(2000). Enzyme-Linked Immunosorbent Assay. Journal of Immunoassay: Vol. 21, No. 2-3, pp. 165-209.

Enzyme-Linked Immunosorbent Assay

Immunoassays are now very widely used in the clinical laboratory, either because no other type of assay system is feasible or because they are often the most effective and suitable of the possible analytical methods. The last decade has seen the development and refinement of many new immunoassay reagents and systems. The major trend has been away from liquid-phase assays involving radioisotopic labels, towards fast homogeneous or solid-phase assays capable of operation anywhere; and towards precise and reliable nonisotopic, automated or semi-automated laboratory assays, often with detection limits measured in pico- or attomoles. The use of monoclonal antibodies is now widespread, and the methodologies of labels and of solid-phase components are much more sophisticated. New assay formulations, novel homogeneous systems, immunosensors, free-analyte assays, the importance of thorough validation and of interfering substances, and future trends are discussed.

A Decade of Development in Immunoassay Methodology

A novel separation-free sandwich-type enzyme immunoassay for proteins is performed by designing an electrochemical detection system that enables preferential measurement of surface-bound enzyme-labeled antibody relative to the excess enzyme-labeled reagent in the bulk sample solution. In this initial model system, the assay is carried out using gold-coated microporous nylon membranes (pore size 0.2 micron) which are mounted between two chambers of a diffusion cell. The membrane serves as both a solid phase for the sandwich assay and the working electrode in the three-electrode amperometric detection system. The capture monoclonal antibody is immobilized covalently on the gold side of the membrane via a self-assembled monolayer of thioctic acid. In the separation-free sandwich assay, both model analyte protein (human chorionic gonadotropin; hCG) and alkaline phosphatase labeled anti-hCG (ALP-Ab) are incubated simultaneously with the immobilized capture anti-hCG antibody. Surface-bound ALP-Ab is spatially resolved from the excess conjugate in the bulk sample solution by introducing the enzyme substrate (4-aminophenyl phosphate) through the back side of the porous membrane. The substrate diffuses rapidly through the porous membrane where it first encounters bound ALP-Ab at the gold surface. The enzymatically generated product, aminophenol, is detected immediately by oxidation at the gold electrode (at +0.19 V vs Ag/AgCl), and the magnitude of current is directly proportional to the concentration of hCG in the sample. The response time after substrate addition is less than 1 min, although maximum response toward the analyte protein requires a sample/conjugate preincubation time of 30 min with the porous electrode.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation-free sandwich enzyme immunoassays using microporous gold electrodes and self-assembled monolayer/immobilized capture antibodies

Irregular-shaped platinum nanoparticles as peroxidase mimics for highly efficient colorimetric immunoassay.

Immunochemical assays for small molecules such as pesticides are rapidly gaining acceptance among analytical chemists. These techniques are rapid, sensitive, cost effective and can easily cope with large sample loads. This review lists the advantages and disadvantages of the technique and describes the steps in assay development using examples from this laboratory, particularly the thiocarbamate herbicide molinate and the triazine herbicides. The focus is primarily on hapten synthesis strategies, assay format considerations, sample preparation and assay validation.

Use of Immunochemical Techniques for the Analysis of Pesticides

Enzyme Mediated Immunoassay: An Overview.- Separation-Free (Homogeneous) Enzyme Mediated Immunoassays.- Separation-Free Enzyme Immunoassay for Haptens.- Enzyme Modulator as Label in Separation-Free Immunoassays: Enzyme Modulator Mediated Immunoassay (EMMIA).- Prosthetic Group Labeled Enzyme Immunoassay.- Fluorogenic Substrate Labeled Separation-Free Enzyme Mediated Immunoassays for Haptens and Macromolecules.- Immunoassays Based on Conformational Restriction of the Enzyme Label Induced by the Antibody.- Nonseparation Enzyme Immunoassays for Macromolecules.- Liposome-Entrapped Enzyme Mediated Immunoassays.- Test Strip Enzyme Immunoassay.- Separation-Free Enzyme Fluorescence Immunoassay by Continuous Flow Injection Analysis.- Seperation-Required (Heterogenous) Enzyme Immunoassay.- Separation-Required (Heterogeneous) Enzyme Immunoassay for Haptens and Antigens.- Enzyme Immunoassay of Antibody.- The Amplified Enzyme-Linked Immunosorbent Assay (a-ELISA).- Affinity Column Mediated Immunoenzymetric Assays.- Steric Hindrance Enzyme Immunoassay (SHEIA).- Maleimide Derivative of Hapten for Enzyme Coupling in EIA.- Enzyme Immunoassay Using Tagged Enzyme-Ligand Conjugates.- Fluorimetric Measurements in Enzyme Immunoassays.- Radial Partition Enzyme Immunoassay.- Development of Immunochemical Enzyme Assays for Cardiac Isoenzymes.- Enzyme-Linked Immunoelectrotransfer Blot (EITB).- Enzyme Immunoassays for Tropical Parasitic Diseases.- Monoclonal Antibodies in Immunoenzymetric Assays.- The Ngo-Lenhoff (MBTH-DMAB) Peroxidase Assay.- Immunoperoxidase Techniques Using the Avidin-Biotin System.- Contributors.

Enzyme-mediated immunoassay

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Enzyme-mediated immunoassay