A monoclonal-antibody-based ELISA for the detection of human FADD (Fas-associated death domain).
FADD (Fas-associated death domain) has been widely expressed in various tissues and its expression has been recently demonstrated to correlate with tumour progression and prognosis. Currently, measurement of FADD expression mainly depends on Western-blot or immunohistochemical approaches. To develop a conventional sandwich ELISA avenue for the detection of FADD protein to supplement Western blotting or immunohistochemistry, a series of mAbs (monoclonal antibodies) specific for FADD protein, designated 3A3, 3F9, 3G4, 4B9, 4G1, 7A8, 7B8 and 7F4, were produced by fusing mouse s/p20 myeloma cells with the spleen cells of a mouse immunized with the Escherichia coli-expressed recombinant His(6)-FADD protein. On the basis of the characterization of these mAbs, purified 3F9 was selected as the capture antibody and the biotin-conjugated 3A3 was selected as the detection antibody in sandwich ELISA. The limit of detection for the ELISA was 0.3 ng of purified His(6)-FADD (FADD tagged with hexahistidine), and it could detect both recombinant and native human FADD protein. Furthermore, the positive reaction of the ELISA could be blocked by rabbit anti-FADD sera. All of these results indicated that the ELISA developed in the present paper could be a promising tool for detection of FADD protein.
Ye
Jianqiang
J
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People's Republic of China.
Shao
Hongxia
H
Ma
Dingyuan
D
Qin
Aijian
A
Han
Bing
B
Marikar
Faiz M M T
FM
Cheng
Wei
W
Huang
Xiaofeng
X
Qiu
Yudong
Y
Hu
Qingang
Q
Hua
Zichun
Z
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D053200:Fas-Associated Death Domain Protein / Q000032:analysis* / Q000276:immunology*; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007668:Kidney / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein