Affinity-purified antibodies

Nakamura KC, Kameda H, Koshimizu Y, et al. Production and histological application of affinity-purified antibodies to heat-denatured green fluorescent protein. J. Histochem. Cytochem. 2008 56 647-657. 

Osborn, M., and Weber, K. (1982) Immunofluorescence and immunocytochemical procedures with affinity purified antibodies Tubulin-containing structures. Meth. Cell Biol. 24, 97-132. 

Affinity-purified antibodies are isolated from antisera by immunoaffinity chromatography using antigens coupled to agarose beads. 

The purity of the reagents is critical. Always use the highest quality available, i.e., affinity-purified antibodies, EM-grade glutaraldehyde, distilled... 

Dialyze the affinity-purified antibody against two changes of 2 L of phosphate/ saline buffer, pass through a 0.22-pm filter, and store at 4°C in the presence of 0.02% (w/v) NaN3... 

Dialyze the affinity-purified antibody-toxin conjugate into PBSA, filter-sterilize in a sterile hood, and store at 4°C or, freeze rapidly and store at-70°C (see Notes 15 and 16). 

The eluted antibody is then neutralized and dialyzed to remove the salts. Such antibody usually represents only 1% of the total IgG in the serum. This procedure is different from the so-called affinity-purified antibody in which protein A or protein G is used the latter purifies IgG only and has no meaning in relation to selective antigen reactivity. 

FIGURE 6.14. Effect of pretreatment with 1% sodium dodecyl sulfate (SDS) on immunostaining of cultured epithelial cells with anti-AE1/2 affinity-purified antibody at a final concentration of 0.45 i,g/ml. (A) In the absence of SDS treatment, AE2 is barely detectable in MDCK cell cultures. (B) After SDS treatment a bright basolateral membrane staining is visible. Reproduced, with permission, from Brown et al. (1996). Copyright 1996 Springer-Verlag. 

Antibody this should be the pure IgG fraction or, better, affinity-purified antibody from an antiserum or pure monoclonal antibody (see Chapter 7). 

B22. Boutjdir, M.,Chen, L.,Zhang, Z. H., Tseng,C.E., DiDonato, F., etal, Arrhythmogenicity of IgG and anti-52-kD SSA/Ro affinity-purified antibodies from mothers of children with congenital heart block. Circ. Res. 80, 354-362 (1997). 

Fig- ( ) Gel electrophoresis patterns of immune serum and affinity purified antibodies [A], Density gradient ultracentrifugation (B), top pattern glucose oxidase, lower pattern mesquite antibody. 

Cross-reactivity of antigens from related species is a common problem in multi-staining. This can often be avoided by using affinity purified antibodies, sub-type specific antibodies or site/ region specific antibodies. For more detail on cross-reactivity, see Chapter 1, Antibodies. 

The capacity of immunoaffinity HPLC columns prepared using affinity-purified antibody against ricin A chain, abrin A chain, gelonin, and momordin varies between 400 and 600 pg/mL of bed vol for the appropriate RIP. In the case of the antibody-toxin conjugates made with these RIPs, the capacity is at least 2 mg/mL of bed vol. 

The method presented may be easily modified to accommodate primary polyclonal heteroantisera prepared in goats, rabbits, or other species. The advantages of monoclonal antibodies are the high specificity of these reagents and their standardization from laboratory to laboratory. Monoclonal antibodies will react only with specific and crossreacting epitopes. Serum-derived antibodies (including some sold as affinity-purified antibodies) may be reactive with many irrelevant antigens. 

Blakely, R.D., Ory-Lavollee, L., Grzanna, R., Koller, K.J. and Coyle, J.T. (1987) Selective immunocytochem-ical staining of mitral cells in rat olfactory bulb with affinity purified antibodies against N-acetyl-aspartyl-glutamate. Brain Re.v, 402, 373-378. 

Besides choosing the appropriate Ab, the assay method can be designed or manipulated to improve assay specificity using (a) protein precipitation, (b) liquid/liquid or solid-phase extraction, (c) HPLC separation of the analyte from the interfering compounds, (d) sample dilution with buffer or control matrix, or (e) an affinity solid phase (e.g., antibody-coated microtiter plate or polystyrene beads) to capture the analyte followed by wash steps. Affinity-purified antibodies and protein blockers are used in EIA to decrease nonspecific binding in plate assays. Increasing incubation time to reach equilibrium also improves binding specificity. 

Where possible, the antibodies used for labeling should be pure (see Chapters 2-5). Fluorochrome-labeled, affinity-purified antibodies produce less background and lower non-specific fluorescence than are obtained with fluorescent antiserum or... 

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