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Chapter 8 : Antigen-Antibody Reactions

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Abstract:

Reactions between antigens (Ags) and antibodies (Abs) are usefully exploited in many areas of life science research. The monoclonal Ab (MAb) technology developed by Köhler and Milstein allows for the production of unlimited quantities of Abs against virtually any molecule. As there is an immense volume of information concerning all aspects of Ag-Ab reactions in the literature, the objective of this chapter is to provide simple and useful protocols and an introduction to some of the more novel techniques. The routes of injection commonly used on rabbits, mice and rats include intradermal (i.d.), subcutaneous (s.c.), intramuscular (i.m.), intraperitoneal (i.p.), and intravenous (i.v.). Nucleic acid (DNA or RNA) immunization and genetic vaccines introduced in the early 1990s are some of the most important discoveries and novel strategies in vaccine development. The essential features of a DNA vaccine are a bacterial plasmid vector engineered to carry a DNA insert encoding the protein immunogen(s) of interest, a eukaryote gene promoter, and a poly(A) site to enable expression of the protein in mammalian cells. The vectors are usually maintained in and purified from . The polyclonal serum is expressed from the blood clot by centrifugation, and approximately 52% of its volume can be collected as serum. The serum can be used directly in Ag-Ab reactions, absorbed to deplete it of nonspecific or cross-reactive Abs, or fractionated to purify Abs free of other serum proteins.

Citation: Mutharia L, Lam J. 2007. Antigen-Antibody Reactions, p 138-167. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch08

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Outer Membrane Proteins
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Agarose Gel Electrophoresis
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Enzyme-Linked Immunosorbent Assay
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Figures

Image of FIGURE 1
FIGURE 1

HPLC separation of IgM MAb from ascites fluid. The first peak represents unbound material, and the second peak represents pure IgM Abs eluted after the addition of elution buffer 1. The arrow indicates the time at which the elution buffer was added.

Citation: Mutharia L, Lam J. 2007. Antigen-Antibody Reactions, p 138-167. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch08
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Image of FIGURE 2
FIGURE 2

SDS-PAGE profiles of IgM MAb purification by HPLC. Lanes: 1, molecular mass standards; 2, ascites fluid before purification (note the large number of bands that are normally found in animal serum proteins or body fluids); and 3, purified IgM MAb eluted from the column (the second major peak in Fig. 1 ). Note the presence of only two major bands representing the heavy and light chains at apparent molecular masses of 60 and 28 kDa, respectively.

Citation: Mutharia L, Lam J. 2007. Antigen-Antibody Reactions, p 138-167. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch08
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Image of FIGURE 3
FIGURE 3

Double immunodiffusion profiles of antisera to proteases of . Each well was filled with 10 μl of either an Ag or an antiserum preparation. Wells: A, rabbit antiserum to protease A; B, rabbit antiserum to protease B; C, a 1:1 (vol/vol) mixture of antiserum to protease A and antiserum to protease B, respectively. The Ags were placed into the outer wells. Well 1, protease B from strain 1; well 2, protease A from strain 2; well 3, extracellular products of strain 3. (Courtesy of K. Y. Leung and R. M. W. Stevenson.)

Citation: Mutharia L, Lam J. 2007. Antigen-Antibody Reactions, p 138-167. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch08
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Image of FIGURE 4
FIGURE 4

XIE of cell lysate and homologous polyclonal antiserum. A 10-μl portion of the lysate was loaded in the first-dimension gel (1D), the intermediate gel (IG) contained NaCl, and the second-dimension gel (2D) contained antiserum.

Citation: Mutharia L, Lam J. 2007. Antigen-Antibody Reactions, p 138-167. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch08
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Tables

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TABLE 1

Volumes of Ag-Ab mixtures for injection

Freund's adjuvants should not be used with i.v. injections.

Citation: Mutharia L, Lam J. 2007. Antigen-Antibody Reactions, p 138-167. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch08
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TABLE 2

Adjuvants and their characteristics

Citation: Mutharia L, Lam J. 2007. Antigen-Antibody Reactions, p 138-167. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch08
Generic image for table
TABLE 3

Commercial sources of immunoglobulin isotype determination reagents

Citation: Mutharia L, Lam J. 2007. Antigen-Antibody Reactions, p 138-167. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch08
Generic image for table
TABLE 4

Conditions for HPLC purification of various immunoglobulin isotypes

Citation: Mutharia L, Lam J. 2007. Antigen-Antibody Reactions, p 138-167. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch08

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