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Techniques in immunology Vol. 1. Immunoenzymatic techniques
700
Book Reviews
Techniques in Immunology Vol. 1. Immunoenzymatic Techniques,
by T. TERNINCKand S. AVRAMEAS. Elsevier, Amsterdam, 131 pp.
1990.
This is the first volume of a series of handbooks introducing techniques based on immunological reactions. This manual is the outcome of a Technological Workshop organized by the French Society of Immunology, translated into English by D. Jarvis. It brings together a series of experimental procedures, starting from the preparation of basic reagents used in these methods, continuing with the practical application of immunoenzymatic techniques on a variety of fields including immunocytochemistry, the quantitative determination of the amount of antigen or antibody in biological fluids, and the detection of immunoprecipitates on gels or nitrocellulose blots. Each procedure is preceded by a description of the principles involved. Often technical notes are added in order to facilitate their application. Each chapter includes comments based on the authors own experience reflecting their personal view and the list of materials, buffers and solutions used in the particular procedure. Preparation of stock solutions and sources of materials are given in the Appendix. The description of the procedures contains all the details which enable the users, even beginners to carry it out on a correct and most successful way. The first chapter deals with the purification of antibodies using chemical or immunochemical methods. It describes the simple desalting method, the use of ion exchange chromatography and proceeds to the fractionation on high performance ion exchange columns, which gives a higher resolution in a shorter time. The paragraph of immunochemical purification of antibodies includes the preparation of immunoabsorbents, and the use of protein A beads as well as the details of affinity purification of immunoglobulins. The next paragraph describes the methods of fragmentation of immunoglobulins and the purification of the Fab and F(ab’), fragments. Chapter 2 deals with the coupling of antibodies to enzymes. The coupling sodium periodate and p-benzoagents (glutaraldehyde, quinone) are introduced, then an example is given for each coupling procedures, followed by the description of purification of conjugates. In Chapter 3 the quantitative enzyme immunoassays are described. This chapter deals with the different enzyme immunoassays, where a solid support is used to immobilize the antigen or antibody. This is probably the most widely used version of the technique, named enzymelinked immunoadsorbent assay. The methods of both antigen and antibody determinations using non-competitive and competitive assays and the specification of materials, the solid phase, the enzyme, the conjugate and the substrate are given. This chapter also shows practical examples for different types of determinations using both soluble and particular antigens (intact cells), explains how to: (1) build up the experimental system; (2) calibrate it and (3) evaluate the results. Chapter 4
General Immunology, by HERMANN. EISEN. J. B. Lippincott
Company,
Philadelphia,
1990. 259 pp. Essential Immunology, 7th edn, by IVANM. ROITT. Blackwell Sci. Publications, Oxford, 1991. 356 pp. f14.95. Immunology: a Synthesis, 2nd edn, by EDWARDS. GOLUB and DOUGLASR. GREEN. Sinauer Associates, MA, 1991. 744 pp. $39.95. The situation in contemporary immunology might be properly described by the apt remark of Prof. Roitt: “You just cannot
describes enzyme-immunocytochemical methods. Methods of this type make use of antibodies labeled with enzymes in order to localize constituents present in tissues or cells, alternatively, to demonstrate the presence of antibodies against such constituents in sera of patients. The basic principles of these methods are the same as for the immunofluorescent techniques, except that a coloured cytochemical reaction is seen instead of fluorescence at the site of antigen-antibody reaction. Direct and indirect procedures are shown, in the latter case the second antibody is labeled with the enzyme. This chapter describes the preparation of tissues and cells, the most widely used enzymes and conjugates, and the various chromogenic substrates, which are different from those applied in ELISA, since these must give rise to coloured insoluble product for visualizing the location of the reaction. Examples for application of immunocytochemical methods are given for detection of both cell surface and intracellular antigens. The authors give advices and general comments for a variety of applications. Chapter 5 deals with the detection of proteins after transfer to nitrocellulose. Immobilisation of the proteins may be performed using proteins in solution (dot procedure) or proteins which have been electrophoretically separated in gels and subsequently transfered to membranes (blotting procedure). This latter procedure enables characterization of proteins present in a mixture by the use of an enzyme-coupled antibody specifically recognizing them. The most widely used membrane is nitrocellulose, having a high capacity to irreversibly bind large amount of proteins. The protein transfer can be performed by simple diffusion or electrophoretically as well. The localisation of proteins is accomplished directly on the nitrocellulose sheet after the binding of the enzyme-linked antibody and the coloured substrate. In the 6th chapter the authors describe the advantages and disadvantages of the use of monoclonal antibodies in enzyme immunoassays. The 7th chapter introduces the different amplification systems for immunoenzymatic methods. Two main approaches are described, one is suitable for betagalactosidase labeled conjugates in which the visualisation is achieved by fluorogenic substrate. The second approach is based on the amplification of the signal by increasing the quantity of enzymes in the antigen-antibodyxnzyme complex, the avidin-biotin and peroxidase-anti-peroxidase systems are examples of this. References are given for each chapter enabling the users to get further informations. This manual is a valuable tool for researchers working not only on immunological fields, but in other areas like histochemistry or biochemistry as well, since it gives detailed informations for non-specialists to use immunological detection systems in a variety of cases.
L. E&v& University Gild, Hungary
GABRIELLASARMAY
stop these immunologists from making exciting new advances” Fortunately, you also can’t stop them from creating update comprehensive textbooks, summarizing these advances into harmonious system of modern immunological knowledge. A good corroboration of this is recent publication of the new versions of three well-known basic textbooks: “Essential Immunology” by Roitt, “General Immunology” by Eisen and “Immunology: a Synthesis” by Golub and Green. Evidently, these books have several common features. Firstly, they turn to be almost completely rewritten editions of
Book Reviews
Techniques in Immunology Vol. 1. Immunoenzymatic Techniques,
by T. TERNINCKand S. AVRAMEAS. Elsevier, Amsterdam, 131 pp.
1990.
This is the first volume of a series of handbooks introducing techniques based on immunological reactions. This manual is the outcome of a Technological Workshop organized by the French Society of Immunology, translated into English by D. Jarvis. It brings together a series of experimental procedures, starting from the preparation of basic reagents used in these methods, continuing with the practical application of immunoenzymatic techniques on a variety of fields including immunocytochemistry, the quantitative determination of the amount of antigen or antibody in biological fluids, and the detection of immunoprecipitates on gels or nitrocellulose blots. Each procedure is preceded by a description of the principles involved. Often technical notes are added in order to facilitate their application. Each chapter includes comments based on the authors own experience reflecting their personal view and the list of materials, buffers and solutions used in the particular procedure. Preparation of stock solutions and sources of materials are given in the Appendix. The description of the procedures contains all the details which enable the users, even beginners to carry it out on a correct and most successful way. The first chapter deals with the purification of antibodies using chemical or immunochemical methods. It describes the simple desalting method, the use of ion exchange chromatography and proceeds to the fractionation on high performance ion exchange columns, which gives a higher resolution in a shorter time. The paragraph of immunochemical purification of antibodies includes the preparation of immunoabsorbents, and the use of protein A beads as well as the details of affinity purification of immunoglobulins. The next paragraph describes the methods of fragmentation of immunoglobulins and the purification of the Fab and F(ab’), fragments. Chapter 2 deals with the coupling of antibodies to enzymes. The coupling sodium periodate and p-benzoagents (glutaraldehyde, quinone) are introduced, then an example is given for each coupling procedures, followed by the description of purification of conjugates. In Chapter 3 the quantitative enzyme immunoassays are described. This chapter deals with the different enzyme immunoassays, where a solid support is used to immobilize the antigen or antibody. This is probably the most widely used version of the technique, named enzymelinked immunoadsorbent assay. The methods of both antigen and antibody determinations using non-competitive and competitive assays and the specification of materials, the solid phase, the enzyme, the conjugate and the substrate are given. This chapter also shows practical examples for different types of determinations using both soluble and particular antigens (intact cells), explains how to: (1) build up the experimental system; (2) calibrate it and (3) evaluate the results. Chapter 4
General Immunology, by HERMANN. EISEN. J. B. Lippincott
Company,
Philadelphia,
1990. 259 pp. Essential Immunology, 7th edn, by IVANM. ROITT. Blackwell Sci. Publications, Oxford, 1991. 356 pp. f14.95. Immunology: a Synthesis, 2nd edn, by EDWARDS. GOLUB and DOUGLASR. GREEN. Sinauer Associates, MA, 1991. 744 pp. $39.95. The situation in contemporary immunology might be properly described by the apt remark of Prof. Roitt: “You just cannot
describes enzyme-immunocytochemical methods. Methods of this type make use of antibodies labeled with enzymes in order to localize constituents present in tissues or cells, alternatively, to demonstrate the presence of antibodies against such constituents in sera of patients. The basic principles of these methods are the same as for the immunofluorescent techniques, except that a coloured cytochemical reaction is seen instead of fluorescence at the site of antigen-antibody reaction. Direct and indirect procedures are shown, in the latter case the second antibody is labeled with the enzyme. This chapter describes the preparation of tissues and cells, the most widely used enzymes and conjugates, and the various chromogenic substrates, which are different from those applied in ELISA, since these must give rise to coloured insoluble product for visualizing the location of the reaction. Examples for application of immunocytochemical methods are given for detection of both cell surface and intracellular antigens. The authors give advices and general comments for a variety of applications. Chapter 5 deals with the detection of proteins after transfer to nitrocellulose. Immobilisation of the proteins may be performed using proteins in solution (dot procedure) or proteins which have been electrophoretically separated in gels and subsequently transfered to membranes (blotting procedure). This latter procedure enables characterization of proteins present in a mixture by the use of an enzyme-coupled antibody specifically recognizing them. The most widely used membrane is nitrocellulose, having a high capacity to irreversibly bind large amount of proteins. The protein transfer can be performed by simple diffusion or electrophoretically as well. The localisation of proteins is accomplished directly on the nitrocellulose sheet after the binding of the enzyme-linked antibody and the coloured substrate. In the 6th chapter the authors describe the advantages and disadvantages of the use of monoclonal antibodies in enzyme immunoassays. The 7th chapter introduces the different amplification systems for immunoenzymatic methods. Two main approaches are described, one is suitable for betagalactosidase labeled conjugates in which the visualisation is achieved by fluorogenic substrate. The second approach is based on the amplification of the signal by increasing the quantity of enzymes in the antigen-antibodyxnzyme complex, the avidin-biotin and peroxidase-anti-peroxidase systems are examples of this. References are given for each chapter enabling the users to get further informations. This manual is a valuable tool for researchers working not only on immunological fields, but in other areas like histochemistry or biochemistry as well, since it gives detailed informations for non-specialists to use immunological detection systems in a variety of cases.
L. E&v& University Gild, Hungary
GABRIELLASARMAY
stop these immunologists from making exciting new advances” Fortunately, you also can’t stop them from creating update comprehensive textbooks, summarizing these advances into harmonious system of modern immunological knowledge. A good corroboration of this is recent publication of the new versions of three well-known basic textbooks: “Essential Immunology” by Roitt, “General Immunology” by Eisen and “Immunology: a Synthesis” by Golub and Green. Evidently, these books have several common features. Firstly, they turn to be almost completely rewritten editions of