Bypassing hybridomas and cloning of Mab genes.
genes for antibodies can be amplified through polymerase chain reaction (PCR)
using 'universal primers'
(universal primers will carry conserved sequences for most antibodies). By building restriction sites in the above primers, the amplified VH
and VL genes
can also be cloned directly for expression in mammalian cells or bacteria (Fig. 43.3). The raw material for PCR may be hybridomas or B cells, which may be homogenous (if derived from single cells) or heterogeneous. In the latter case, a variety of VH
genes will be amplified and will combine at random to produce as many as 106
clones for antibody genes (from 1000 different VH
and 1000 different VL genes).
These genes will be cloned in λphage and their products (particularly Fab fragments) can be screened for antigen binding activities. From such a large number of combinations in a combinatorial library, it is very difficult to recover the original pairs of V genes (e.g. VHa
is a original pair; VHa
is a new combination). However, the, complexity may be reduced by using antigen-selected B lymphocytes (filters coated with antigen can be used for screening). Different steps involved in cloning paired VH
genes are outlined in Figure 43.4.
Designing and building of Mab genes.
|Fig. 43.3. PCR cloning of rearranged Vh and genes into expression vectors.
|Fig. 43.4. Strategies for cloning paired Vh and Vl genes from lymphocytes of an immunized animal.
The antigen binding sites of antibodies have been studied in some detail in recent years. This led to modelling of entirely new antibodies, sometimes for their use as enzymes. This modelling through computer graphics can be used for alteration of antibody genes or for synthesis of entirely new genes. These genes can be cloned and expressed in bacteria. The antibodies produced can be tested for their specificity and affinity for specific antigens. Primary and
secondary libraries for
antibody genes. In this method a repertoire of antibody genes can be prepared by using genes that can be obtained from a number of different sources including the following : (i) rearranged V genes from animals obtained through the use of PCR (with universal primers); (ii) new V genes obtained through gene conversion, a process adopted in birds; (iii) rearranged genes obtained from mRNA through reverse transcription; (iv) designing entirely new V genes or D segments. The next step is to allow the expression of library in bacteria and screen antibodies for antigen binding activities. The screening can be done on membrane filters coated with antigen. In future, the screening procedures may be replaced by methods of selection. In either case the selected VH
genes can be subjected to mutations to increase the affinity of an antibody for a specific antigen. A variety of methods for the above strategy are being developed, so that in future monoclonal antibodies will be produced without hybridomas and lymphocytes.