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Immunochemical Studies on Human Serum¹

II. In Vitro Studies on the Stability of Human Complement and Its Components

From the Institute of Pathology, Western Reserve University, and the University Hospitals, Cleveland, Ohio

Abstract

1. Whole complement is preserved for at least one year by freezing and storage at –35 C with about one–third loss in activity. The addition of 10 per cent solid NaCl together with 4 per cent solid boric acid decreases the loss in activity.

2. The precipitate which separates when human serum is dialyzed against phosphate buffer of pH = 5.4 and µ 0.02 is best preserved at 1 C in 5:1 concentration in pH = 6.6 phosphate buffer of ionic strength 0.3.

3. The "pH = 5.4—µ 0.02 supernate" is best preserved at 1 C in 1:1.2 dilution in isotonic saline solution, or in 1:2.5 dilution in phosphate buffer of pH = 6.6 and ionic strength 0.3. For periods longer than 2 months it is best preserved by drying from the frozen state.

4. Both the "pH = 5.4—µ 0.02 precipitate" and supernate may be passed through a micro-Seitz filter without impairment of their component activity.

5. C'2 is that component of whole serum and of the supernate which is most susceptible to heat. C'4 is the most heat-susceptible component in the "pH = 5.4—µ 0.02 precipitate," and the least heat-susceptible component in the "pH = 5.4—µ 0.02 supernate."

6. C'4 is the most susceptible component to pH changes in both supernate and precipitate, being most stable at pH between 6.0 and 6.8.

7. C'3 and C'4 are those components of whole complement most susceptible to destruction upon standing under various conditions of temperature and dilution.

8. The fact that C'4 is the least stable of the components in the "pH = 5.4—µ 0.02 precipitate," as evidenced by its greater susceptibility to aging, pH variation, and heat, would indicate that the precipitate lacks certain protective substances which enhance the greater heat stability of C'4 in whole serum and in the "pH = 5.4—µ 0.02 supernate," as well as the greater resistance of C'4 in whole serum to alkali. However, as yet there is no experimental evidence to warrant the conclusion that C'2 is the component necessary for the stabilization of C'4, or is the "carrier" component of C'4, as was concluded in the case of guinea-pig complement (4).

Footnotes

¹ Aided by a grant from the Commonwealth Fund.