Non-immune libraries

Non-immune libraries are produced in a similar fashion, but using B-lymphocytes fromnon-im-munized donors as a source of antibody genes. This approach becomes necessary if initial immunization with the antigen of interest is not possible (e.g. due to ethical considerations). Although such... 

Two types of antibody libraries can be constructed, immune or non-immune. Immune libraries are constructed by immunizing the animal of interest with an antigen(s). In the case of humans, the source can be volunteers with the disease or condition under study (Persson et al., 1991). Human antibodies have also been obtained from severe combined immunodeficiency mice populated with human peripheral blood... 

Vaughan, T. J., Williams, A. J., Pritchard, K., Osbourn, J. K., Pope, A. R., Eamshaw, J. C., McCafferty, J., Hodits, R. A., Wilton, J., and Johnson, K. S. (1996). Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat. Biotechnol. 14, 309-314. 

De Haardt, H. J., van Neer, N., Reurst, A., Hufton, S. E., Roovers, R. C., Henderikx, P., et al. (1999) A large non-immunized human fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J. Biol. Chem. 274, 18,218-18,230. 

Sheets MD, et al., Efficient construction of a large non-immune phage antibody library the production of panels of high affinity human single-chain antibodies to protein antigens, Proc. Natl. Acad. Sci. USA., 95 6157-6162, 1998. 

Vaughan, T.J., et al., Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library, Nat. BiotechnoL, 14, 309, 1996. 

Soderlind, E. Carlsson, R. Borrebaeck, C. A. Ohlin, M., The immune diversity in a test tube-non-immunised antibody libraries and functional variability in defined protein scaffolds, Comb. Chem. High Throughput Screen. 2001, 4, 409-416... 

The quality of mRNA is important in generating recombinant antibodies. It is particularly important to ensure minimal ribosomal RNA contamination when cDNA is random primed for library construction to reduce the proportion of clones which represent non-mRNA species. The spleen mRNA of immunized mice is presumably derived mainly from plasma cells, as the level of Ig mRNA in these cells is up to f000-fold greater than in resting B cells. For spleens, extraction of total RNA followed by mRNA isolation on oligo(dT)-cellulosc is recommended in order to effectively process the large number of cells present in a spleen. Cytoplasmic RNA Extraction Kit (see Protocol 2) followed by mRNA Purification Kit is highly recommended. 

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