Development of a Sensitive ELISA for Gastric Intrinsic Factor and Detection of Intrinsic Factor Immunoreactivity in Human Serum

Abstract

Gastric Intrinsic Factor (IF) is produced by the parietal cells of the stomach and secreted into the gastrointestinal tract where it ensures the active absorption of vitamin B12. We hypothesized that a small amount of IF ends up in the circulation and can be measured in serum. The aim of this study was to develop an assay for measuring human IF and to demonstrate its presence in serum. We designed a sensitive ELISA for measurement of human IF using a commercial monoclonal antibody and an in-house polyclonal antibody as capture and detecting antibody, respectively. Imprecision, accuracy, and linearity of the assay were examined. We established a reference interval based on serum samples from 240 healthy donors, and explored the daily IF fluctuations in 20 healthy subjects. Employing a prototype IF ELISA and size exclusion chromatography experiments, we demonstrated the presence of IF in human serum. In its final design, the IF ELISA has a measurement range of 0.2 to 50 pmol/L. The intra-assay and total imprecision were 7.9% and 15%, respectively. The 95% reference interval (18–65 years) was 1.7–11.6 pmol/L. No diurnal fluctuation or notable sex differences were observed. Our results suggest that the assay is capable of detecting and quantifying human IF in the circulation and may prove useful in the characterization of patients with impaired IF production.

Keywords

• intrinsic factor
• ELISA
• vitamin B12
• cobalamin
• pernicious anemia
• autoimmune disease