A rapid and efficient method for the generation and screening of monoclonal antibodies specific for cell surface antigens

被引:19
作者
Mesci, Aruz [1 ]
Carlyle, James R. [1 ]
机构
[1] Univ Toronto, Sunnybrook Res Inst, Dept Immunol, Toronto, ON M4N 3M5, Canada
基金
加拿大健康研究院;
关键词
BWZ reporter-cell assay; monoclonal antibodies; primary screening; CD3 zeta-fusion receptor; beta-galactosidase;
D O I
10.1016/j.jim.2007.02.007
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The generation of monoclonal antibodies (mAb) of desired specificity to cell surface antigens can serve as a valuable tool to study protein expression and function. However, traditional approaches to mAb generation usually involve large-scale protein purification and intensive screening, and may not result in mAb specificities to the native protein of interest. We describe a simple, inexpensive, high-throughput method for the generation and screening of hybridomas secreting mAb specific for cell surface receptors. Intact reporter cells expressing a CD3 xi-fusion receptor of the protein of interest are plated in 96-well arrays of captured, plate-bound hybridoma supernatants. A mAb to the protein of interest generates a signal leading to reporter-cell expression of beta-galactosidase, and enzyme activity can be screened in a single day using a non-radioactive substrate. Importantly, a single cell line can be used for immunization, screening, semi-quantitative affinity comparisons, and subsequent screening for physiological ligand expression, if the protein of interest is a receptor. We describe an application of this approach to generate mAb specific for a protein of previously unknown expression and undocumented function. (C) 2007 Elsevier B.V. All rights reserved.
引用
收藏
页码:78 / 87
页数:10
相关论文
共 10 条
[1]   Molecular and genetic basis for strain-dependent NK1.1 alloreactivity of mouse NK cells [J].
Carlyle, James R. ;
Mesci, Aruz ;
Ljutic, Belma ;
Belanger, Simon ;
Tai, Lee-Hwa ;
Rousselle, Etienne ;
Troke, Angela D. ;
Proteau, Marie-France ;
Makrigiannis, Andrew P. .
JOURNAL OF IMMUNOLOGY, 2006, 176 (12) :7511-7524
[2]   Missing self-recognition of Ocil/Cir-b by inhibitory NKR-P1 natural killer cell receptors [J].
Carlyle, JR ;
Jamieson, AM ;
Gasser, S ;
Clingan, CS ;
Arase, H ;
Raulet, DH .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2004, 101 (10) :3527-3532
[3]  
Carlyle JR, 1998, J IMMUNOL, V160, P744
[4]   The role of the NKG2D immunoreceptor in immune cell activation and natural killing [J].
Jamieson, AM ;
Diefenbach, A ;
McMahon, CW ;
Xiong, N ;
Carlyle, JR ;
Raulet, DH .
IMMUNITY, 2002, 17 (01) :19-29
[5]   T cell responses modulated through interaction between CD8αα and the nonclassical MHC class I molecule, TL [J].
Leishman, AJ ;
Naidenko, OV ;
Attinger, A ;
Koning, F ;
Lena, CJ ;
Xiong, Y ;
Chang, HC ;
Reinherz, E ;
Kronenberg, M ;
Cheroutre, H .
SCIENCE, 2001, 294 (5548) :1936-1939
[6]   LACZ INDUCIBLE, ANTIGEN MHC-SPECIFIC T-CELL HYBRIDS [J].
SANDERSON, S ;
SHASTRI, N .
INTERNATIONAL IMMUNOLOGY, 1994, 6 (03) :369-376
[7]   CLONING AND CHARACTERIZATION OF 5E6(LY-49C), A RECEPTOR MOLECULE EXPRESSED ON A SUBSET OF MURINE NATURAL-KILLER-CELLS [J].
STONEMAN, ER ;
BENNETT, M ;
AN, JB ;
CHESNUT, KA ;
WAKELAND, EK ;
SCHEERER, JB ;
SICILIANO, MJ ;
KUMAR, V ;
MATHEW, PA .
JOURNAL OF EXPERIMENTAL MEDICINE, 1995, 182 (02) :305-313
[8]   Bacterial cell microarrays for the detection and characterization of antibodies against surface antigens [J].
Thirumalapura, NR ;
Ramachandran, A ;
Morton, RJ ;
Malayer, JR .
JOURNAL OF IMMUNOLOGICAL METHODS, 2006, 309 (1-2) :48-54
[9]   Identification and characterization of a spliced C-type lectin-like gene encoded by rat cytomegalovirus [J].
Voigt, S ;
Sandford, GR ;
Ding, LJ ;
Burns, WH .
JOURNAL OF VIROLOGY, 2001, 75 (02) :603-611
[10]  
VOIGT S, IN PRESS IMMUNITY