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Figure 2 Constructing antibody phage display libraries. Immunized libraries are constructed from the B lymphocytes of an immunized individual (A) whereas nonimmunized repertoires are obtained from the B cells of an individual not undergoing an immune response (B). The lymphocytes are used to prepare RNA and cDNA from which the VH and VL genes are polymerase chain reaction (PCR) amplified using specific oligonucleotides. In a synthetic library (C), previously cloned V„ segments are used to construct functional VH genes by adding randomized CDR3 sequences to the immunoglobulin genes by PCR. Completed scFv genes are cloned in phagemid vectors which are used to produce the phage antibody particles.

production of phage antbodies In Escherichia con

Figure 2 Constructing antibody phage display libraries. Immunized libraries are constructed from the B lymphocytes of an immunized individual (A) whereas nonimmunized repertoires are obtained from the B cells of an individual not undergoing an immune response (B). The lymphocytes are used to prepare RNA and cDNA from which the VH and VL genes are polymerase chain reaction (PCR) amplified using specific oligonucleotides. In a synthetic library (C), previously cloned V„ segments are used to construct functional VH genes by adding randomized CDR3 sequences to the immunoglobulin genes by PCR. Completed scFv genes are cloned in phagemid vectors which are used to produce the phage antibody particles.

structed from the V regions expressed by the B cells of 'nonimmunized' individuals in an attempt to recruit all the diversity present in the pool of IgM-bearing B cells (Figure 2B). These so-called 'naive' antibody phage display libraries harbor a high proportion of germline V regions and are expected to encode a different collection of antibodies as compared to the immunized libraries. A third approach to create diverse libraries exploits the use of large collections of cloned V gene segments to which randomized CDR3 and JH regions are fused in vitro by the polymerase chain reaction (Figure 2C). It may be anticipated that such semisynthetic libraries contain

'novel' specificities not present in the actual antibody repertoires of humans. Phage antibody display libraries of various sizes, constructed according to the above-mentioned strategies have been described. As a general rule, it appears that large-sized libraries (>1010 clones) permit the direct selection of high-affinity antibodies, even from naive or semisynthetic repertoires.

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