Production of horseradish peroxidase conjugated monoclonal anti-heparan sulfate proteoglycans antibody for immunologic assay

Abstract

Heparan sulfate proteoglycans (HSPGs) significantly involves in fundamental cell activities and acts as the co-receptor of many certain molecules with heparin binding region. HSPGs express on membrane of many cell types. Moreover, HSPGs are main composition on hepatocyte membrane of liver. In effort to study about relationship and role of HSPGs, human liver HSPGs was isolated and purified and specific monoclonal antibody (mAbs) was produced to use as a tool to educate the characteristics and function of liver HSPGs. Previous study showed that mAbs clone 1E4-1C2 made specific with cell membrane molecules of acute myeloid leukemia (AML). Since, anti-human liver HSPGs has not been reported and avialable, our study was aimed to prepare horseradish peroxidase (HRP) conjugated mAbs clone 1E4-1C2. This product can be used in immunologic assay with no need of secondary antibody, that is expensive and may cause cross reaction with other protein in serum. Two-step glutaraldehyde technique was used to conjugate mAbs clone 1E4-1C2 with HRP. Glutaraldehyde enhanced HRP to produce aldehyde group which can be caught primary amine of mAbs. After that molecular weight of mAbs conjugated with HRP was investigated by SDS-PAGE. The results showed 195 kDa of mAbs conjugated with HRP. Monoclonal antibody clone 1E4-1C2 specificity was investigated by direct ELISA. The results showed that HRP conjugated anti liver HSPG has its own activity both in antigen specific and peroxidase pattern. Bull Chiang Mai Assoc Med Sci 2008; 41: 167-174.