Multiplexed PrEST immunization for high-throughput affinity proteomics.
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.
Larsson
Karin
K
Department of Proteomics, AlbaNova University Center, Royal Institute of Technology (KTH), Stockholm, Sweden.
Wester
Kenneth
K
Nilsson
Peter
P
Uhlén
Mathias
M
Hober
Sophia
S
Wernérus
Henrik
H
D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000302:isolation & purification; D000918:Antibody Specificity; D020224:Expressed Sequence Tags / Q000737:chemistry*; D007114:Immunization / Q000379:methods*; D007150:Immunohistochemistry; D040081:Protein Array Analysis / Q000379:methods; D040901:Proteomics / Q000379:methods*; D011817:Rabbits
D000906:Antibodies