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Assay of Radiolabeled VIP Binding and Hydrolysis by Antibodies

High-specific-activity radiolabeled polypeptide probes permit sensitive detection of antigen-antibody interactions. In the case of small polypeptides labeled with 125I, various iodinated forms can be separated by reversed-phase HPLC and resolved peaks characterized by ...

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Molecular Modeling of Antibody-Combining Sites

Antibodies possess a vast repertoire of specificity and affinity. To understand the molecular basis of antibody function, we require high-resolution X-ray crystallographic structures and good solution structures of free and antigen-bound antibodies. The number of reported an ...

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Radiolabeling of Antibodies for Therapy and Diagnosis

Radioimmunotherapeutic and radioimmunodiagnostic procedures have been the subjects of nearly 200 clinical trials since 1978. Applications range from diagnosis of tumor deposits to systemic radiation therapy. Originally, murine IgG was the most common antibody form used cli ...

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Crystallographic and Chromatographic Methods for Study of Antibody Light Chains and Other Proteins

A three-dimensional structure of reasonably high resolution is the starting point of any protein engineering project, whether intended to improve the affinity of an antibody, change the specificity of an enzyme, or to test a hypothesis regarding the role of a particular residue in determin ...

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Structure and Properties of Human Immunoglobulin Light-Chain Dimers

Antibodies are multisubunit proteins, but unlike many other multisubunit proteins, it is possible to study structural and functional properties of an antibody subunit free of the others. Thus, antibodies are singular proteins in that they are highly amenable to analysis by a fundamental ...

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Purification of Reduced and Alkylated Antibody Subunits

Antigen-binding sites in antibodies are formed by the variable regions of light (L) and heavy (H) chains. Delineation of antigen interactions with the individual subunits of antibodies is of interest for several reasons, including: 1. F

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Murine Monoclonal Antibody Development

Major advances in the analysis of biomolecular structure have emerged from the development of highly specific monoclonal antibodies (MAb) by K�hler and Milstein in 1975 (1). MAb are produced from hybridoma cell lines that secrete a single species of antibody to a unique antigen. The advantag ...

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Comparative Properties of Polyclonal and Monoclonal Antibodies

This chapter was written with the express goal of introducing several basic concepts involved when antigens and antibodies interact in vitro to the novice reader and to serve as a review of these concepts to the more advanced reader. Most of these concepts also apply to in vivo reactivity. Antigen- ...

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Measurement of Endotoxin

Bacterial endotoxin is probably the most common significant contaminant that might be found in antibody preparations. It is found ubiquitously in normal environments but its pyrogenic effects in vivo can be lethal. It is absolutely vital to control the level of endotoxin in therapeutic pr ...

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Aseptic Vial Filling

Ideally, injectable drugs are sterilized in their final containers by a foolproof method like autoclaving. This is not possible for biologicals like monoclonal antibodies (mAbs), so they must be manufactured aseptically, sterilized by filtration and then filled into sterile vials or ...

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Measurement of Antibody Concentrations by Hemagglutination

During every stage of development and production of diagnostic or therapeutic antibodies, it is necessary to have an assay to measure antibody concentration. Several techniques are routine in virtually all antibody laboratories. High-performance liquid chromatograpy (HPLC) ...

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Enzymatic Digestion of Monoclonal Antibodies

Originally, digestion of antibodies by proteolytic enzymes was used to study their structure. Many diverse structures can be obtained by fragmentation of the different classes of antibody with different enzymes, or by using the same enzyme and changing the conditions Fig. 1). Not all the frag ...

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How to Make Bispecific Antibodies

This protocol describes the production of bispecific F(ab’)2 antibody derivatives (BsAbs) by the linking of two Fab’ fragments via their hinge region SH groups using the bifunctional crosslinker o-phenylenedimaleimide (o-PDM) as described by Glennie et al. (1,2). The procedure is illus ...

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Determination of the Immunoreactivity of Radiolabeled Monoclonal Antibodies

Radiolabeled monoclonal antibodies (mAbs) are in use for numerous immunoassays and localization studies both in vitro and in vivo. It is often important to determine to what extent the radiolabeling procedure has affected the immunoreactivity of the antibody. If, for example, the radio ...

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Radiolabeling of Monoclonal Antibodies

Radiolabeling of monoclonal antibodies (mAbs) is often one of the principal methods for the in vitro and in vivo assessment of these reagents, whether this is determining the affinity of a new reagent, preparing for preclinical testing, or clinically for diagnostic or therapeutic use.

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How to Set Up an ELISA

The enzyme-linked immunosorbent assay (ELISA) represents a simple and sensitive technique for specific quantitative detection of molecules to which an antibody is available (1,2). Although there are a huge number of variations based on the original ELISA principle, this chapter will f ...

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Use of Biosensors to Measure the Kinetics of Antibody-Antigen Interactions

The affinity and kinetics of antibody-antigen interactions are increasingly realized to be important parameters in determining the usefulness of antibodies in both in vivo and in vitro settings (1). As discussed in Chapter 1, the affinity of an antibody (given by the association or dissoci ...

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SDS-PAGE and Western Blotting

Proteins can be separated according to their molecular sizes and charges, since these factors will determine the speed at which they will travel through a gel. The SDS-PAGE method involves the denaturation of proteins with the detergent sodium dodecyl sulfate (SDS) and the use of an electric cur ...

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Measurement of HAMA and Anti-Idiotypic Antibodies

Murine monoclonal antibodies (mAbs) when administered to patients are perceived much as any other foreign antigen: an immune response is usually mounted against them, the result of which is the generation of human antimouse antibodies (HAMA). Even in the absence of such immunization some p ...

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Flow Cytometric Analysis

Flow cytometry, as the name suggests, is the analysis of cells (which carry one or more fluorescent labels) moving in a fluid flow (1,2). This technique has become widely used because of the enormous increase in the number and range of specificities of antibodies to cell determinants. Monoclonal and ...

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