[Preparation and activity detection of monoclonal antibody against anti-CD3 ScFv].
OBJECTIVE
To prepare monoclonal antibody (McAb) against anti-CD3 ScFv for purifying and detecting serum anti-CD3 antibody concentration.


METHODS
McAb against anti-CD3 ScFv was prepared by hybridoma technique and used to prepare affinity chromatography column, which was used to purify anti-CD3 ScFv and Diabody [CD3 x Pgp] without E-tag. The binding activities of anti-CD3 ScFv, Diabody [CD3 x Pgp] without E-tag, and Diabody [CD3 x Pgp] purified by anti-CD3 affinity chromatography column or anti-E-tag affinity chromatography column against K562/A02 cell and Jurket cells were detected by fluorescence activated cell sorting (FACS) method. ELISA was used to identify the specificity of the McAb.


RESULTS
McAb against anti-CD3 ScFv specifically detected serum anti-CD3 ScFv without reacting with sera. The anti-CD3 ScFv purified by anti-CD3 affinity chromatography column and purified by anti-E-tag affinity chromatography column had the same specific binding activity with Jurkat cells. The positive binding rates of Diabody [CD3 x Pgp] without E-tag to K562/A02 and Jurkat cells were 89.87% and 83.95%, respectively. In the competitive binding experiments with K562/A02 and Jurkat cells, the binding rates of Diabody [CD3 x Pgp] without E-tag decreased to 56.30% and 43.78%, respectively.


CONCLUSIONS
The McAb against anti-CD3 ScFv prepared in our lab can be used to purify and detect serum anti-CD3 antibody concentration.
Shao
Xiao-Feng
XF
State Key Laboratory of Experimental Hematology, Institute of Hematology, CAMS and PUMC, Tianjin 300020, China.
Gao
Ying-Dai
YD
Liu
Juan-Ni
JN
Wang
Jin-Hong
JH
Xu
Yuan-Fu
YF
Fan
Dong-Mei
DM
Yang
Chun-Zheng
CZ
Xiong
Dong-Sheng
DS
D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology* / Q000302:isolation & purification; D017252:CD3 Complex / Q000276:immunology*; D002460:Cell Line; D002846:Chromatography, Affinity; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D019169:Jurkat Cells; D020014:K562 Cells
D000911:Antibodies, Monoclonal; D017252:CD3 Complex