This book gives a comprehensive account of the practical aspects of Real time PCR and its application to veterinary diagnostic laboratories. The optimisation of assays to help diagnose livestock diseases is stressed and exemplified through assembling standard operating procedures from many laboratory sources. Theoretical aspects of PCR are dealt with as well as quality control features necessary to maintain an assured testing system. The book will be helpful to all scientists involved in diagnostic applications of molecular techniques, but is designed primarily to offer developing country scientists a collection of working methods in a single source. The book is an adjunct to the Molecular Diagnostic PCR Handbook published in 2005.

There have been very few developments that markedly affect the need to greatly revise the text from the last version of this book. This is testament to the fact that hetero- neous enzyme-linked immunosorbent assays (ELISA) provide ideal systems for dealing with a wide range of studies in many biological areas. The main reason for this success is test flexibility, whereby reactants can be used in different combinations, either attached passively to a solid phase support or in the liquid phase. The exploitation of the ELISA has been increased through continued development of specifically produced reagents, for example, monoclonal and polyclonal antibodies and peptide antigens coupled with the improvement and expansion of commercial products such as enzyme-linked conjugates, substrates and chromogens, plastics technology and design of microwell plates, inst- mentation advances and robotics. However, the principles of the ELISA remain the same. There has been some rearrangement of chapters plus addition of three new ones dealing with charting methods for assessing the indirect ELISA, ruggedness and robustness of tests-aspects of kit use and validation, and internal quality control and external quality management of data, respectively. These reflect the need to control what you are doing with ELISA and to exploit the method to its full extent. I do not apologize for dealing with the same areas in different ways a number of times, as it is imperative that principles are understood to allow planning, operation, and control of ELISA.

PREFACE The Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture is involved in agricultural research and development and assists Member States of FAO and IAEA in improving strategies to ensure food security through the use of nuclear techniques and related biotechnologies, where such techniques have a valuable and often unique role. In particular, molecular diagnostic methods have rapidly evolved in the past twenty years, since the advent of the Polymerase Chain Reaction (PCR). They are used in a wide range of agricultural areas such as, improving soil and water management; producing better crop varieties; diagnosing plant and animal diseases; controlling insect pests and improving food quality and safety. The uses of nucleic acid-directed methods have increased significantly in the past five years and have made important contributions to disease control country programmes for improving national and international trade. These developments include the more routine use of PCR as a diagnostic tool in veterinary diagnostic laboratories. However, there are many problems associated with the transfer and particularly, the application of this technology. These include lack of consideration of: the establishment of quality-assured procedures, the required set-up of the laboratory and the proper training of staff. This can lead to a situation where results are not assured. This book gives a comprehensive account of the practical aspects of PCR and strong consideration is given to ensure its optimal use in a laboratory environment. This includes the setting-up of a PCR laboratory; Good Laboratory Practice and standardised of PCR protocols.

This book gives a comprehensive account of the practical aspects of Real time PCR and its application to veterinary diagnostic laboratories. The optimisation of assays to help diagnose livestock diseases is stressed and exemplified through assembling standard operating procedures from many laboratory sources. Theoretical aspects of PCR are dealt with as well as quality control features necessary to maintain an assured testing system. The book will be helpful to all scientists involved in diagnostic applications of molecular techniques, but is designed primarily to offer developing country scientists a collection of working methods in a single source. The book is an adjunct to the Molecular Diagnostic PCR Handbook published in 2005.

The Enzyme Linked Immunosorbent Assay (ELISA) continues, as predicted, to be the predominant assay used in biomedical and agricultural research. While little has changed in the principles of the ELISAs used since the publication of The ELISA Guidebook, many technological advantages, including the increasing number of reagents available for exploitation and the larger spectrum of instruments to measure the coloured products of ELISAs, have greatly benefited the field. Other testing technologies using molecular techniques, predominantly the conventional and more and more often, real time PCR, are rapidly evolving to help diagnose and differentiate disease agents and identify substances present in exquisitely small amounts. The ELISA is still needed either alone in mass screening, such as serological assays, or used in tandem with molecular techniques to fully evaluate the molecular and antigenic nature of analytes.

John R. Crowther provides today s premier practical guide to the understanding and application of ELISA. This Edition enlarges on charting methods for assessing the indirect ELISA, ruggedness and robustness of tests, aspects of kit use and validation, and internal quality control and external quality management of data. The author describes each method in great detail to ensure experimental success and includes advice on equipment choice, maintenance, and calibration. Wherever possible, helpful written explanations are provided along with copious diagrams.

With its numerous worked examples, detailed instructions, and extensive illustrations, The ELISA Guidebook, Second Edition offers a powerful synthesis of all the basic concepts and practical experimental details investigators need to understand, develop, and apply ELISA methodology successfully in day-to-day basic and clinical research.

PREFACE The Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture is involved in agricultural research and development and assists Member States of FAO and IAEA in improving strategies to ensure food security through the use of nuclear techniques and related biotechnologies, where such techniques have a valuable and often unique role. In particular, molecular diagnostic methods have rapidly evolved in the past twenty years, since the advent of the Polymerase Chain Reaction (PCR). They are used in a wide range of agricultural areas such as, improving soil and water management; producing better crop varieties; diagnosing plant and animal diseases; controlling insect pests and improving food quality and safety. The uses of nucleic acid-directed methods have increased significantly in the past five years and have made important contributions to disease control country programmes for improving national and international trade. These developments include the more routine use of PCR as a diagnostic tool in veterinary diagnostic laboratories. However, there are many problems associated with the transfer and particularly, the application of this technology. These include lack of consideration of: the establishment of quality-assured procedures, the required set-up of the laboratory and the proper training of staff. This can lead to a situation where results are not assured. This book gives a comprehensive account of the practical aspects of PCR and strong consideration is given to ensure its optimal use in a laboratory environment. This includes the setting-up of a PCR laboratory; Good Laboratory Practice and standardised of PCR protocols.

John R. Crowther provides today's premier practical guide to the understanding and application of ELISA. Updating and greatly expanding his widely appreciated earlier publication, ELISA Theory and Practice (1995), this important work introduces chapters on such major new topics as checkerboard titrations, quality control of testing, kit production and control, novel monoclonal antibodies, validation of assays, statistical requirements for data examination, and epidemiological considerations. With its numerous worked examples, detailed instructions, and extensive illustrations, The ELISA Guidebook offers a powerful synthesis of all the basic concepts and practical experimental details investigators need to understand, develop, and apply the new ELISA methodology successfully in day-to-day basic and clinical research.

7he a1m 0f EL15A: 7he0ry and Pract1ce t0 1ntr0duce the expe- menter t0 the techn0109y kn0wn the En2yme L1nked 1mmun050r6ent A55ay (EL15A). 7he 600k d1rected at w0rker5 w1th vary1n9 de9ree5 0f exper1ence, 6ut f0r th05e w1th0ut any prev10u5 c0ntact w1th the a55ay. 1mmun0a55ay5 f0rm the 6ack60ne 0f u5ed te5t5 the 5tudy 0f 1nf- d15ea5e5 t10u5 and end0cr1n0109y, and hence can 6e u5ed t01d- a9ent5 cau5at1ve t1fy 0f the extent and nature (ep1dem10109y) 0f d15ea5e, and mea5ure h0rm0ne5 at EL15A5 pr0v1de h19h1y and prec15e meth0d5 f0r the mat10n 0f parameter5, w1th the added advanta9e that they can hand1e 1ar9e num6er5 0f 5amp1e5 that may then 6e ana1y2ed rap1d1y. 7he5e a55ay5 are ver5at11e, hav1n9 6een app11ed t0 a w1de ran9e 0f e.9., v1ru5e5, 6acter1a, fun91, and pr0t020an and meta- 20an 51nce para51te5. 1971, when the effect1ve en2yme-1a6e1ed a55ay wa5 de5cr16ed (1), th0u5and5 0f have 6een pu6115hed de- w1th the 4uant1f1cat10n 0f ant19en5 and ant160d1e5 f0r re5earch and app11ed purp05e5. EL15A and re1ated a55ay5 1nv01v1n9 u5e 0f en2yme5 t0 06ta1n c010r1metr1c re5u1t5 have n0w rep1aced rad101mmun0a55ay (R1A) f0r m05t d1a9n05t1c purp05e5, 51nce the f0rmer 0ffer5 a p0tent1a1 w1th 1ncrea5ed ver5at111ty t0 a w1der 9r0up 0f Examp1e5 0f 5pec1f1c area5 t0 wh1ch EL15A5 have 6een app11ed re5earch and are: Detect10n and 0f wh01e 0r part5 0f a9ent5, e.9., typ1n9; 2. 0f a9ent5, e.9., 5u6typ1n9; d15ea5e D15cr1m1nat10n d15ea5e 1dent1f1cat10n 1. d1a9n0515 1n 5c1ent15t5.