The production of polyclonal antibodies by the immunization of animals is a method that has been used for more than a century (1). Hybridoma technology was the next development, allowing the production of monoclonal antibodies (2). However, hybridoma technology has some limitations, some resulting from the instability of the aneuploid cell lines, but most of all, difficulties in producing human antibodies especially to toxic or highly conserved antigens (3).
From: Methods in Molecular Biology, vol. 295: Immunochemical Protocols, Third Edition. Edited by: R. Burns © Humana Press Inc., Totowa, NJ
In the past decade, the in vitro selection of antibodies from recombinant antibody repertoires has proven to bypass these limitations. In vivo libraries offer the capability of generating human antibodies in transgenic mice carrying the human IgG loci. Here, antigens are injected into the humanized mouse, followed by conventional hybridoma technology for selection and production of antibodies (4-6).
To be able to raise human antibodies against toxic, highly pathogenic, or nonimmunogenic antigens, the method of choice is the use of libraries and an in vitro selection process. The first antibody repertoires were generated and screened in phage Lambda (7,8) with limited success. The phage display method most commonly used today is based on the groundbreaking work of Smith (9). Here, the genotype and phenotype of a polypeptide are linked by fusing short gene fragments to the minor coat protein III gene of the filamentous bacteriophage M13. This results in the expression of this fusion protein on the surface of phage and allows affinity purification of the gene by the peptide binding. Shortly after this breakthrough, another method was developed in which antibodies were presented on the surface of M13, fused to pIII (10-15). By uncoupling antibody gene replication and expression from the phage life cycle by locating them on a separate plasmid (phagemid), genetic stability, propagation, and screening of antibody libraries was greatly improved (11,12,14,15). To date, "single-pot" antibody libraries with a theoretical diversity of up to 1011 independent clones have been assembled (16) to serve as molecular repertoires for phage display selections. An overview of antibody libraries is given by Hust and Dübel (17).
The novel way of isolating antibodies by their binding activity in vitro is called "panning," which refers to the method used by gold prospectors (18). The antigen is immobilized to a solid surface, such as nitrocellulose (19), magnetic beads (20), a column matrix (12) or, the most widely used, plastic surfaces as polystyrole tubes (21) or 96-well plates (11). The antibody phage are incubated with the surface-bound antigen, followed by thorough washing to remove the excess nonbinders. The bound antibody phage can subsequently be eluted and amplified by infection of Escherichia coli. This method allows the detection of a single antibody phage by panning, and as it can be selected by its resistance marker, it can give rise to a bacterial colony after elution. This illustrates the tremendous sensitivity of the method. This selection cycle can be repeated, by infecting the resulting E. coli colonies with a helper phage to produce new antibody phage, which can then be used for a new round of panning, and so on. The number of antigen specific antibodies will increase with every panning round. Usually two to six panning rounds are necessary to get specifically binding antibodies.
Other display techniques have been developed and successfully applied for antibody selection. Techniques requiring intracellular antibody expression, such as the two-hybrid system (22), are of restricted use because of the limited folding of antibody fragments in the reducing milieu of the cytoplasm (23). Bacterial surface display techniques were developed, which rely on fusing antibodies to peptidoglycan-associated lipoprotein (24,25). Recently, fusions to lipoprotein NlpA have been used (26), but a universal method has not yet been developed. To display antibodies on yeast, antibodies were fused with the Aga2p mating adhesion receptor embedded in the cell wall (27). An extravagant technique completely avoiding the use of living cells in the process uses the linkage of messenger ribonucleic acid (mRNA) to the translation product (protein). In ribosomal display, mRNA and the related protein (antibody) complexed to the ribosome are selected by antigen binding of the attached antibody fragment. Then, the antibody mRNA is amplified by reverse transcription polymerase chain reaction and a further round of selection can be started (28,29). A related concept is the puromycin linked display ("profusion"). The 3' end of the mRNA is fused to puromycin, which serves as a peptidyl acceptor. After translation, the resultant polypeptide is covalently linked to mRNA by the puromycin (30), allowing selection as previously described.
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