Enhanced antigen detection in immunohistochemical staining using a 'digitized' chimeric antibody.
The immunohistochemical (IHC) staining of mouse tissue sections using antibodies of mouse origin can result in high nonspecific background due to the staining of endogenous immunoglobulins (Igs) by enzyme-conjugated secondary antibodies. In order to obviate this issue, we developed a chimeric mouse-human anti-p53 monoclonal antibody (MH242) by grafting the variable regions of a known mouse antibody into a human Ig scaffold. This facilitated use of an anti-human secondary antibody, and resulted in near-zero background when compared with its parental mouse monoclonal antibody (PAb242). Furthermore, the chimeric antibody enabled reproducible detection of mutant p53 (homozygous R172H) expression in mouse tissue, an observation hitherto largely equivocal based on the use of existing antibodies. The approach we describe leads to the generation of tractable antibody reagents, whose integrity can be readily verified through DNA sequencing of expressor plasmids. The wide-spread adoption of such 'digitized' antibodies should reduce experimental disparities that can commonly arise through variations in antibody quality.
p53 Laboratory, 8A Biomedical Grove, Immunos #06-06, Singapore 138648, Singapore.
Hui-Yan
HY
Eng
Singapore Immunology Network, 8A Biomedical Grove, Singapore 138648, Singapore.
Cheng-I
CI
Wang
p53 Laboratory, 8A Biomedical Grove, Immunos #06-06, Singapore 138648, Singapore.
Yuezhen
Y
Xue
Singapore Immunology Network, 8A Biomedical Grove, Singapore 138648, Singapore.
Chia-Yin
CY
Lee
Institute of Medical Biology Microscopy Unit, 8A Biomedical Grove, Singapore 138648, Singapore.
Sarah Binte
SB
Zulkifli
p53 Laboratory, 8A Biomedical Grove, Immunos #06-06, Singapore 138648, Singapore.
Poh-Cheang
PC
Chiam
p53 Laboratory, 8A Biomedical Grove, Immunos #06-06, Singapore 138648, Singapore dplane@p53lab.a-star.edu.sg fghadessy@p53lab.a-star.edu.sg.
Farid J
FJ
Ghadessy
p53 Laboratory, 8A Biomedical Grove, Immunos #06-06, Singapore 138648, Singapore dplane@p53lab.a-star.edu.sg fghadessy@p53lab.a-star.edu.sg.
David P
DP
Lane
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics; D007150:Immunohistochemistry / Q000379:methods*; D007422:Intestines / Q000737:chemistry; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53
PAb242; anti-mouse p53; antibody; chimeric