Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA

被引：11

作者：

Kuhne, Maren ^{[1
]}

Dippong, Martin ^{[1
,2
]}

Flemig, Sabine ^{[1
]}

Hoffmann, Katrin ^{[1
]}

Petsch, Kristin ^{[1
]}

Schenk, Joerg A. ^{[3
,4
]}

Kunte, Hans-Joerg ^{[1
]}

Schneider, Rudolf J. ^{[1
]}

机构：

[1] BAM Fed Inst Mat Res & Testing, D-12489 Berlin, Germany

[2] Univ Potsdam, Inst Biochem & Biol, D-14476 Potsdam, Germany

[3] UP Transfer GmbH, D-14469 Potsdam, Germany

[4] Hybrotec GmbH, D-14476 Potsdam, Germany

来源：

JOURNAL OF IMMUNOLOGICAL METHODS | 2014年 / 413卷

关键词：

Immunization; Hapten; Monoclonal antibodies; Hybridoma; Flow cytometry; ELISA; FLUORESCENCE POLARIZATION IMMUNOASSAY; LINKED IMMUNOSORBENT ASSAYS; LIMITING DILUTION TECHNIQUE; MONOCLONAL-ANTIBODIES; MOLECULAR CHARACTERIZATION; STATISTICAL-ANALYSIS; SURFACE EXPRESSION; AFFINITY CONSTANT; B-CELLS; CLONING;

D O I：

10.1016/j.jim.2014.07.004

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

A novel method that optimizes the screening for antibody-secreting hapten-specific hybridoma cells by using flow cytometry is described. Cell clones specific for five different haptens were analyzed. We selectively double stained and analyzed fixed hybridoma cells with fluorophore-labeled haptens to demonstrate the target-selectivity, and with a fluorophore-labeled anti-mouse IgG antibody to characterize the level of surface expression of membrane-bound IgGs. ELISA measurements with the supernatants of the individual hybridoma clones revealed that antibodies from those cells, which showed the highest fluorescence intensities in the flow cytometric analysis, also displayed the highest affinities for the target antigens. The fluorescence intensity of antibody-producing cells corresponded well with the produced antibodies' affinities toward their respective antigens. Immunohistochemical staining verified the successful double labeling of the cells. Our method makes it possible to perform a high-throughput screening for hybridoma cells, which have both an adequate IgG production rate and a high target affinity. (C) 2014 Elsevier B.V. All rights reserved.

引用

页码：45 / 56

页数：12

共 51 条

[1] Monitoring carbamazepine in surface and wastewaters by an immunoassay based on a monoclonal antibody
Bahlmann, Arnold
Weller, Michael G.
Panne, Ulrich
Schneider, Rudolf J.
[J]. ANALYTICAL AND BIOANALYTICAL CHEMISTRY, 2009, 395 (06) : 1809 - 1820
[2] Borth N, 2000, BIOTECHNOL BIOENG, V71, P266, DOI 10.1002/1097-0290(2000)71:4<266::AID-BIT1016>3.0.CO
[3] 2-2
[4] Selection methods for high-producing mammalian cell lines
Browne, Susan M.
Al-Rubeai, Mohamed
[J]. TRENDS IN BIOTECHNOLOGY, 2007, 25 (09) : 425 - 432
[5] Comparison of hybridoma screening methods for the efficient detection of high-affinity hapten-specific monoclonal antibodies
Cervino, Christian
Weber, Ekkehard
Knopp, Dietmar
Niessner, Reinhard
[J]. JOURNAL OF IMMUNOLOGICAL METHODS, 2008, 329 (1-2) : 184 - 193
[6] SURFACE IGG CONTENT OF MURINE HYBRIDOMAS - DIRECT EVIDENCE FOR VARIATION OF ANTIBODY SECRETION RATES DURING THE CELL-CYCLE
CHERLET, M
KROMENAKER, SJ
SRIENC, F
[J]. BIOTECHNOLOGY AND BIOENGINEERING, 1995, 47 (05) : 535 - 540
[7] Mouse monoclonal antibodies in biological research: strategies for high-throughput production
Chiarella, Pieranna
Fazio, Vito Michele
[J]. BIOTECHNOLOGY LETTERS, 2008, 30 (08) : 1303 - 1310
[8] A fluorescence polarization immunoassay for the detection of zearalenone in corn
Chun, Hyang Sook
Choi, Eun Hye
Chang, Hyun-Joo
Choi, Sung-Wook
Eremin, Sergey A.
[J]. ANALYTICA CHIMICA ACTA, 2009, 639 (1-2) : 83 - 89
[9] Clementi M E, 1991, Ann Ist Super Sanita, V27, P139
[10] STATISTICAL-ANALYSIS OF REPETITIVE SUBCLONING BY THE LIMITING DILUTION TECHNIQUE WITH A VIEW TOWARD ENSURING HYBRIDOMA MONOCLONALITY
COLLER, HA
COLLER, BS
[J]. HYBRIDOMA, 1983, 2 (01): : 91 - 96

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