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Polyclonal antibody production in mammals is generally associated with multiple injections of antigens and adjuvant and repeated blood sampling procedures. In the production of second polyclonal antibodies, a number of critical steps can be identified that may influence the outcome of the animal experiment (immunological results and the pain and suffering of the animals). The goal of this work was to evaluate critical steps in the production of these antibodies, and to optimize production protocols that will ultimately result in effective antibody responses. This work was achieved through immunization of two healthy sheep by purified Alpha feto protein (AFP) antigen. Also, the present study involved the preparation of AFP standards from human cord blood. Furthermore, preparation of a radiolabeled AFP tracer of a high specific activity using 125I isotope by chloramine T method was undertaken. Moreover, this study provides that there was no observable difference between the Scottish Antibody Production Unit (SAPU) and Sigma SAM IgG and the two second antibodies obtained from the local sheep sera.