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More Enzyme-Linked ImmunoSorbent Assay protocols

Immunoassays: biological tools for high throughput screening and characterisation of combinatorial l
(Comb Chem High Throughput Screen. 2008 May;11(4):325-35)
Taipa MA.
IBB - Institute for Biotechnology and Bioengineering, Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais 1049 - 001, Lisboa, Portugal. angela.taipa@ist.utl.pt.

In the demanding field of proteomics, there is an urgent need for affinity-catcher molecules to implement effective and high throughput methods for analysing the human proteome or parts of it. Antibodies have an essential role in this endeavour, and selection, isolation and characterisation of specific antibodies represent a key issue to meet success. Alternatively, it is expected that new, well-characterised affinity reagents generated in rapid and cost-effective manners will also be used to facilitate the deciphering of the function, location and interactions of the high number of encoded protein products. Combinatorial approaches combined with high throughput screening (HTS) technologies have become essential for the generation and identification of robust affinity reagents from biological combinatorial libraries and the lead discovery of active/mimic molecules in large chemical libraries. Phage and yeast display provide the means for engineering a multitude of antibody-like molecules against any desired antigen. The construction of peptide libraries is commonly used for the identification and characterisation of ligand-receptor specific interactions, and the search for novel ligands for protein purification. Further improvement of chemical and biological resistance of affinity ligands encouraged the "intelligent" design and synthesis of chemical libraries of low-molecular-weight bio-inspired mimic compounds. No matter what the ligand source, selection and characterisation of leads is a most relevant task. Immunological assays, in microtiter plates, biosensors or microarrays, are a biological tool of inestimable value for the iterative screening of combinatorial ligand libraries for tailored specificities, and improved affinities. Particularly, enzyme-linked immunosorbent assays are frequently the method of choice in a large number of screening strategies, for both biological and chemical libraries.

ELISA protocol (pdf)

ELISA protocol (pdf)

Competition ELISA protocol

Enzyme-linked immunosorbent assays.
(Curr Protoc Immunol. 2001 May;Chapter 2:Unit 2.1.)
Hornbeck P.

University of Maryland, Baltimore, Maryland, USA.

This unit describes six different ELISA systems for the detection of specific antibodies, soluble antigens, or cell-surface antigens. In all six systems, soluble reactants are removed from solution after specifically binding to solid-phase reactants. In the first four protocols, solid-phase reactants are prepared by adsorbing an antigen or antibody onto plastic microtiter plates; in the next two protocols, the solid-phase reactants are cell-associated molecules. In all protocols, the solid-phase reagents are incubated with secondary or tertiary reactants covalently coupled to an enzyme. Unbound conjugates are washed out and a chromogenic or fluorogenic substrate is added. As the substrate is hydrolyzed by the bound enzyme conjugate, a colored or fluorescent product is generated. Finally, the product is detected visually or with a microtiter plate reader. The amount of product generated is proportional to the amount of analysate in the test mixture. The Support Protocol can be used to optimize the different ELISAs. The second support protocol presents a method for preparing alkaline phosphatase conjugates.

Proteins Can Be Detected and Quantitated by Using an Enzyme-Linked Immunosorbent Assay
I. The Molecular Design of Life 4. Exploring Proteins

Figure. Indirect ELISA and Sandwich ELISA
I. The Molecular Design of Life 4. Exploring Proteins 4.3. Immunology Provides Important Techniques with Which to Investigate Proteins

Figure. The principle of the enzyme-linked immunosorbent assay (ELISA)
Appendix I. Immunologists' Toolbox The detection, measurement, and characterization of antibodies and their use as research and diagnostic tools.

ELISA weak reaction
measure the cytokine level by Sandwich ELISA

Estradiol ELISA
standard curve problem with estradiol standards

Biotinylated Antibodies used in ELISA
sandwich ELISA for the detection of Protein G in samples

ELISA High Background Signal
typical sandwich ELISA troubleshooting

ELISA with fresh frozen plasma
upon thawing out FFP, cryoprecipitate forms

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