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After separation and recombination of chains derived from antibody molecules, a combination of specific heavy (H) and light (L) chains usually provides the most avid binding sites, although isolated specific H and L chains may have some affinity for the antigen. The assembly of H and L chains of different specificities gives molecules with reduced antibody activity.'-7 Experiments on myeloma cells indicate that H and L chains are made on different classes of polyribosomes,8' 9 but the mechanisms which bring them together are not clear. If, as is generally believed, one plasma cell synthesizes antibodies of but a single specificity, there is no possibility of assembling chains of different specificities. However, the situation might be different in a cell-free system in which the protein-synthesizing apparatus of cells of different specificities are no longer kept apart. In the absence of a special mechanism for assembling chains of like specificity, the resulting antibody activity might be minimal or absent. In this case, a cell-free system would be of no use for exploring the roles of such factors as messenger RNA, transfer RNA, or ribosomal surface in the acquisition of antibody specificity. Evaluation of these factors, modifiable only under cell-free conditions, requires a system in which synthesis of protein with definite and easily measured antibody activity occurs. The present study, using a system previously described,10 shows that H and L chains can be completed in cell-free conditions, and combined to form molecules with significant antibody activity, located within the microsomal vesicles. This may indicate the cell-free assembly of H and L chains of the same specificity, made possible because the microsomal vesicles, whose structure is well preserved in the cell-free system used, are the site of assembly of immunoglobulin chains in the intact cell. Materials and Methods.-Rabbit, rat, and bovine gamma globulin (BGG), and bovine serum albumin (BSA) were obtained from Pentex, Inc., and 2,4-dinitrophenyl-lysine (DNP-lysine) and streptomycin sulfate from Mann Research Laboratories. DNP-BGG (50 hapten groups/mole of protein), DNP-BSA (10 groups/mole), and p-toluenesulfonyl-e-aminocaproic acid (tosyl-EACA) were prepared." Bromoacetylcellulose (BAC) was prepared, coupled to DNP-BSA or BSA, and washed according to Robbins et al.;'2 the coupled adsorbent was kept in 9 vol of phosphate-buffered saline (0.15 M NaCl-0.01 M phosphate, pH 7.0) (PBS). H3-leucine (5 c/mM) and a C"4amino acid mixture were purchased from New England Nuclear Corp. Rats were injected in each footpad with 2 X 1() killed H. pertussis (Eli Lilly and Co.), alone or mixed with 50 mug DNPBGG.13 After 1 week the rats were killed and the lymph nodes excised. Cell-free incubation: Preparation of microsomes from lymph nodes and conditions of cell-free incubation were as described previously,'0 except that the concentration of sucrose in the incubation medium was 0.25 or 0.44 M. One mg protein/ml of lymph node microsomes and 2 mg/ml of liver pH 5 fraction were incubated in the presence of either 10 M&c/ml of H3-leucine or a mixture of C'4-amino acids (the latter only for the autoradiographic studies) in a total volume of 20 ml. After 30 min, 10 or 20 ml of ice-cold incubation medium were added to the cell-free system, which was kept in ice for 5 min and centrifuged for 60 min at 105,000 gay. Aliquots of the supernatant were kept for determination of protein radioactivity.'0 The microsomal pellet was resuspended

Studies on cell-free synthesis of rat immunoglobulins, II. Synthesis of immunoglobulin and of antibody to the dinitrophenyl hapten.