The use of commercial ELISA kits in measuring CA6 concentration of saliva

Abstract

Introduction: Carbonic anhydrase 6 (CA6) is the only secreted member of the carbonic anhydrase enzyme family. This enzyme catalyzes the reversible hydration of carbon dioxide: CO2 + H2O ⇔ H+ + HCO3-. The dehydration reaction contributes to net acidification in several tissues and biological fluids. Salivary CA6 attaches to the enamel and predisposes to caries probably by acidifying the dental biofilm by catalyzing carbon dioxide dehydration. Enzyme-linked immunosorbent assay (ELISA) is an assay technique commonly used to measure protein concentrations in solubilized samples. Commercial ELISA kits for specific antigens are available from several companies. The quality of an ELISA kit can vary in terms of sensitivity, specificity, detection range and intra-assay variation. Although commercial ELISA kits for CA6 have been used in several studies, the basic quality parameters of the kits have not been reported by independent research groups. The main objective of this study was to determine whether the two different commercially available ELISA kits are reliable for CA6 concentration measuring. We hypothesized that the two commercial ELISA kits are reliable for measuring CA6 concentration. Material and Methods: We obtained two kits from two companies that we from now on call «Company X» and “Company Y”. One experiment was run with the “Composite X” kit (competitive ELISA) and three experiments were run with the “Company Y” kits (sandwich ELISA) from three different batches (Batch 1, 2 and 3). For ELISA assay procedure, we followed the manufacturers’ instructions. We collected saliva samples from the same two participants in all the experiments, and both of the samples was present in every experiment. In addition, we used purified CA6 which we received as a kind gift from Professor Seppo Parkkila’s group (Tampere, Finland). Results: None of the kits detected the purified CA6 (Table 2). Only the “Company X” kit and the batch 3 of “Company Y” kits gave a positive reaction in saliva samples. Discussion: The findings of this study indicate that one should be vary of studies where commercial non-certified kits have been used. It might be that the results of these studies are based on unspecific binding. This is to our knowledge the only study examining the quality of commercial CA6 ELISA kits. The results obtained in this study indicate that quality assessment of commercial ELISA kits is necessary, at least for CA6 kits if not to other kits as well.