Protein Protocols Protein Protocols

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confirming that it is immiscible and thus oil-phase continuous.
3.7. Immunization Procedure
The emulsified conjugate can be administered in a variety of ways. For rabbits, the
most frequently used are multiple (30–50) intradermal injections in the neck or back
region, by four subcutaneous injections into each groin and axilla (0.5) (8). The latter is
the method we have successfully adopted for more than 20 yr. Injection into the footpads,
which was at one time commonly employed, provides no advantage in terms of
antibody response and should be avoided to prevent distress to the animals.
The procedure is as follows:
1. Bleed the animals and collect preimmune serum for later comparison with antisera produced
by the immunization procedure.
2. Primary inoculations are given in complete Freund’s adjuvant, 0.5 mL of emulsion into
each groin and axilla.
3. Booster injections are given at 2–4-wk intervals, in the same manner but with incomplete
Freund’s adjuvant. The optimum is probably about 4 wk, but time constraints and cost
may necessitate a shorter immunization schedule. With small synthetic haptens three to
five, or even more boosts may be required to produce the desired high titer or high avidity
antibody (see Notes 7 and 8).
4. After the first and subsequent boosts blood should be collected from an ear vein to test for
the antibody titer and avidity.
3.8. Antibody Characterization for Radioimmunoassay
1. Serial dilutions of antisera are incubated with radiolabeled peptide under routine assay
conditions to determine a working dilution.
2. The maximum displacement of radioactively labeled hormone from the antibody by the
minimum amount of unlabeled peptide (the maximum displacement slope) is one of the
main criteria for radioimmunoassay sensitivity.
3. Rapid screening for slope can be achieved by the addition of small amounts (usually about
10 fmol) of standard peptide to one set of a series of replicate antiserum dilutions set up to
determine the antiserum titer. The amount of standard used should reflect the useful range
(e.g., the concentration at which a hormone circulates).
4. Antibody heterogeneity may be due to use of nonhomogeneous antigens for immunization,
polymerization or degradation of the hapten or carrier after coupling, or individual
differences in the lymphocytic response to the antigen (9).
5. Existence of heterogeneity can be revealed by Scatchard analysis. However, for high titer
antisera there is frequently effectively only a single class of antibody that predominates in
the reaction. Other populations of lower concentrations and avidities make insignificant
contributions.
6. Specificity should be tested using related peptides.
4. Notes
1. The ratio of hapten reactive with protein is usually arranged to be in excess of 41 to
achieve better antigenicity with respect to the hapten. Some authorities prefer ratios as
high as 401 but in our experience this gives a lower affinity antibody response. This is
presumably due to conjugation between hapten molecules rather between hapten and carrier.
The hapten and protein carriers should be both present in high concentration to
increase the efficiency of the cross-linking between the molecules.
2. Coupling of a hapten at a specific site gives more chance of governing which part of the