Antiserum, affinity separation

Individual animals immunized with the same substance can produce antibodies that may differ in affinities, titer, and specificities. " Such differences are appeu-ent with antibodies studied by the more classical physical chemical procedures. However, even among anti-enzyme sera harvested from individual rabbits that had been immunized with -lactamase, some were found to neutralize the activity of the enzyme, others to stimulate its activity, and still others were stimulatory and then inhibitory at higher concentrations." In radioimmunoassay, each antiserum from an individual animal must be characterized separately to select those that have the proper affinities and specificities. The production of monoclonal antibodies by hybridoma technology can yield molecules with defined specificities and affinities. As this technology becomes affordable for the average research laboratory, problems associated with antibody heterogeneity are due to diminish. 

Aaltonen et al. (61) compared RRA and RIA for atropine. These workers obtained preparations of receptor from rat brain and lyophilized them to a stable, dry form, They used the tritium-labeled quinuclidinyl benzilate at 35 Ci/mmol. The affinity constant was 0.48 nM, and by analysis of 25- xL serum samples they could obtain a sensitivity down to 1.25 ng/mL in serum. Nonspecific binding was again quite reasonable (4%) and a filtration-type separation was used. The d isomer of an atropine did not bind, and therefore, the cross-reaction of the d,l compound was 50% that of the ( isomer. For comparison they used RIA developed by the method of Virtanen et al, (37). The immunogen was an /-hyoscyamine-bovine serum albumin conjugate, but the antiserum was sensitive to both d,l and I isomers. Racemic tritium-labeled atropine was used as the radioligand. 

Monoclonal antibodies can be raised to defined antigens and provide very specific probes (17) practical protocols are provided by Cuello and Cote (18). It is important to keep in mind that monoclonal antibodies— in spite of their monospecificity—do not circumvent the problem of crossreactivity with imi-dentified tissue antigens. The same rigorous testing needs to be performed for monoclonals. To increase the specificity of polyclonal antisera, one can perform affinity purification and/or separate IgGs from the whole serum. For such purifications, commercial columns are available. Details on antiserum/antibody production have been given in many accounts fe.g., 13,18-23). 

High-performance resins can act as supports for specialized functional groups. For example, the separation of isoenzymes by affinity chromatography was accomplished on silica Glycophase to which adenosine monophosphate had been coupled (Ohlson et a/., 1978). The same investigators prepared specific immunoabsorbents by coupling antiserum to the Glycophase. 

The course of the immune response may be followed by measuring the titre of the antiserum, defined as the dilution giving 50% binding of the radioactive tracer. When the titre of the serum was high enough, its sensitivity, affinity and specificity were tested. The best bleedings were kept separately or pooled. They were stored either at +4 C after addition of 0.02% sodium azide, or at -20°C after dilution in an equal volume of glycerol. Table 2 shows the specificity of certain selected antisera. 

Figure 13 Purification of monospecific rabbit antibody against annexin VI. The rabbit polyclonal antibody against rat annexin Iv (CCBP 65/67) reacts against other annexins (CBP 35 and CBP 33). Monospecific polyclonal antibodies against annexin VI were prepared by immobilized antigen affinity chromatography. Purified annexin VI is immobilized on an epoxy activated CIM disk (BIA Separations) and 1 mL of antiserum is diluted 1 5 in PBS and applied to the disk (flow rate 3mL/min). Bound antibodies were eluted by 0.1 M glycine HCI, pH 3.0, in a step gradient. The Western blot analysis of EDTA extracts of Morris hepatoma rat plasma membranes with the antiserum before and after the affinity purification is shown.

One of the most careful investigations of this question is that by Haber and Richards (13), who utilized purified rabbit antibodies specific for the 2,4-dinitrophenyl (Dnp) and 2,4,6-trinitrophenyl (Tnp) groups. In contrast to many studies of this type, precautions were taken to ensure that the separated H chains were free of L chains, and analyses for purity were carried out. The H chains were recycled through Sephadex G-lOO until they were essentially free of L chains by two criteria failure to react with antiserum to L chains and the absence of the N-terminal alanine characteristic of rabbit L chains, as measured by a sensitive isotope-dilution assay. Light chains of antibody, either alone or combined with nonspecific H chains, possessed no activity detectable by the method of fluorescence quenching. By contrast, a large percentage of the H chains, polyalanylated or in combination with nonspecific L chains, were able to bind hapten specifically, but with reduced affinity. Some of... 

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