Preparation of rabbit monoclonal antibody against cGMP and development of competitive ELISA for cGMP.

Xing-mei Tan; Hai-feng HU; Shu-qing Chen

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Preparation of rabbit monoclonal antibody against cGMP and development of competitive ELISA for cGMP.

Author: Xing-mei TAN ^{1
,

2} ; Hai-feng HU ³ ; Shu-qing CHEN ⁴
Author Information

1. Institute of Pharmacology, Toxicology and Biological Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
2. 2. Abcam(Hangzhou)Biotechnology Co. Ltd., Hangzhou 310015, China.
3. Abcam(Hangzhou)Biotechnology Co. Ltd., Hangzhou 310015, China.
4. Institute of Pharmacology, Toxicology and Biological Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
Publication Type:Journal Article
MeSH: Animals; Antibodies, Monoclonal; biosynthesis; Antibody Specificity; Cross Reactions; Cyclic GMP; immunology; Enzyme-Linked Immunosorbent Assay; Rabbits
From: Journal of Zhejiang University. Medical sciences 2015;44(4):423-428
CountryChina
Language:Chinese
Abstract:
OBJECTIVETo prepare rabbit monoclonal antibody (RabMab) against guanosine 3', 5'-cyclic monophosphate (cGMP) and to develop a competitive ELISA for the detection of cGMP.
METHODSNew Zealand white rabbits were immunized with synthesized cGMP-keyhole limpet hemoeyanin (cGMP-KLH) to prepared a RabMAb with monoclonal antibody technique of Epitomics. A competitive ELISA kit was produced with cGMP RabMAb. The specificity, the precision and the recoveries of the method were determined.
RESULTSThe RabMAb with high sensitivity towards cGMP were prepared with an antibody timer of 3.1 ng/mL and 50% inhibitive concentration (IC50) of 12.57 ng/mL. The cGMP RabMAb had 33% cross-reactivity to inosine 3', 5'-cyclic monophosphate (cIMP) and little or no cross-reactivity to other compounds. A competitive ELISA was developed for detection of cGMP. The range of detection was 0~120 ng/mL with a minimal limit of 1.95 ng/mL. The recovery of assay was 89%~103%. The inter-assay and intra-assay coefficient variations were below 11.68% and 13.85%, respectively.
CONCLUSIONThe RabMab against cGMP with high affinity and high specificity has been generated successfully, and a competitive ELISA for detection of cGMP has been developed with the prepared cGMP RabMAb.