Chondroitinase ABC (ChABC) is a bacterial enzyme that degrades sugar chains attached to the core protein of chondroitin sulfate proteoglycans (CSPGs). CSPGs are involved in a variety of biological processes in higher organisms, including humans. ChABC has a well-known potential for therapeutic application and extensive testing is underway. Therefore, the ELISA has an important application to determine ChABC distribution and content in tissues and bodily fluids.

A panel of monoclonal antibodies was produced that specifically bind ChABC. Additional screening identified a pair of complimentary monoclonal antibodies, a capture antibody and detection antibody, and a Sandwich ELISA was established.

A working protocol for the Sandwich ELISA was established and validated. Extensive testing documents the accuracy, precision, selectivity, sensitivity and reproducibility of the ChABC ELISA methodology for quantifying and detecting ChABC in buffers and biological samples. The monoclonal anti-ChABC antibodies bind ChABC from prominent vendors. In addition, both antibodies are effective in Western immunoblotting and immunohistochemistry. Detailed methods and assay specifications are available upon request.

Market:

Presently, there is no commercial source of monoclonal antibody to ChABC. Efforts to quantify ChABC rely on assay of its enzymatic activity. This approach is complicated and lacks specificity when applied to complex biological samples and fails to detect inactive ChABC protein residuals. No quantitative immunoassays are available commercially for ChABC. We are seeking licensing partners to commercialize the ChABC antibodies and Sandwich ELISA. License of the hybridomas or purified antibodies will be considered.

Vector and method to visually genotype transgenic animals

A reporter transgene encoding enhanced green fluorescent protein (eGFP) has been designed to be co-injected with various transgene constructs. The eGFP reporter was constructed by inserting the cDNA into a vector harboring a keratin-14 (K14) promoter to drive expression of eGFP in skin. The K14 vector was created by removing the promoter element of the MoPrP.Xho vector and replacing it with a 2 kb fragment of the human keratin-14 gene. Previous studies have shown that co-injection into single cell embryos of two independent transgenes results in co-integration of the transgenes at a single locus. In our experience, the two transgenes are so closely integrated that recombination to segregate the genes virtually never occurs. The transgene construct to be tested is co-injected with the K14-eGFP construct. Using goggles fitted with the appropriate filters to visualize eGFP expression, we could reliably distinguish transgenic mice from nontransgenic mice. The expression of eGFP persists into adulthood and is most visible in ears, paws and tail skin. Mice harboring the K14-eGFP transgene alone appeared normal throughout their lifespan (more than 530 days).

The Keratin14-eGFP expression vector was made by amplifying the Keratin14 promoter using the following primers: 5’-CCGGATCCGCGGCCGCCTCCGGAGCTTCTATTCC-3’ and 5’-CCGGATCCTAAATTGGAAAGGGATGCGAGTGC-3’. The 2 kb amplicon was digested with BamHI (underlined sequence) and ligated into the PrP-Tet promoter vector, which is a variation of the MoPrP.Xho vector in which the PrP promoter elements have been replaced by the tetracycline operator. The tetracycline operator is flanked by BamHI restriction sites, allowing for easy excision and replacement. The eGFP cDNA was excised from its vector (Clontech #6086-1, Accession #U55761) and ligated into the XhoI cloning site of the new K14.PrP.Xho vector.