[Immunological and physicochemical properties of Cry j II, the second major allergen of Japanese cedar pollen (Cryptomeria japonica)].

Cry j II, the second major allergen of Japanese cedar (sugi, Cryptomeria japonica) pollen was examined for the allergenicity by intradermal test and RAST. Nineteen of the 25 allergic patients examined, showed positive reaction to the Cry j II. Contents of Cry j II in the extracts of the pollen collected in various regions from 1977 to 1991 showed yearly variation ranging from 2.9 to 14 mg/100 g pollen, whereas the amount of Cry j I in the extract was comparatively stable at about 35 mg/100 g pollen. Physicochemical treatments of Cry j I and Cry j II suggested that specific human IgE antibodies and some mAbs bind to conformational epitopes which are denatured and destroyed by certain treatments.

[Granule-accumulation in VERO cells used for determination of antitoxin titration by micro cell culture method: II. Nature of granules and influence on the potency testing of diphtheria toxoid].

Accumulation of granules occurs in VERO cells when mouse serum was added in high concentration. The nature of the granule and influence of the granule-accumulation on the potency testing of diphtheria antitoxin were studied. The concentration of triglyceride in VERO cells increased 15 folds after 24 hours of incubation by the addition of undiluted mouse serum. Kinetical study of the increase of triglyceride and electron microscope observation suggested that the accumulated granules might be oil droplets with an average diameter of 0.27 micron. The end point reaction of neutralization of diphtheria toxin by the standard antitoxin was analyzed by the addition of mouse serum to the micro cell culture using VERO cells. The mean values of the end point reactions in the cell cultures added with high concentrations of mouse serum were -2.324, -2.338 and -2.346, while that of the control group was -2.343. The variance of these mean values in the experiment and control groups were 0.0017, 0.0024, 0.0017 and 0.0021, respectively. A test of homogeneity of variance for these data showed no significant difference between them. Furthermore, antitoxin titer of the serum specimens of mouse immunized with diphtheria toxoids determined by the micro cell culture method were compared with that obtained by rabbit skin test; a high correlation between the antitoxin titers by the two techniques was recognized. The results were comparable with those of guinea-pig sera. From these results it is evident that little influence, if any, was observed in the potency testing of diphtheria antitoxin using VERO cells, in spite of the remarkable accumulation of granules.

[Granule-accumulation in VERO cells used for determination of antitoxin titration by micro cell culture method: I. Influence of mouse serum].

When the titration of diphtheria antitoxin of mouse serum was carried out by micro cell culture method using VERO cells, a large number of granules were observed in the cells. In order to examine the influence of this phenomenon on the titration of antitoxin, kinetics of the granule-accumulation was investigated. The granule-accumulation occurred in the cells in the culture medium to which either immunized or normal mouse serum was added. The granule-rich cells appeared at the dilution of the serum less than 1:32 and increased in number with the concentration of the serum. After 4 days of incubation 88% of the cells showed granule-accumulation when undiluted serum was added. Besides the mouse serum, those from guinea-pigs, horses, fetal calves and humans were examined. However, intensive accumulation of granules such as shown with mouse serum was not observed. From these results it was suggested that mouse serum might have some unknown mechanism which caused the remarkable accumulation of granules in VERO cells. The nature of granule and influence of this phenomenon on the titration of diphtheria antitoxin will be presented in an accompanying paper.