Rescue Strategy for Nonviable Hybridomas: Amplifying Heavy- and Light-Chain Sequences from Genomic DNA by Polymerase Chain Reaction.

Many hybridoma researchers have lost an important hybridoma because of instability, mechanical breakdown of a freezer, or contamination. In many cases, however, it is possible to retrieve the heavy- and light-chain sequences from those hybridomas. Although most cloning methods associated with isolation of VH or VL gene sequences require the isolation of intact, nondegraded mRNA, alternative approaches based on genomic DNA have been reported.

Modification of Antibody Function by Mutagenesis.

The ability to "fine-tune" recombinant antibodies by mutagenesis separates recombinant antibodies from hybridoma-derived antibodies because the latter are locked with respect to their properties. Recombinant antibodies can be modified to suit the application: Changes in isotype, format (e.g.

Using Phage Display to Create Recombinant Antibodies.

A variety of phage display technologies have been developed since the approach was first described for antibodies. The most widely used approaches incorporate antibody sequences into the minor coat protein pIII of the nonlytic filamentous phage fd or M13. Libraries of variable gene sequences, encoding either scFv or Fab fragments, are made by incorporating sequences into phagemid vectors.

Creation of Recombinant Antibodies: Using 5'-RACE to Amplify Immunoglobulin Sequences.

Modern molecular biology techniques have been applied to the production of therapeutic antibodies. Nonetheless, recombinant antibodies remain the exception rather than the rule for the antibodies used in most research and diagnostic applications. The lack of penetration of recombinant antibodies into the research arena can be attributed largely to the fact that the Ig gene families are large and the variable region gene segments are, indeed, variable, precluding the use of polymerase chain reaction (PCR) with two simple primers to amplify the heavy and light chain gene segments.

Creation of Recombinant Antibodies: Using Degenerate Oligonucleotides to Amplify Heavy- and Light-Chain Sequences.

Recombinant antibodies can be modified to suit the application: Changes in isotype, format (e.g., scFv, Fab, bispecific antibodies), and specificity can be made once the heavy- and light-chain sequences are available.