ELISA Essentials: Surfaces, Antibodies, Enzymes, and Substrates.

The enzyme-linked immunosorbent assay is a powerful analytical tool for the assessment of the kind and quantity of specific analytes found within a biological sample. It is based upon the exceptional specificity of antibody recognition of its cognate antigen and the power of enzyme-mediated signal amplification for sensitivity. However, development of the assay is not without challenges. Here, we describe the essential components and features necessary to successfully prepare and carry out the ELISA format.

Antibody Immobilization.

In the ELISA format(s), the capture antibody is usually affixed to a solid phase, commonly referred to as the immunosorbent. How to tether the antibody most effectively will depend upon the physical properties of the support (plate well, latex bead, flow cell, etc.) as well as its chemical nature (hydrophobic, hydrophilic, the presence of reactive groups such as epoxide, etc.). Of course, it is ultimately the suitability of the antibody to withstand the linking process while preserving its antigen-binding efficiency that must be determined. In this chapter, the antibody immobilization processes and their consequences are described.

Interference in ELISA.

ELISA is a well-established technique used worldwide to quantify analytes present in a diverse milieu of biological samplings. It is especially important to clinicians who rely on the accuracy and precision of the test to administer patient care. Those results are to be held with great scrutiny since the assay is subject to error caused by interfering substances found in the sample matrix. In this chapter, we examine the nature of such interferences and discuss approaches to identify and offer remedies to remove the interference and validate the assay.

Lateral Flow Microarray-Based ELISA for Cytokines.

Cytokines are well known to be involved in numerous biological responses with diverse mechanisms of action, including the inflammatory process. The so-called "cytokine storm" has recently been associated with cases of severe COVID-19 infection.Lateral flow microarray (LFM) devices have been constructed for multiplex detection of cytokines. The LFM-cytokine rapid test involves the immobilization of an array of capture anti-cytokine antibodies. Here, we describe the methods to create and use multiplex lateral flow-based immunoassays based upon the enzyme-linked immunosorbent assay (ELISA).

Well-Based Multiplex Food Allergen Colorimetric ELISA.

Food allergy is a well-recognized and significant health hazard around the world. At least 160 food groups have been identified that present allergenic reactions or other sensitivities and intolerance in humans. Enzyme-linked immunosorbent assay (ELISA) is an accepted platform for identifying the nature of the food allergy and its severity. It is now possible to simultaneously screen patients for allergic sensitivity and intolerance to multiple allergens using multiplex immunoassays. This chapter describes the preparation and utility of a multiplex allergen ELISA for the assessment of food allergy and sensitivity in patients.

A Unique Multiplex ELISA to Profile Growth Factors and Cytokines in Cerebrospinal Fluid.

Multiplex arrays designed for enzyme-linked immunosorbent assays (ELISAs) are robust and cost-effective for profiling biomarkers. Identification of relevant biomarkers in biological matrices or fluids helps in the understanding of disease pathogenesis. Here, we describe a sandwich ELISA-based multiplex assay to assess growth factor and cytokine levels in cerebrospinal fluid (CSF) samples derived from multiple sclerosis patients, amyotrophic lateral sclerosis patients, and control subjects without any neurological disorder. Results indicate that multiplex assay designed for the sandwich ELISA method is a unique, robust, and cost-effective method for profiling growth factors and cytokines present in CSF samples.

Mycotoxin Quantification by Competitive ELISA.

Enzyme-linked immunosorbent assays (ELISAs) are useful in the quantification of antigens like mycotoxins. The zearalenone (ZEA) mycotoxin is commonly found in cereal crops such as corn and wheat, which are used in domestic and farm animal feed. When consumed by farm animals, ZEA can cause harmful reproductive effects. The procedure used to prepare corn and wheat samples for quantification is described in this chapter. An automated procedure was developed to prepare samples from known levels of ZEA in corn and wheat. The final corn and wheat samples were analyzed using a competitive ELISA specific to ZEA.

ELISA-Based Biosensors.

Enzyme-linked immunosorbent assay (ELISA) is by definition a biosensor. However, not all immuno-biosensors involve the use of enzymes, while other biosensors incorporate ELISA as a key signaling component. In this chapter, we review the role of ELISA in signal amplification, integration with microfluidic systems, digital labeling, and electrochemical detection.

Well-Based Antibody Arrays.

Multiplex immunoassays are important tools in basic research and diagnostics. The ability to accurately quantify the presence of several antigens within an individual sample all at once has been useful in developing a proteomics view of biology. This in turn has enabled the development of disease-associated immunodiagnostic panels for better prognosis and well-being. Moreover, it is well understood that such multiplexing approaches lend themselves to automation, thereby reducing labor while providing the ability to dramatically conserve both reagent and sample all of which will reduce the cost per test. Here we describe various methods to create and use multiplex immunoassays in the wells of microtiter plates or similar formats.

Multiplex ELISA Using Oligonucleotide Tethered Antibodies.

Multiplex assays represent a new paradigm for diagnostics. The simultaneous measure of multiple analytes from a single sample is advantageous in creating disease-associated panels that enable more accurate prognosis or diagnosis of the disease state. Furthermore, multiplexing may reduce reagent consumption, sample requirements and labor thereby lowering the cost per test. Here we describe a novel multiplex immunoassay technology based upon creating microarrays in microtiter plates that are formed upon the self-assembly of oligonucleotide-antibody conjugates.

Construction of in situ oligonucleotide arrays on plastic.

The concept of DNA arrays was first introduced in the early 1980s, by Sir Edwin Southern. Since then, many research institutions and biotechnology companies have investigated the potential use of arrays in fields ranging from genetic diagnostics to forensics investigations. A 64-channel automated chemical delivery system, known as the Southern Array Maker, which synthesizes oligonucleotides directly onto an aminated polypropylene substrate has been constructed. Many different arrays have been synthesized for the purpose of detecting single point mutations, which might be either indicators of, or directly responsible for, many different types of genetic diseases and cancers. These include cystic fibrosis, H-ras, K-ras, and other mutations. In addition to the synthesized arrays, we are also looking into various alternative methods of producing both high-and low-density DNA arrays. This chapter is intended to demonstrate the synthesis of oligoarrays by in situ method using standard phosphoramidite chemistry. Phosphoramidate linkage to the aminated polypropylene is quite stable under oligo cleavage and deprotection conditions. Oligonucleotide density is approx 3 pmole or 10(1)2 molecules/mm(2).

Hybridization analysis using oligonucleotide probe arrays.

This chapter describes methodology for the labeling, hybridization, and detection of amplicon target DNA to arrays of oligonucleotide probes attached to plastic substrates. A systematic approach to target discrimination based on both hybridization and wash stringency is provided.

Printing low density protein arrays in microplates.

Here, we provide methods for the creation of protein microarrays in microplates. The microplate consists of 96 wells with each well capable of holding a protein microarray at a spot density of up to 400 (20 x 20) individual elements. Arrays of capture monoclonal antibodies, corresponding to specific interleukins, were printed onto the bottom of the wells which had been surface activated for covalent attachment. A Biomek 2000 laboratory automation workstation (Beckman Coulter, Inc., Fullerton, CA) equipped with a high-density replicating tool was used for printing low density 3 x 3 to 5 x 5 arrays. For higher density arrays, a microarrayer system (Cartesian PS7200, Genomic Solutions, Inc., Ann Arbor, MI) was employed. Multiple antigens were simultaneously analyzed without detectable cross-reactivity associated with capture antibody or secondary antibody interactions. Detection of interleukin antigens, spiked into cell culture media containing 10% fetal calf serum, was specific and sensitive.

Overprint immunoassay using protein A microarrays.

The ability to perform microarray-based immunoassays without the need for wells or other fluid barriers were demonstrated. Both contact and noncontact microarray printing technology is used to prepare spotted arrays of analyte binding sites, as well as, to deliver samples, secondary antibodies and other signal development reagents directly to these sites in a parallel fashion are called as overprint immunoassays. A micro-ELISA is demonstrated based upon the use of Protein A as a universal microarray. All components of the assay (capture antibody, antigen, and signal development reagents) were site-specifically dispensed in parallel fashion to the surface in nanoliter volumes. This represents a 1000-fold reduction in reagent consumption from that used in a conventional 96-well microtiter plate assay. Overprinting nanoliter volumes directly onto 200-300 microm spots yields similar levels of sensitivity achieved with the bulk dispensing of microliter volumes into wells.

Are multiple markers the future of prostate cancer diagnostics?

Prostate specific antigen (PSA) is the most successful and widely employed cancer serum marker in use today. There is growing evidence that the introduction of wide PSA screening and earlier detection can result in decreased cancer mortality associated with a decline in metastatic disease. PSA circulates in a number of distinct forms. Measurement of these in addition to total PSA significantly increases diagnostic utility. Diagnostic utility is likely to be further increased by adding kallikreins, cytokines, growth factors, receptors and cellular adhesion factors to the biomarker panel. The need for multiple markers reflects the multidimensional nature of prostate disease which ranges from metastatic cancer to indolent cancer to benign hyperplasia and inflammation, all of which require distinct treatments and medical interventions.