AV-1 protein is a membrane-localized glycoprotein which is expressed in a stage- and position-specific manner during limb development and is a candidate pattern formation molecule. In this study, we have purified the AV-1 protein using an affinity column coupled with the anti-AV-l monoclonal antibody. Using the purified protein, we produced a rat anti-AV-1 antiserum. Characterization of the antiserum by immunoblotting and immunostaining showed highly specific reactivity which was identical to that of the anti-AV-1 monoclonal antibody. The rat anti-AV-1 antiserum was injected into the vitelline vein of stage 20 chick embryos and these embryos were allowed to develop further to examine the effect of the antibody on limb pattern. The injected embryos developed deformed limbs. Many of the abnormalities in limb cartilage pattern were related to the normal pattern of the AV-1 expression, suggesting an involvement of the AV-1 protein in the process of limb pattern formation. (C) 1996 Academic Press, Inc.