Labelling of mouse alloantibody with tritiated dl-alanine

Abstract

Radio-iodination is not a satisfactory method of labelling mouse antibody, which is peculiarly susceptible to destruction during the process of iodination. An alternative which causes very little loss of antibody is the attachment of a tritium-labelled amino acid to mouse γG by peptide linkage. This is accomplished by reaction of γG with DL-alanine N-carboxy anhydride under mild conditions. The method is applicable to estimation in vitro of the relative amounts of H-2 antibody absorbed by viable cells, and of the relative amounts of H-2 antigen on viable cells. Non-specific uptake is virtually eliminated by: (i) prior absorption of the labelled product in vivo, (ii) pre-incubation of the cells in 20 per cent foetal bovine serum and its inclusion in the suspending medium, and (iii) performance of absorption procedures in the cold.