Purification of G protein-coupled receptor 35 (GPR35) and evaluation of reactivity and specificity of GPR35 antibodies

Abstract

Identification of cancers at an early stage increase the probability of successful treatment and reduce morbidity and mortality. Measurements of specific biomarkers may provide useful insight regarding cancer identification, prognosis, and prediction. G protein-coupled receptor 35 (GPR35) is found expressed at increased levels in several cancers and can be a possible biomarker for e.g. colorectal cancer. A T108M polymorphism in GPR35 cause a hypermorphic protein variant. This polymorphism is associated with primary sclerosing cholangitis, a severe liver disease that affects the bile ducts and it is associated with increased risk of colorectal cancer and cholangiocarcinoma. An aim of the study was to develop immunological methods to detect and measure expression of GPR35 in cell lines. Other aims were to purify GPR35, optimise the purification method, and evaluate reactivity and specificity of GPR35 antibodies in various methodologies. To detect GPR35 in colorectal cancer cells (Caco-2) and in human embryonic kidney 293 (HEK293) cells transfected with the gene for GPR35, cells were analysed with enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining. These techniques were also used to evaluate the reactivity and specificity of GPR35 antibodies. GPR35 was purified with immobilised metal affinity chromatography. Results indicated that GPR35 was specifically detected in transfected HEK293 cells, but not in Caco-2 cells, by ELISA. Immunofluorescence staining of cells showed non-specific binding of the antibodies. The purification method of GPR35 was optimised. Antibodies tested showed variable affinity to purified protein. The antibodies used for detection of GPR35 in Caco-2 and HEK293 cells exhibited non-specific binding. Thus, novel antibodies with specific affinity to GPR35 are required before a method to detect and quantify GPR35 in cell lines can be established.