Immuno and Receptor-Assays in Theory and Practice.

Places the subject of the book in its historical and practical context.

Defines and classifies the assay components. Presents the many research and industrial uses of immuno- and receptor-assays. Delineates the physiological roles of antibodies and receptors, and the types of signal transduction pathways occuring upon ligand-receptor interaction in vivo.

Places antibodies and receptors in the cross-talk context between cells in pluricellular organisms. Discusses the cellular origin of various receptors and their structure relationship.

Describes how receptors are synthesized within the cell and their overall and binding site structure, how they interact with the ligands and what are the mechanisms by which their effector function occurs. This is illustrated with three receptor examples: the steroid and thyroid receptor superfamily, the circulating steroid-binding proteins and the immunoglobulin-like superfamily.

Outlines the techniques available to produce receptors: cell culture, extraction from organs or body fluids, recombinant DNA technology.

The contents of this subchapter raises and answers the following questions: What are antigens and haptens? What renders them immunogenic? How to prepare immunogens?

Ways and recipes for immunization are provided in this part of the chapter. The production of polyclonal and monoclonal antibodies is emphasized. The various methods for antibody titration and titer follow-up as well as ways to improve the specificity of antibodies during immunization are discussed.

This part of the chapter discusses the strategies available for the purification of (active) receptors and antibodies: extraction by surfactants, salting-out, various types of chromatographic techniques. The last part of this subchapter discusses the many ways these proteins can experience degradation during purification and storage as well as the means to avoid it.

This is a further discussion complementing that of chapter 2.2. and focusing on the mechanisms underlying the molecular recognition between receptors and their specific ligands. The molecular recognition process is then discussed in terms of QSAR and drug design.

This part of the chapter details the various kinetic and binding mathematical models describing the ligand-receptor interaction. Examples of binding raw data are used throughout and introduced into the mathematical equations which are then solved for the rate or binding constants.

In this part of the chapter, the thermodynamic aspects of the interaction are presented with a particular emphasis on the way the free energy contributes to the strength of interaction. Two practical examples are detailed showing how the analysis of free energy contributions can lead to mapping binding sites.

The effects of temperature, pH, salt concentration...on the interaction are detailed by use of practical examples.

This part of the chapter discusses respectively the differences, advantages or disadvantages between competitive and non-competitive assays in various formats. The discussion is illustrated by practical examples and a decision tree is presented so as to help the reader decide what type of assay he should best select for his application.

This subchapter discusses respectively the differences, advantages or disadvantages between homogeneous and heterogeneous assays in various formats. The use as labels in homogeneous assays of new "intelligent" materials experiencing physical transitions upon ligand-receptor interactions is introduced.

Practical examples of development and optimization of agglutination and precipitation assays are provided so as to support the discussion in this subchapter.

Implications resulting from the introduction of a label in the reagents for homogeneous and heterogeneous assays are presented with practical examples of assay development. In particular, the consequences on the affinity for the receptor of labeling a ligand is emphasized.

This short historical discussion shows how new technologies and end-user requirements have contributed to the design of commercialized assays over the last decades.

The many labels currently available for assay development are presented along with the measurement methods allowing to detect their signal. A complete discussion with many examples of assay development emphasizes the use of new "intelligent" labels such as conductive polymers and colloidal metals. A practical label selection guide is also provided.

Chemical, physical and biochemical methods available for the internal or external introduction of a label into a proteinic or haptenic tracer are presented. Labeling with the new "intelligent" labels is emphasized.

This part of the chapter presents the ways tracers can be purified with practical examples. Their further stabilization for long-term storage is also discussed.

Direct (with a label) and indirect (without label) biosensors are presented and illustrated with examples from the literature.

The instruments available in the immuno-and receptor-assay field as well as their mode of automation and label detection are presented in this subchapter.

The various forms of sample and analyte as found in nature are presented. The implications of the type of sample on the assay performance and development is discussed as well as the possible extraction or purification means for analyte separation before assaying.

Constant and proportional effects on assay performance resulting from abnormal substances in the sample or reagents are discussed. Practical examples showing how to detect these effects and how to reduce or suppress them are presented.

This part of the chapter presents definitions for assay accuracy, precision, reproducibility and repeatability and provides practical informations on reference materials and reagents which allow for assay standardization against reference methods.

This part of the chapter explains what a reference method is and the advantages of confronting to it a newly developed assay. Copy of a computer program is given with a practical example of method comparison.

This part of the chapter discusses assay specificity, cross-reactions, sensitivity and linearity with practical examples. The methods available for checking the specificity of assays for different types of analytes are further illustrated.

Substances leading to systematic and random inteferences which can alter the performances of an assay are presented. Methods for the detection and quantification of interferences are further illustrated with practical examples.

This subchapter illustrates with practical examples the best among the many ways available for presenting a dose-response relationship, depending on the informations it is intended to provide the analyst.

This part of the chapter illustrates with practical examples the most informative among the many ways available for presenting and calculating binding data. Computer programs are provided so as to familiarize the reader with their application.

This subchapter discusses the advantages and disadvantages of using respectively accelerated degradation tests or real-time follow-up for monitoring reagent stability. The discussion is further supported by practical examples.

This last part of the book presents a check-list containing all the components which should be validated during the development of new assay. The chapters of the book where the information regarding the validation process for each individual component can be found are indicated in a summary table.