Sedimentation rate of HB Ag was measured by the moving boundary method and proved to be 40.
Serum HB Ag was banded in sucrose-in-water density gradient ultracentrifugation using Backman L 2-65 B ultracentrifuge with SW 50.1 rotor at 5?C, 192,000 g for 15 hours, and HB Ag collected from the initial 40% sucrose fraction of density around 1.16 when used with pepsin digested samples, and from both the initial 40% fraction and the pellet when used with native samples.
With use of purified HB Ag and HB Ag treated by proteolytic enzyme, acid and 2-mercaptoethanol.
The following evidences were obtained:
1) Immunoreactivity of HB Ag was not changed by the digestion with gastric juice.
2) Pellet HB Ag from sucrose-in-water density gradient ultracentrifugation was not obtained when the HB Ag sample treated with acid solution, pH 2.0 or 2.3, poteolytic enzymes, and 2-mercaptoethanol followed by dialysis against 5M urea solution was recentrifuged, while pellet HB Ag with serum IgM were identified as with recentrifuged using HB Ag sample treated by nuclease, 5M urea dialysis alone, and acid solution of pH over 3.0.
3) When the pellet HB Ag of untreated serum was treated with acid or pepsin digestion, and banded again by sucrose-in-water density gradient sedimentation, HB Ag was found in the 40% sucrose fraction. In addition, when the HB Ag positive 40% sucrose fraction of untreated serum was treated with acid or pepsin digestion, and banded again in the sucrose-in-water density gradient sedimentation, HB Ag was again found only in the 40% sucrose fraction.
4), When the purified HB Ag was rebanded in sucrose-in-water density gradient sedimentation and also when the purified HB Ag mixed with supernate proteins of macroglobulin pelleting and incubated was rebanded, HB Ag was found again in the, 40% sucrose fraction.
5) On the other hand, when the macroglobulin pellet obtained by ultracentrifugation of the serum from the patients who had initially been positive for HB Ag but had become HB Ag negative at the time the sample was drawn was added to the purified HB Ag, incubated and rebanded, the HB Ag at this time was found mostly in the pellet fraction. And it was demonstrated that the only found pellet protein was IgM globulin.
6) Treatment of pellet HB Ag of sucrose-in-water density gradient ultracentrifugation with 2-mercaptoethanol followed by dialysis against 5M urea solution dissociated the IgM protein from HB Ag and HB Ag was found in the 40% sucrose fraction when rebanded.
It is assumed from the above data that the serum HB Ag immune complex may be that with IgM, i.e., HB Ag IgM complex and disulfide bonding may be Important to the stability of the complex and perhaps in the structure of the IgM.
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