7.2 Making antibodies
All methods used in immunochemistry rely on the antibody molecule or derivatives of
it. Antibodies can be made in various ways and the choice of which method to use is
very much dependent on the final assay format. For an antibody to be of use it has to
have a defined specificity, affinity and avidity as these are the qualities that determine
its usefulness in the method to be used. There are considerable cost differences in
producing the various antibody types and it is important to remember that the most
expensive product is not always the best.7.2.1 Polyclonal antibody production
Polyclonal antibodies are raised in appropriate donor animals, generally rabbits for
smaller amounts and sheep or goats for larger quantities. Occasionally rats or mice
can be used for small research quantities of antibody. It is important that animals are
sourced from reputable suppliers and that they are housed and managed according to
domestic welfare legislation.
Usually antigens are mixed with an appropriateadjuvantprior to immunising the
animals. Adjuvants are substances which increase the immunogenicity of the antigen
and are used to reduce the amount of antigen required as well as stimulate specific
immunity to it. Adjuvants may be chemicals such as detergents and oils or complex
proprietary products containing bacterial cell walls or preparations of them. Pre-
immune blood samples are taken to provide baseline IgG levels (Fig. 7.4). Immunisations
are spaced at intervals to maximise antibody production usually at least 4–6 weeks
apart although the first two may be given within 14 days. Blood samples are taken 10
days after the immunisation programme is complete and the serum tested for specific
activity to antigen by a method suchenzyme-linked immunosorbent assay (ELISA)
(see Section 7.3). Usually a rangeof doubling serum dilutions are made (1/100–1/12 800)
and tested against the antigen. Serum from a satisfactory course of immunisations will
detect antigen at 1/6400 dilution indicating high levels of circulating antibody. Once
a high level of circulating antibody is detected in test bleeds then donations can be
taken. Animal welfare legislation governs permissible amounts and frequency of bleeds.
Donations can be taken untilthe antibody titre begins to drop and if necessary the
animal can be immunised again and a second round of donations taken.
Blood donations are allowed to clot and the serum collected. Individual bleeds may
be kept separate or pooled to provide a larger volume of standard product. Serum can
be stored at 4C or lower for longer periods.
It is also possible to produce antibodies in chicken eggs. Avian immunoglobulin is
known asimmunoglobulin Y (IgY)and chickens secrete it into eggs to provide
protection for the developing embryo. This can be utilised for effective polyclonal
antibody production. The chickens are immunised three or four times with the antigen
and the immune status monitored by test bleeds. Eggs are collected and can yield up to
50 mg antibody per yolk. The antibody has to be purified from the egg yolks prior to
use and a number of proprietary kits can be used to do this. Occasionally antigens that
give a poor response in mammals can give much higher yields in chickens.273 7.2 Making antibodies