Characterization of transferrin receptor in an immortalized cell line of rat brain endothelial cells, RBE4

被引:16
作者
Huwyler, J [1 ]
Froidevaux, S
Roux, F
Eberle, AN
机构
[1] Univ Basel Hosp, Dept Res, ZLF, CH-4031 Basel, Switzerland
[2] Univ Basel, Childrens Hosp, CH-4031 Basel, Switzerland
[3] Hop Fernand Widal, INSERM U26, F-75475 Paris, France
来源
JOURNAL OF RECEPTOR AND SIGNAL TRANSDUCTION RESEARCH | 1999年 / 19卷 / 1-4期
关键词
D O I
10.3109/10799899909036683
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The content and distribution of transferrin receptors in an immortalized cell line, RBE4, derived from rat cerebral capillary endothelial cells was investigated using the monoclonal antibody MRC OX-26 (OX-26 mAb) specific for the rat transferrin receptor. An ELISA assay was developed with which the OX-26 mAb can be determined quantiatively. The detection limit of the assay was 10 pg or 0.07 fmol of murine antibody. With this technique accurate measurement of native antibody is now possible without the need for isotope labeling (iodination). Immunostaining of confluent monolayers of RBE4 cells using an antibody directed against the tight junction associated protein ZO-1 was indicative for structural intactness of RBE4 cell monolayers. OX-26 immunostaining demonstrated localization of the transferrin receptor at the plasma membrane and/or in the cytosol. Binding studies showed saturation of OX-26 mAb binding. The antibody binding analysis gave a dissociation constant (KD) of 17.1+/-1.2 nmol/l. The total amount of transferrin receptors present per cell was 70,800 +/- 17,000. Our results indicate that receptor binding of OX-26 mAb can be studied using an in vitro cell culture model of rat brain mircrovessel endothelium in conjunction with an ELISA technique for detection of native antibody. This approach will be used to investigate mechanisms of transendothelial transport of OX-26 in vitro.
引用
收藏
页码:729 / 739
页数:11
相关论文
共 20 条
[11]  
PARDRIDGE WM, 1991, J PHARMACOL EXP THER, V259, P66
[12]   VECTOR-MEDIATED DELIVERY OF A POLYAMIDE (PEPTIDE) NUCLEIC-ACID ANALOG THROUGH THE BLOOD-BRAIN-BARRIER IN-VIVO [J].
PARDRIDGE, WM ;
BOADO, RJ ;
KANG, YS .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1995, 92 (12) :5592-5596
[13]  
RAUB TJ, 1991, J CELLULAR PHYSL, V149
[14]   REGULATION OF GAMMA-GLUTAMYL-TRANSPEPTIDASE AND ALKALINE-PHOSPHATASE ACTIVITIES IN IMMORTALIZED RAT-BRAIN MICROVESSEL ENDOTHELIAL-CELLS [J].
ROUX, F ;
DURIEUTRAUTMANN, O ;
CHAVEROT, N ;
CLAIRE, M ;
MAILLY, P ;
BOURRE, JM ;
STROSBERG, AD ;
COURAUD, PO .
JOURNAL OF CELLULAR PHYSIOLOGY, 1994, 159 (01) :101-113
[15]   QUANTIFICATION OF RAT HEPATOCYTE TRANSFERRIN RECEPTORS WITH POLYCLONAL AND MONOCLONAL-ANTIBODIES AND PROTEIN-A [J].
RUDOLPH, JR ;
REGOECZI, E ;
SOUTHWARD, S .
HISTOCHEMISTRY, 1988, 88 (02) :187-192
[16]   A METHOD TO DETERMINE THE ABILITY OF DRUGS TO DIFFUSE THROUGH THE BLOOD-BRAIN-BARRIER [J].
SEELIG, A ;
GOTTSCHLICH, R ;
DEVANT, RM .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (01) :68-72
[17]   Transcellular delivery of an insulin-transferrin conjugate in enterocyte-like Caco-2 cells [J].
Shah, D ;
Shen, WC .
JOURNAL OF PHARMACEUTICAL SCIENCES, 1996, 85 (12) :1306-1311
[18]   TRANSPORT OF [I-125] TRANSFERRIN THROUGH THE RAT BLOOD-BRAIN-BARRIER [J].
SKARLATOS, S ;
YOSHIKAWA, T ;
PARDRIDGE, WM .
BRAIN RESEARCH, 1995, 683 (02) :164-171
[19]  
TRAUTMANN OD, 1993, J CELL PHYSL, V155, P104
[20]  
VANGELDER W, 1995, J NEUROCHEM, V64, P2708