P. J. MOLONEY AND L. GOLDSMITH
Manuscript received June 13, 1956.
Contribution from Connaught Medical Research Laboratories, University of Toronto, Toronto, Ontario.
Abstract
On the basis of body weight, mice showed a higher percentage survival to large doses of insulin than did rabbits and these in turn were more resistant to insulin than guinea pigs. Antibodies to insulin were induced in five species of animals. Anti-insulins produced by the guinea pig, rabbit, sheep, and horse can each neutralize insulin extracted from the pancreas of the rabbit, sheep, horse, pig, ox, and monkey (Macacus rhesus), but none of the four can neutralize insulin extracted from guinea-pig pancreas. Guinea-pig anti-insulin can neutralize endogenous mouse insulin; horse anti-insulin cannot. The problem of the relative effectiveness of insulin as an antigen is discussed.
It has been shown by Moloney and Coval (5) that insulin injected parenterally can elicit antibodies in the guinea pig and sheep; that such antibodies can neutralize, in vitro, extracted insulins of a number of species; and that guinea-pig anti-insulin can neutralize the endogenous insulin of the
mouse.
This paper deals with anti-insulin production in the mouse, guinea pig, rabbit, sheep, and horse; with the problem of neutralization of endogenous insulin in the mouse; and with the relative effectiveness of insulin as an antigen.
Materials and Methods
Preparation of Antigen
Insulin in Freund's adjuvant was prepared as previously described (5) and injections were made subcutaneously. The maximum amount injected in one site was 0.5 ml. for mice, 1.0 ml. for guinea pigs, 2.0 ml. for rabbits, 25 ml. for sheep, and 50 ml. for horses. In general, animals were inoculated at monthly intervals.
Titration of Anti-insulin in Animal Sera
Neutralizing activity of serum was tested by a mouse-convulsion procedure under the same conditions of temperature and mouse-starvation as are laid down for insulin assay (3). Undiluted serum or dilutions of serum in buffered saline were mixed with equal volumes of a solution of crystalline ox or pig insulin in acid water (pH 2.6) containing 0.2 units per milliliter (resulting pH 6.0-7.5). A group of six starved mice was used to test each mixture. Each mouse was injected with 0.5 ml. When a mixture contained insulin plus normal serum or buffered saline, six of six mice usually convulsed; occasionally only five of six convulsed. With a mixture containing insulin plus immune serum of sufficiently high anti-insulin titer, none of the injected mice convulsed. When unknown sera were being tested, a serum of known neutralizing power (standard immune serum) was included. Table I gives typical results. [end of text]
Can. J. Biochem. Physiol. 35 (1957)