of Pathogen Infections, Bioconjugate Chem., 2004, 15 (2), pp 307–31

Table II. Costs of Immunoassays/sample

Costs for every immunoassay will depend on the desired sensitivity, the labeling/detection system and particularly the cost of the primary antibody. For assays with abundantly available antibodies, the costs are lower.

Assay

Approximate Costs /sample/analyte

 

Assumptions

Western*

$1.50-10.00

1-2-plex

Depends on digital or film imaging, labeling and detection method.

ELISA**

$5-30

Monoplex

Cost based on efficient use of 96 well plate and commercially available kit.

Microarray

$0.1-30

Multiplex

(up to 100s for discovery, up to 10s for clinical)

Cost based on detection of multiple analytes/slide and use of multiple samples for same assay (same slide content). Depends on labeling and detection (color vs. fluorescence).

 *Li-Cor Corp.

**Udeh et al, 2008 BMC Infectious Diseases 8:174