That T. Ngo – Författare

The development of radioimmunoassay (RIA) by R.S. Yalow and S.A. Berson in 1959 opens up a new avenue in ultra­ sensitive analysis of trace substances in complex biological systems. In recognition of the enormous contributions of RIA to basic research in biology and to routine clinical tests in laboratory medicine, R.S. Yalow, the co-developer of RIA, was awarded, in 1977, the Nobel Prize for Medicine and Physiology. The basic principle of RIA is elegantly simple. It is based on a specific, competitive binding reaction between the analyte and the radio-labeled analog of the analyte for the specific antibody raised to the analyte. The combination of high specificity and affinity of an antibody molecule makes it a very versatile analytical reagent capable of reacting specifically with analytes at a very low concentration in a complex solution such as serum. The sensitivity of RIA is provided by using a radioactive tracer.

Rapid advances in molecular biology have accelerated the production of a great number of protein-based therapeutic agents. The major cost in producing these proteins appears to be associated with their purification from the complex mixture of the crude extract. A major challenge to the protein biochemist and the biochemical engineer is the development of rapid, efficient, and cost-effective purification systems. This volume presents state-of-the-art reviews of current methods used in the purifica­ tion of biological macromolecules that are based on molecular interactions. Thus, the major emphasis is placed on affinity-related techniques. Part I provides a general introduction to affinity chromatography and includes a chapter describing an interesting new technique called "slalom chromatography" for DNA fractionations. Affinity chromatography using molecules of biological origin as the affinity ligand is covered in Part II. Part III describes the use of a special class of biomolecules, antibodies, as affinity ligands. Affinity chromatography with biomimetic ligands is discussed in Part IV. Newer concepts and their applications in bioseparation are presented in Part V. Part VI covers affinity-related techniques such as affinity-based extracorporeal shunts, affinity electrophoresis, affinity precipitation, and affinity extraction. I would like to express my sincere thanks to all the authors, who are recognized experts in their respective fields, for their cooperation and contributions. I thank the editorial staff of Plenum Press for their professionalism, and Mary Phillips Born, Senior Editor, for her encouragement. The support of my family (Ping and Peilin) made it possible to complete editing this book. That T.

A biosensor is a device in which a bioactive layer lies in direct contact with a transducer whose responses to change in the bioactive layer generate eloctronic signals for interpretation. The bioactive layer may consist of membrane-bound enzymes, anti-bodies, or receptors. The potential of this blend of electronics and biotechnology includes the direct assay of clinically important substrates (e.g. blood glucose) and of substances too unstable for storage or whose concentrations fluctuate rapidly. Written by the leading researchers in the field, this book reflects the most current developments in successfully constructing a biosensor. Major applications are in the fields of pharmacology, molecular biology, virology and electronics.

A biosensor is a device in which a bioactive layer lies in direct contact with a transducer whose responses to change in the bioactive layer generate eloctronic signals for interpretation. The bioactive layer may consist of membrane-bound enzymes, anti-bodies, or receptors. The potential of this blend of electronics and biotechnology includes the direct assay of clinically important substrates (e.g. blood glucose) and of substances too unstable for storage or whose concentrations fluctuate rapidly. Written by the leading researchers in the field, this book reflects the most current developments in successfully constructing a biosensor. Major applications are in the fields of pharmacology, molecular biology, virology and electronics.

T. T. Ngo and H. M. Lenhoff Department of Developmental and Cell Biology University of California, Irvine, CA 92717 In 1959, Yalow and Berson used insulin labeled with radioactive iodine to develop a quantitative immunological method for determining the amount of insulin in human plasma. Their method depends upon ~ competition between insulin labeled with radioactive iodine (II 1) and unlabeled insulin from plasma for a fixed and limited number of specific binding sites on the antibody to insulin. The amount of the labeled insulin bound to the antibody is inversely proportional to the amount of insulin in the plasma sample. Their method, which is so elegantly simple in concept, is made possible by the ability to detect with ease extremely low levels of radioactivity, and by the exquisite specificity of an antibody capable of specifically binding the analyte. Such a combination of sensitivity and specificity is the basis of this versatile analytical tool called radioimmunoassay (RIA). Twelve years later, Engvall and Perlmann (1971) and Van Weemen and Schuurs (1971) independently introduced the use of enzymes as another category of sensitive and even more versatile labels for use in immunoassays. Engvall and Perlmann (l971) coined the term ELISA, which stands for Enzyme Linked Immunosorbent Assay.

The basis of all immunoassays is the interaction of antibodies with antigens. Indeed the last fifteen years have seen the development of a great number of ingenious non-isotopic labels in immunoassay so that a whole new industry capitalizing on the potential market for non­ isotopic immunoassays has appeared.

Affinity chromatography using molecules of biological origin as the affinity ligand is covered in Part II. Part VI covers affinity-related techniques such as affinity-based extracorporeal shunts, affinity electrophoresis, affinity precipitation, and affinity extraction.