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Studies on Antireticular Cytotoxic Serum¹

II. Preparation and Titration of the Serum and Study of Its Serological Properties

From the Department of Pathology and Research of the Cedars of Lebanon Hospital, Los Angeles 27, California

Abstract

Antigens for the preparation of ACS have been prepared with a clean but not aseptic technic from fresh spleen and bone marrow. The antigens were freshly prepared for each injection.

The serums were prepared in rabbits and goats by repeated intravenous injections of progressively increasing doses of the antigens. To avoid anaphylaxis the animals were injected every third day, instead of every fourth or fifth day, with excellent results.

ACS was similarly prepared against human tissue in rabbits and goats, against rabbit tissue only in goats, and against rat and mouse tissue in rabbits. The peak of antibody production is not reached until the fourth to the seventh day after the last injection of antigen. Higher titers of anti-human ACS were secured in rabbits than in goats.

In titrating the unit of complement, we recommend that the test be subjected to one and one-quarter hours of incubation at 37 C instead of three-fourths hour as required by Marchuk. This tends to make the unit of complement larger, the titer of ACS lower, and allows for the loss of complement in the process of serum titration which is a source of error.

Providing an excess of antigen is present, the dilution of antigen used in titration of the immune serum is immaterial except in high concentrations where it may be anticomplementary. It is important however that the antigen be fresh (not older than three days) for both titration of immune sera and for preparation of ACS in animals.

It is apparent that ACS is species specific and that a slight cross reaction in the lower dilutions of the sera may be due to a foreign antibody, possibly one of the Forssmann type. Hemolysins are usually not present in the serum in titers greater than 1:20 but hemagglutinins may be present to titers of 1:640 or more. These may be selectively absorbed out without materially affecting the titer of the ACS.

Liquid ACS may be kept at 4 C. or at room temperature for several months to a year with very gradual deterioration. Serum prepared in our laboratory, sent refrigerated to the USSR possessed adequate titers of ACS upon being checked by A. A. Bogomolets.

Footnotes

¹ These studies were supported by funds established by Mr. Harry Warner, Mr. Harry F. Leveson, Dr. C. Emery, Mr. J. W. Drown, Mr. B. Bard, Mr. R. Dechter, and an anonymous donor.