Preface

The concept of monoclonal antibodies was first .suggested more than 100 yr ago by Paul Ehrlich, who envisioned them as u "magic bullet" for the treatment of various diseases. Early on, immunization of animals was used mainly to generate tumor-specific polyclonal antibodies. Generation of monoclonal antibodies fur use in laboratory research and medical Icsls commenced in the mid-1970s. Not until the mid 1990s did technological advances allow the generation of monoclonal antibodies that were clinically useful as therapeutic agents. Monoclonal antibodies now constitute an integral component of laboratory diagnostics and are used in most laboratories that offer clinical testing.

The goal of Monoclonal Amihodies: Methods and Protocols is to provide a collection of methods that employ monoclonal antibodies in the clinical setting. The book starts with a chapter describing the gold standard method for generating mouse monoclonal antibodies through hybrldoma technology. Chapter 2 takes us to the future and describes methods for engineering recombinant and humanized antibodies. The authors describe the use of phage display and bacterial systems to generate monoclonal antibodies without the need for animal immunization. They also present methods for using these antibodies not only as therapeutic agents, but in laboratory experiments and clinical testing as well. Along the same lines, Chapter J describes a method for engineering soluble Fc fusion proteins. The Fc portion of monoclonal antibodies can be used to generate and purify large quantities of proteins (hat have the potential to be used for immunization as well as in elinicitl testing and patient monitoring.

The next chapters focus on laboratoiy methods that employ monoclonal antibodies. Chapter 4 describes the use of monoclonal antibodies in the contest of flow cytometry for the diagnosis and classification of hematological disease^ The authors provide detailed methods for staining and detecting specific antigens using flow cytometry, and discuss the use of algorithms to diagnose and classify hematological diseases. Chapter 5 provides methods for flow cytometric quantification of antigens present on the surface of cells. Quantifying the expression levels of these eel!-surface antigens allow s for ii belter understanding of the biology underlying antibody-based treatments, and has the potent i;i I to improve monitoring of such therapy. Chapter 6 discusses methods for quantifying CD 13 and CD 10 expression on granulocytes using the same quantitative flow cytometry approach, and shows how these levels can be used to muniiijr immune system function.

The use of flow cytometry has recently expanded from cell surface analysis to intracellular protein analy.sK. Measuring intracellular protein iv partieularly important for therapies that target the activity of specific oncoproteins (i.e., targeted therapy). These intracellular proteins must be monitored and quantified before and during therapy. Chapter 7 describes methods for the permeabilizutinn and quanLitati ve monitoring ofintraeellular proteins and their iit^ii^. iry (phosphorylation).

Changing direction from flow cytometry to immunohistochemistry, Chapter 8 depicls. methods that have transformed morphological evaluation and anatomical pathology from guesswork to obj^eiive science. The authors describe how monoclonal antibodies can be used in immu no histochemistry to diagnose hematological diseases. This approach is also applicable to other tumors and reactive diseases (hat can manifest with histological changes.

Although monoclonal antibodies have been used extensively in standard enzyme-1 inked immunosorbent assays (ELlSAs), the recent adaptation of polystyrene beads in place of microliter plates as the solid surface for capture of antibody binding has allowed the introduclion of new classes of testing with flow cytometry or Lumine.v platforms. This bead-based technology has several advantages over routing LLlSAs, mosl impnrtanlly the ability [n multiples and analyze several analytes simultaneously, Chapter 9 illustrates methods for the use of beads to measure cytokines, chemokines, and other soluble proteins. Similarly, Chapter 10, whiirb explores (he detection of HLT-3 and iis phosphorylation. represents a method for immunoprecipitating cellular proteins on beads and using flow cytometry to measure these proteins and their phosphorylation. Chapter 11, which focuses oil measuring levels of alineLujuiinah, a humanixed rat aniilxidy used for treatment of lymphoid cells thai express CD 52, describes a be ad -based method for quantifying the humanized antibody, CD¬°>2, and the almetuyumab/CD.Sl! iinmune enmplexes that ean be seen free in circulation. The most recent innovation using bead-based immunoassays with How cytometry in detecting chromosomal lranslocations is presented in Chapter 12 witlt the quantitation of the BCR-ABL fusion protein, which results from a l ran sloe at ion between ehnomosomes 9 and 22. The authors also describe a method for quantifying BCR-ABL phosphorylation, which may have clinical implications when patients with chronic myeloid leukemia are treated with ABL-specille kinase inhibitors.

This volume also contains chapters selected to provide additional examples of methodologies that employ monoclonal antibodies, Chapter 13 concentrates on detecting ma I lid rug-re si stance (MDR)in(umorcells presenting an approach for not only detecting (he MDR protein, bul also for demonstrating the active efflux that allows cytotoxic drugs to be pumped out of cells. Chapter 14 presents a method IW detecting human an [¬°bodies dine eted against therapeutic mouse antibodies; detection of these antibodies has significant clinical implications for the course of therapy. Although humanization of therapeutic antibodies has reduced the incidence of developing anti-mouse antibodies, the i^ue remains ;i serious concern, Finally, Chapter 15 illusirates methods that depend on highly automated instruments for detection of [gE in patients suffering from an allergy,

In summary, Monoclonal Antibodies: Methods tmtl Protocols pmv idiis descriptions of methods that cover a wide spetlrum of application* in the field of monoclonal antibodies. This field will continue to expand and provide new and innovative techniques, not only in the laboratory, but also as a basis that complements targeted therapy,

Mahcr Albitar

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