Abstract
The present study was undertaken to quantity the human gastric intrinsic factor (IF) with charcoal radioimmunoassay using heterologus antisera against human IF instead of anti-IF autoantibody (PA-A-IF) from the patients with pernicious anemia (PA).
For this purpose, purification of the human gastric IF was performed. A large amount of lyophilized gastric juice was dissolved in small amount of pH 9.0 borate buffer solution, and then fractionated with Sephadex G 200 and DEAE cellulose chromatography. B12 binding capacity of each fraction, before and after adding to it PA-A-IF, was determined by the method of Gottlieb et al. The fractions, with great B12 binding capacity and which strikingly reduces its capacity after adding PA-A-IF, was concentrated and used for immunization as IF antigen. Guiner pigs were used as immune animals for preparation of anti-IF antisera, because their serum showed remarkably lower unsaturated B12 binding capacity than a rabbit's serum.
The antisera thus prepared (GP-A-IF) showed antibody activities which were similar to the type I blocking and the type II binding antibody found in the patients with PA. No evidence were found about the existence of antibody activities against non-IF binder in these antisera.
The amount of human gastric IF determined by charcoal radioimmunoassay using GP-A-IF was generally equal to that determined using PA-A-IF.
In further experiments, IF-B12 complex was fractionated by the same method from gastric juice presaturated with a small amount of 57CoB12 and excess amount of non-radioactive CN-B12. Guinea pigs were immunized with this IFB12 complex (GP-A-IFB12) in order to compare antigenic activities of IF alone with those of IFB12 complex.
These GP-A-IFB12 also revealed the same two types of antibody activities as were observed in the patient's serum, but showed a tendency that was weaker in the type I antibody and stronger in the type II than GP-A-IF.
