In situ phage screening. A method for identification of subnanogram tissue components in situ.
We have established a novel method, in situ phage screening (ISPS), to identify proteins in tissue microstructures. The method is based on the selection of repertoires of phage-displayed antibody fragments with small samples of tissues microdissected using a laser. Using a human muscle frozen section with an area of 4800 microm2 as a model target, we successfully selected monoclonal antibody fragments directed against three major (myosin heavy chain, actin, and tropomyosin-alpha) and one minor (alpha-actinin 2) muscle constituent proteins. These proteins were present in the sample in amounts less than one nanogram, and the antibodies were used to visualize the proteins in situ. This shows that the use of ISPS can obtain monoclonal antibodies for histochemical and biochemical purposes against minute amounts of proteins from microstructures with no requirement for large amounts of samples or biochemical efforts.
Tanaka
Torahiko
T
Department of Cortical Function Disorders, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan.
Ito
Takashi
T
Furuta
Masaru
M
Eguchi
Chikashi
C
Toda
Hiroyuki
H
Wakabayashi-Takai
Eriko
E
Kaneko
Kiyotoshi
K
D000941:Antigens / Q000235:genetics; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics*; D018482:Muscle, Skeletal / Q000378:metabolism
D000941:Antigens; D017931:DNA Primers; D007128:Immunoglobulin Fragments