Multiplexed PrEST immunization for high-throughput affinity proteomics

• Published in: Journal of immunological methods 2006-08, Vol.315 (1), p.110-120
• Main Authors: Larsson, Karin, Wester, Kenneth, Nilsson, Peter, Uhlén, Mathias, Hober, Sophia, Wernérus, Henrik
• Format: Article
• Language: English
• Subjects: Affinity purification; Animals; Antibodies - chemistry; Antibodies - isolation & purification; Antibody Specificity; Bioengineering; Biological and medical sciences; Bioteknik; Expressed Sequence Tags - chemistry; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Immunization - methods; Immunohistochemistry; MEDICIN; MEDICINE; Molecular immunology; Monospecific antibody; Multiplex antigens; PrEST; Protein Array Analysis - methods; Proteomics; Proteomics - methods; Rabbits; Techniques; TECHNOLOGY; TEKNIKVETENSKAP
• ISSN: 0022-1759; 1872-7905; 1872-7905
• DOI: 10.1016/j.jim.2006.07.014

Monospecific antibodies dfdfdfdf (msAbs) generated through antigen specific purification of polyclonal antisera are valuable tools in proteome analyses. However, proteome wide generation of msAbs would require extensive immunization programs. Therefore, it would be desirable to develop efficient immunization and purification methods to reduce the number of animals needed for such antibody-based research. Here we describe a multiplex immunization strategy for generation of msAbs towards recombinantly produced human protein fragments, denoted PrESTs. Antisera from rabbits immunized with a mixture of two, three, five and up to ten different PrESTs have been purified by a two-step immunoaffinity-based protocol and the efficiency of the purification method was analyzed using a two-color protein array concept. The obtained results showed that almost 80% of the animals immunized with antigens composed of two or three different PrESTs yielded antibodies recognizing all the included PrESTs. Furthermore, the modified two-step purification method effectively eliminated all background binding and produced pure antibody pools against individual PrESTs. This indicates that the multiplexed PrEST immunization strategy described here could become useful for high-throughput antibody-based proteomics initiatives, thus significantly reducing the number of animals needed in addition to providing a more cost-efficient method for production of msAbs.