Preparation and characterization of monoclonal antibodies against C-terminal telopeptides of type Ⅱ collagen
- VernacularTitle:Ⅱ型胶原C端肽单克隆抗体制备及鉴定
- Author:
Dan-Hui ZHAO
;
Li-Li ZHANG
;
Fan LAN
;
Na WANG
;
Yue YUAN
;
Yan-Zhuo ZHANG
;
- Publication Type:Journal Article
- Keywords:
Collagen type Ⅱ;
Antibodies,monoclonal;
Specimen handling
- From:
Chinese Journal of Laboratory Medicine
2003;0(12):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prepare,purify and validate specific monoclonal antibodies(McAb) against fragment in C-terminal telopeptides of collagen type Ⅱ(EKGPDP)for clinical diagnostics and related research of osteoarthritis(OA).Methods 8 BALB/c mice were immunized with EKGPDP-KLH antigen complex.McAb against EKGPDP fragements were prepared by hybridoma technique.Immunoglobulin classes and subclasses were determined using an Immuno-Type~(TM)mouse McAb isotyping kit.Ascites were producted and McAb were purified by saturation ammonium sulfate(SAS)precipitation and protein G chromatography.Specificity and immunoactivity of antibodies were detected by indirect enzyme-linked imrnunosorbant assay(ELISA).Cartilage specimens were analyzed by immunohistochemical method.Results The hybridoma cells were obtained.10 IgG and 3 IgM single colonies were picked out by limiting dilution and ELISA kit.The titers of McAb in ascites were from 2.8?10~4 to 5.1?10~5.The McAb showed the characteristics of no cross reactions with KLH,BSA,cell culture supernatant,type Ⅰ,Ⅲ collagens and whole type Ⅱ collagen.The method of SAS could get better recovery,immunoactivity of the McAb than protein G chromatograply(t=25.26;P
