[Preparation of iodine-125 labeled anti-human CD40 monoclonal antibody 5H6 and its binding properties in ovary cancer cell line HO8910 in vitro].
OBJECTIVE
To prepare iodine-125 labeled anti-human CD40 monoclonal antibody 5H6 ((125)I-5H6) and investigate the binding properties of (125)I-5H6 to HO8910 cells in vitro.


METHODS
The mAb 5H6 was labeled with Na (125)I using chloramine-T method. The labeling efficiency and radiochemical purity of (125)I-5H6 were measured by paper chromatography. The stability of (125)I-5H6 in vitro was monitored by trichloroacetic acid (TCA) precipitation. The dissociation constant (K(d)) of (125)I-5H6 from HO8910 cells and the numbers of maximum binding sites (B(max)) were obtained by Scatchard analysis. Immunoreactivity of (125)I-5H6 to HO8910 cells was determined by "Lindmo" assay and its improved method. Internalization and retention of (125)I- 5H6 by HO8910 cells were studied by cell binding experiments.


RESULTS
(1)The labeling efficiency of (125)I-5H6 was (85.4+/-5.2)% and its radiochemical purity was (99.2+/-0.5)%. (2)Radiochemical purity of (125)I-5H6 was (80.3+/-4.7)% after 7 days in 4 Degrees Celsius PB (phosphate buffer) and (95.3+/-0.8)% after 24 hours in 37 Degrees Ceslius plasma. (3)At 4 Degrees Celsius, K(d) for (125)I-5H6 to HO8910 cells was (0.711+/-0.06) nmol/L and Bmax was (2.17+/-0.08)x10(5) sites/cell. (4)Immunoreactivity of (125)I-5H6 was (38.6+/-5.4)%. (5)Retention rate of (125)I-5H6 with HO8910 cells was (89.8+/-6.0)% after 2 hours at 4 Degrees Celsius and internalization rate of (125)I-5H6 by HO8910 cells was (54.9+/-2.6)% after 2 hours at 37 Degrees Celsius.


CONCLUSIONS
(125)I-5H6 possesses good labeling efficiency and perfect radiochemical purity. (125)I-5H6 has suitable stability in vitro and its immunoreactivity was not low. The high affinity of (125)I-5H6 with HO8910 cells will benefit further animal study in vivo.
Institute of Medical Biotechnology, Soochow University, Suzhou, China. lxf150@tom.com
Xian-fu
XF
Luo
Bin
B
Zhang
Hong-bing
HB
Ma
Lin
L
Tang
Xuan
X
Zhou
Ying
Y
Xu
Yi-wei
YW
Wu
Xue-guang
XG
Zhang
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology*; D019013:CD40 Antigens / Q000276:immunology*; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D007457:Iodine Radioisotopes / Q000737:chemistry; D007553:Isotope Labeling; D010051:Ovarian Neoplasms / Q000276:immunology*; D011485:Protein Binding
D000911:Antibodies, Monoclonal; D019013:CD40 Antigens; D007457:Iodine Radioisotopes