Affinity based proteomics research tools

• Listen to the mantra; the mapping of the genome was finished in 2001, and the sequel research challenge is the thorough survey of the corresponding human proteome. This was stated almost a decade ago, it has been repeated over and over, and is still most certainly a hard nut to crack. The workload is daunting, much because there is no protein amplification method and no binary system for the detection of proteins, and because the complexity of the proteome is larger than that of the genome as it seems. Hence, there is a need for high throughput technologies that, at sufficiently low limits of detection and with satisfying sensitivities, may investigate protein content in human samples. With this aim, the Human Proteome Resource (HPR) project was initiated in 2003. All work presented in this thesis relate to protein interactions; binders are either utilized such as for the depletion of high abundant proteins from serum, or analyzed such as in the validation of monospecific antibody specificity, or the epitope mapping of the same. In Paper I, the Gyrolab system is utilized in a setup for the specificity analysis of monospecific antibodies towards their antigen, and the setup is compared to planar protein arrays. Gyrolab technology is used again, in Paper II, where epitope mapping of monospecific antibodies is performed in order to analyze antibody specificity. Also, mapping serves to compare the immune-responses from parallel immunizations using the same antigen, thereby assessing reproducibility in the regeneration of antibodies. Paper III describes a high throughput approach for the depletion of high abundant proteins, in serum and plasma samples, taking advantage of Affibody molecules as binders. The last two papers, IV and V, utilize monospecific antibodies for protein analysis; in Paper IV pull out experiments show that competitive elution using the PrEST antigen can be a fruitful approach to increase specificity. And finally, in Paper V, a setup for the semi-quantitative protein content analysis in fluid samples is suggested. Again, the Gyrolab technology is used, and the setup is tested on a simplified model system.

Ämnesord

NATURVETENSKAP  -- Biologi -- Immunologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Immunology (hsv//eng)

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

Immunology

Immunologi

Molecular biology

Molekylärbiologi

Publikations- och innehållstyp

vet (ämneskategori)

dok (ämneskategori)