Generation of monoclonal antibodies against chromosomal antigens that have a high sequence similarity between human and mouse.
We raised monoclonal antibodies by immunizing mice with total chromosome proteins extracted from isolated human metaphase chromosomes. The indirect immunofluorescence screening of hybridoma cell lines provided 15 monoclonal antibodies against the chromosomal antigens. The antigen proteins of the mAbs were identified by immunoblotting as core histones or by immunoprecipitation followed by a peptide mass fingerprinting method as nuclear mitotic apparatus protein, heterogeneous nuclear ribonucleoprotein A2/B1, ribosomal protein S4, linker histone and beta-actin. During mitosis, localizations of these proteins on chromosomes were clearly observed using the obtained antibodies. These results indicate that the current strategy is effective for obtaining monoclonal antibodies useful for immunoblotting and/or immunofluorescent staining of human proteins, using the antigens with high homology to mouse proteins.
Higashi
Tsunehito
T
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Miyakawa
Shuichi
S
Uchiyama
Susumu
S
Matsunaga
Sachihiro
S
Takata
Hideaki
H
Fujimoto
Satoru
S
Noda
Masanori
M
Terauchi
Akiko
A
Shimizu
Takeyuki
T
Oda
Masayuki
M
Azuma
Takachika
T
Fukui
Kiichi
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology*; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology*; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis* / Q000276:immunology; D002877:Chromosomes, Human / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007116:Immunization, Passive; D015151:Immunoblotting; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology; D013058:Mass Spectrometry; D008677:Metaphase; D051379:Mice; D009101:Multiple Myeloma / Q000276:immunology / Q000473:pathology; D010449:Peptide Mapping; D011233:Precipitin Tests
D000911:Antibodies, Monoclonal; D000941:Antigens; D002868:Chromosomal Proteins, Non-Histone