Electrophoretic studies on normal and tetanus immune horse sera

II. On horse sera hyperimmunized with tetanus toxin

Abstract

I) The electrophoretic analysis was made on sera of 4 horses hyperimmunized with tetanus toxin in several stages of the immunization. The experimental results were shown in Table 1 and 2: An increase of total protein, a decrease of albumin and an increase of β-glob were recognized in all cases of hyperimmune horse sera which showed very high antitoxin titers in the final stage of immunization. However, these phenomena were not recognized in the initial and middle stage of immune course. Alpha-glob or γ-glob did not show any remarkable changes in all cases.
The relative modility of β-glob in the high titer anti-sera was faster than that of γ-glob and slower than β-glob in normal sera. The mobility was almost the same as fibrinogen in the plasma.
It was recognized that amount of β-glob of slow mobility increased in antitoxic horse sera in a final stage of hyperimmune course and it was seemed to contain much antitoxins in the fraction.
II) The relationship of antitoxin to the fractions of horse sera hyperimmunized with the toxin is of importance. In this paper, the serum protein fractions responsible for the antitoxin have been investigated electrophoretically and immunologically.
The serum protein fractions were isolated from antitoxic sera in various immune stages by means of the starch-electrophoresis, and the antitoxin titer of each fraction was measured by the mouse test.
The antitoxin was recognized only in β-glob in the intial stage of immunization, but found in both of β-glob and γ-glob in the middle stage. The majority of antitoxins was found in β-glob and some antitoxins were recognized not only in γ-glob but also in α-glob in the final stage. After the completion of immunization, antitoxins were always found in β-, γ- and α-glob, but albumin did not contain any antitoxin over all the stages.
Alpha-, β- and γ-glob isolated by the starch-electrophoresis were again applied for the same electrophoresis, and separated into fast and slow fractions.
Alpha-glob of fast mobility did not show any antitoxin titer, but slow α-glob showed some titers. Beta-glob of fast and slow mobility were recognized to have much antitoxins. Antitoxins were contained in fast γ-glob, but not in slow γ-glob.
It was discovered thst the tetanus antitoxin is contained in all β-glob fraction but in α- and γ-glob fraction antitoxin is present more near to β-glob peak. Therefore, it was cosidered that the tetanus antitoxin was contained only in β-glob, and antitoxin found in α- and γ-glob would be caused by β-glob contamination.
Albumin did not contain any antitoxin, but it was demonstrated to increase the antitoxic activities of β-glob when it was added to the solution.