Frank Breitling

Production of monoclonal antibodies against epitopes of the main coat protein of filamentous fd phages

Three monoclonal antibodies (MAbs) were produced which react with epitopes of the main structural coat protein (pVIII) of filamentous fd phages as demonstrated by solid-phase fluorometric enzyme immunoassays and by immunoelectron microscopy. The antibodies are of the IgG1, IgG2a and IgG2b immunoglobulin subclasses. Since they also react with recombinant phages expressing antigen fragments in their pIII region they may be suitable reagents for the demonstration and isolation of filamentous phages used in recombinant protein technology.

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.

Recent Developments in Antibody Engineering

Rapid growth in the field of antibody engineering occurred after it was shown that functional antibody fragments could be secreted into the periplasmic space and even into the medium of Escherichia coli by fusing a bacterial signal peptide to the antibodys N-terminus (1,2). These findings allowed scientists to transfer the principles of the immune system for producing specific antibodies to a given antigen into a bacterial system (3). It was now possible to establish antibody libraries in E. coli that could be directly screened for binding to antigen. This was accomplished at first by transforming E coli with plasmids containing polymerase chain reaction (PCR)-amplified immunoglobulin families from the lymphocytes of immunized mice. Immunogen-reactive recombinant antibodies were then selected by an enzyme-linked immunosorbent assay (ELISA) of the bacterial supernatant from isolated bacterial colonies (4). This procedure was subsequently improved upon by inserting the antibody operon into bacteriophage λ. Antibody libraries were then able to be efficiently transfected into E. coli and plaque lift-offs of lysed bacterial colonies on nitrocellulose could be screened for reactivity to a radioactive labeled immunogen (5-7).