Cloning and Expression of Single-Chain Fragments (scFv) from Mouse and Rat Hybridomas.

For cloning and expressing the antigen-binding variable (Fv) portion of an antibody in Escherzchia coli, vectors have been constructed that combine the two variable regions (V(H) and V(L)) with a peptide linker (1-3). The genetic information for V(H) and V(L) is generally amplified from hybridoma cells using the polymerase chain reaction (PCR) with antibody-specific primers A variety of primer sets for the amplification of mouse-variable domams has been developed that are particulary suitable for the generation of complex mouse libraries consisting of more than 10(5) different antibody sequences (4,5). For this purpose, an equimolar amplification of all different antibody genes present in the cDNA mixture should be sought. Therefore, complex sets of primers have been designed Everyone of the target cDNAs should be prrmed with an oltgonucle-otide hybridizing with a standardized affinity to prevent stronger amplification of sequences with a better match to a primer. However, for the amplification of the variable region genes of a particular single hybridoma clone, a much simpler set can be employed. Long primers allowing a high number of mismatches have been successfully used to specifically amplify antibody DNA from a variety of cell lines, including rat hybridomas (6-8). However, some of the restriction sites (in particular P(st)I and BarnHI) introduced for subsequent cloning were found to be frequently present as internal sites in the DNA coding for mouse-antibody-variable domains. Therefore, additional restrtction sites were introduced that occur only rarely. The resulting primer list IS described in Table 1, and a protocol for the amplification of V(H) and V(L) DNA is given in Section 3.1.. Table 1 Oligonucleotides for the Amplification of Mouse and Rat Immunoglobulin-Variable Region DNA γ-Chain CHl domain: Bi4 5 ' -CCAGGGGCCAGTGGATAGACAAGCTTGGGTGTCGTTTT Hind III Heavy-cham variable domain: Bi3f 5 ' -CAGCCGGCCATGGCGCAGGT (C/G) CAGCTGCAG (C/G) AG NcoI PvuII, Pst I κ-Chain constant domain: Bi5c 5 ' -GAAGATGGATCCAGCGGCCGCAGCATCAGC BamHI NotI κ-Chain variable domain Bi8b 5 ' -ATTTTCAGAAGCACGCGTAGATATC (G/T) TG (A/C) T (G/C) ACCCAA (T/A) CTCCA MluI EcoRV.