Systematically generated antibodies against human gene products: high throughput screening on sections from the rat nervous system.
Completion of the Human Genome Project and recent developments in proteomics make it possible to systematically generate affinity reagents to a large portion of the proteome. Recently an antibody-based human protein atlas covering many organs including four areas of the brain has been released (www.proteinatlas.org). Due to the heterogeneity, size, and availability of tissue a more thorough analysis of the human brain is associated with considerable difficulties. Here we applied 120 antibodies raised against 112 human gene products to the smaller rat brain, a rodent animal model, where a single section represents a 'superarray' including many brain areas, and consequently allowing analysis of a huge number of cell types and their neurochemicals. Immunoreactive structures were seen in the investigated brain tissue after incubation with 56 antibodies (46.6%), of which 25 (20.8%) showed a clearly discrete staining pattern that was limited to certain areas, or subsets of brain cells. Bioinformatics, pre-adsorption tests and Western blot analysis were applied to identify non-specific antibodies. Eleven antibodies, including such raised against four 'ambiguous' proteins, passed all validation criteria, and the expression pattern and subcellular distribution of these proteins were studied in detail. To further explore the potential of the systematically generated antibodies, all 11 antibodies that passed validation were used to analyze the spinal cord and lumbar dorsal root ganglia after unilateral transection of the sciatic nerve. Discrete staining patterns were observed for four of the proteins, and injury-induced regulation was found for one of them. In conclusion, the study presented here suggests that a significant portion (10%) of the antibodies generated to a human protein can be used to analyze orthologues present in the rodent brain and to produce a protein-based atlas of the rodent brain. It is hoped that this type of antibody-based, high throughput screening of brain tissue from various rodent disease models will provide new information on the brain chemical neuroanatomy and insights in processes underlying neurological pathologies.
Mulder
J
J
Department of Neuroscience, Karolinska Institutet, Retzius v. 8, S171 77 Stockholm, Sweden. Jan.Mulder@ki.se
Wernérus
H
H
Shi
T-J
TJ
Pontén
F
F
Hober
S
S
Uhlén
M
M
Hökfelt
T
T
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000918:Antibody Specificity; D019771:Axotomy / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D019295:Computational Biology / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008403:Mass Screening; D009420:Nervous System / Q000276:immunology*; D040081:Protein Array Analysis; D020543:Proteome / Q000737:chemistry / Q000276:immunology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
D000906:Antibodies; D020543:Proteome; D011994:Recombinant Proteins