THE PRODUCTION OF MONOCLONAL ANTIBODIES USING RECOMBINANT DNA TECHNOLOGY

Abstract

In recent years a new technology for producing monoclonal antibodies has emerged, that of production through the use of random combinatorial libraries expressed in Escherichia coil. This project was undertaken In order to develop this technology. Spleen cell RNA was isolated from mice previously immunized with ricin. This RNA was converted to RNA:cDNA, which was then amplified using polymerase chain reaction (PCR) to produce two cDNA libraries, one containing immunoglobulin heavy chain cDNA and the other having immunoglobulin light chain cDNA. After screening, the libraries were randomly combined and rescreened. Twenty-two candidate clones from the random combinatorial library were selected on the basis of heavy and light chain expression and ricin-binding activity. Fourteen clones were converted from lambda phage to plasmids via in vivo excision, followed by analysis of induced broth culture supernatants for protein production and ricin-binding activity. Several different assays were performed in an effort to characterize these clones. Results were difficult to interpret due to low levels of antibodies in the culture preparations. Two clones, 3-12 and 6-16, were selected for "chain shuffling" on the basis of collective evidence that they exhibited ricin-binding activity and expressed only heavy chains. Insert DNA from these clones was ligated to DNA from the light chain cDNA library in an attempt to produce Fab fragments having ricin-binding activity. Western blots of protein from culture supernatants of "chain shuffled" clones indicated that one clone, 3-12/L-1, had ricin binding activity. Additional testing using the Pharmacia BlAcore confirmed this activity.