Application and Future of ELISA

Method developed in 1971 as an alternative to radioimmunoassay methods has come a far way. For nearly forty eight years, ELISA based immunoassays have been the mainstay of the diagnostic tests used for the detection of infectious disease. In recent times, researchers have refocused on continuing evolution of ELISA based formats. A miniaturized, highly multiplexed, microarray formats have also began to be explored. ELISA is a popular method for protein detection and quantification. A biochemistry assay which uses a solid-phase enzyme immunoassay (EIA) are simple to carry out and they are designed to rapidly handle a large number of samples in parallel.

ELISA scores over immunofluorescence (IF) and Radioimmunoassay (RIA). IF is  a tedious, time consuming, not easily automated, unsuitable for large batches of tests and suffers from subjective interpretation bias. On the other hand in RIA reagents have a short shelf-life, sophisticated equipment is required and hazard of radioactivity. The theoretical sensitivity of ELISA ranges from 10−4 to 10−16 moles per litre (nanogram quantities per mL) giving it the upper edge in clinical use. ELISA has now been greatly prejudiced by availability of convenient, reliable, commercial kits.

Different formats of ELISA are developed and according to the need of the experiment they can be adopted. In each of the format one of the reactants, either the antigen or the antibody, is immobilised on to a solid phase matrix. It is still is a very popular choice for the evaluation of various research and diagnostic targets. The method has many advantages such as the long lifespan of the reagents used, being free of radiation risks associated with waste substances, enabling the analyses of multiple samples in a short time period in diagnosis and research laboratories, and low cost. The method has been widely used around the world for different purposes like hepatitis screening; parasite analyses, measuring tumor markers; determining hormone quantities as well as in hematology and biochemistry.

The specificity between antigen and antibody is governed by high affinity associations between an epitope on the antigen and its cognate binding site on the Fab portion of an IgG. ELISA by contrast, is a simple yet powerful analytic technique, which can concurrently analyze up to 96 or 384 samples that contain target molecules in very small numbers. The power of ELISA originates from the exquisite Y-shaped antibodies. In this Y-shaped protein, each of the two tips (variable regions) is able to recognize one particular epitope while the base (Fc region) is able to communicate with other biological molecules . Such versatile Y-shaped-antibody based assay is mostly applied to detect antibiotics residues, proteins, and antibodies.

Various methods for the quantitative or qualitative analysis of plant secondary metabolites (1) have also been developed because a lot of marketed drugs are generated from plant secondary metabolites, such as morphine (analgesic drug), vinblastine (antineoplastic drug), paclitaxel (antineoplastic drug), quinine (antimalarial drug), digitoxin (cardiotonic drug) etc. An accurate, sensitive, and selective evaluation of these drugs leads to safe clinical and general usages. Plant secondary metabolites are plant-produced organic compounds that play an important role in the defense of plants against herbivores, pests, and pathogens, as well as in their adaptation to the environment. Most of the useful plant secondary metabolites are low molecular weight compounds (i.e., hapten) with immense structural diversity, which are generally classified on the basis of their biosynthesis pathway. They have diverse functions and so there have been a dramatic increase in their demand in pharmaceuticals, cosmetics, and pesticides, as well as in food additives. ELISA used for their analysis is the competitive type (competitive ELISA or icELISA) using monoclonal or polyclonal. Monoclonals tend to exhibit higher specificity than polyclonals as monoclonals recognize only one epitope. Quality of secondary metabolites affects their potential activity and  so the control of these commercial products containing secondary metabolites is crucial. According to a recent report, 34% of the currently used drugs originate from natural products. Various analytical methods have been developed for monitoring effective concentration, side effects, and metabolism, leading to a better quality of life for patients. ELISA exhibits several advantages over such techniques because of its simplicity, selectivity, and sensitivity. A major barrier is the preparation of specific antibodies against the target hapten and even now for many important plant secondary metabolites antibodies are not available.

In the course of respiratory infections, the efficacy of microbiologic diagnosis has increased years after years, in term of specificity, sensitivity and rapidity. New pathogenic agents have been described such as: Legionella pneumophila, Chlamydia pneumoniae, Hantavirus. Some viruses have been well characterized as responsible for seasonal outbreaks using rapid tools for identification. Needs for efficient diagnostic tools became more obvious when specific antiviral drugs appeared on the market. In the recent advances, a lot of progress has been made in ELISA with recombinant DNA technology, leading to increase in the range of antibodies, probes, and even systems. Microarray based ELISA  is a new addition to the efforts made by researcher in the field of expanding ELISA. One of the main challenges in developing a microarray based ELISA is minimizing nonspecific cross-reactivity between numerous antigen and antibody mixtures to maintain the integrity of the assay. Future applications of this new highthroughput screening (HTS) format include direct cellular protein expression profiling, multiplexed assays for detection of infectious agents and cancer diagnostics. ELISAs based on recombinant proteins have also been developed.

Recently a user friendly glass capillary array based microfluidic ELISA device has been developed (2). The capillary based ELISA device significantly reduced the sample volume to 20 μL and shortened the total assay time to as short as 16 minutes (including detection time). This represent approximately 10-fold and 5-fold reduction in assay time and sample volume, respectively, in comparison with the traditional plate-based method. Furthermore, through the double exposure method, a nearly 10-fold increase in the detection dynamic range was achieved over the traditional well-based ELISA. The device has high expectations to be broadly used in rapid biochemical analysis for biomedicine and research/development laboratories.