[Preparation and characterization of monoclonal antibody against CP4-EPSPS].
OBJECTIVE
To prepare monoclonal antibody (mAb) against CP4-EPSPS, which could be applied to the development of gold colloidal rapid diagnostic kit for the specific detection of GMO.


METHODS
BALB/c mice were immunized with CP4-EPSPS. Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line Sp2/0 cells. The hybridoma cells that secreted CP4-EPSPS mAb antibodies were cloned with limited dilution method. The immunoglobulin (Ig) subtype, the ascites titers, the binding site, and the affinity of the obtained mAbs were determined by indirect ELISA. The specificity of mAbs was tested by ELISA and Western blot analysis.


RESULTS
From over 80 positive hybridomas which secreted anti-cp4-EPSPS mAbs, a pair of hybridomas were screened out, designated III5A3 and III13A2. Chromosome analysis revealed that the obtained hybridomas were with the universal characteristics of the monoclonal hybridoma cells which secreted mAb, and the Ig subtype of III5A3 and III13A2 mAb was both IgG1. The ascites titers of III5A3 and III13A2 mAb were 1:10(6) and 1:10(8), respectively. Our study also demonstrated that these two mAbs, which recognized the same epitope, could both specifically bind to the CP4-EPSPS protein. The relative affinity constant of III5A3 and III13A2 mAb was determined as 10(5) and 10(6) respectively.


CONCLUSIONS
A pair of high titer, specific mAbs against CP4-EPSPS have been successfully prepared and primarily identified, which may be useful in the development of a rapid and convenient diagnostic kit for detection of GMO.
Jing
Ling-xia
LX
The Institute for Viral Hepatitis, Key Laboratory of Molecular Biology on Infectious Disease, Chongqing University of Medical Sciences, Chongqing 400016, China. jinglxcm@163.com
Cai
Xue-fei
XF
Mu
Sheng-zhi
SZ
Liu
Xiang
X
Zhang
Jun
J
Tang
Ni
N
Zheng
Jian
J
Huang
Ai-long
AL
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000235:genetics / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase