the vigorous antibody response. Immune function peaks at puberty and primary responses to new antigens decline with age. Female rabbits are generally preferred because they are more docile and are reported to mount a more vigorous immune response than males. At least two animals per antigen should be used when using outbred animals. This principle reduces potential total failure resulting from non-responsiveness to antigens of individual animals. Antigen preparation The size, extent of aggregation and relative nativity of protein antigens can all dramatically affect the quality and quantity of antibody produced. Small polypeptides (<10 ku) and non-protein antigens generally need to be conjugated or crosslinked to larger, immunogenic, carrier proteins to increase immunogenicity and provide T cell epitopes. Generally, the larger the immunogenic protein the better. Larger proteins, even in smaller amounts, usually result in better engagement of antigen presenting antigen processing cells for a satisfactory immune response. Injection of soluble, non-aggregated proteins has a higher probability of inducing tolerance rather than a satisfactory antibody response. Keyhole limpet hemocyanin (KLH) and bovine serum albumin are two widely used carrier proteins. Poly-L-lysine has also been used successfully as a backbone for peptides. Although the use of Poly-L-lysine reduces or eliminates production of antibodies to foreign proteins, it may result in failure of peptide-induced antibody production. Recently, liposomes have also been successfully used for delivery of small peptides and this technique is an alternative to delivery with oily emulsion adjuvants. Antigen quantity Selection of antigen quantity for immunization varies with the properties of the antigen and the adjuvant selected. In general, microgram to milligram quantities of protein in adjuvant are necessary to elicit high titer antibodies. Antigen dosage is generally species, rather than body weight, associated. The so-called “window” of immunogenicity in each species is broad but too much or too little antigen can induce tolerance, suppression or immune deviation towards cellular immunity rather than a satisfactory humoral response. Optimal and usual protein antigen levels for immunizing specific species have been reported in the following ranges: rabbit, 50–1000 µg; mouse, 10–50 µg; guinea pig, 50–500 µg; and goat, 250–5000 µg. Optimal “priming” doses are reported to be at the low end of each range. The affinity of serum antibodies increases with time (months) after injection of antigen-adjuvant mixtures and as antigen in the system decreases. Widely used antigen dosages for “booster” or secondary immunizations are usually one half to equal the priming