The importance of characterization of FITC-labeled antibodies used in tissue cross-reactivity studies.
Fluorescein isothiocyanate (FITC)-labeled antibodies are widely used as primary antibodies in the tissue cross-reactivity (TCR) studies for the development of therapeutic antibodies. However, the effects of FITC-labeling on the characteristics of an antibody are poorly understood. The present study was performed to examine the effect of FITC-labeling on the binding affinity and immunohistochemical staining profile of an antibody, using several antibodies with different FITC-labeling indices. The results showed that the FITC-labeling index in antibody was negatively correlated with the binding affinity for its target antigen. Immunohistochemically, an antibody with a higher labeling index had a tendency to be more sensitive, but was also more likely to yield non-specific staining. Based on these findings, we recommend that a FITC-labeled antibody used as a primary antibody in a TCR study should be carefully selected from several differently labeled antibodies to minimize the decrease in the binding affinity and achieve the appropriate sensitivity and interpretation of the immunohistochemistry.
Takai
Hirotake
H
Safety Assessment Department, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan. takaihrt@chugai-pharm.co.jp
Kato
Atsuhiko
A
Nakamura
Teruo
T
Tachibana
Tatsuhiko
T
Sakurai
Takayuki
T
Nanami
Masahiko
M
Suzuki
Masami
M
D000345:Affinity Labels; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D000918:Antibody Specificity; D003429:Cross Reactions; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006801:Humans; D009928:Organ Specificity
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D016650:Fluorescein-5-isothiocyanate