Titration of Cytolytic Antitumor Antibody Utilizing Absorbance at 260 m{micro} to Measure the Leakage of Cell Constituents
Abstract
Ehrlich ascites tumor cells were incubated first with rabbit serum and guinea pig complement and then with trypsin to promote leakage of constituents from the damaged cells. The system then was treated with ribonuclease and deoxyribonuclease to free the fibrinous material from the cells by solubilizing it. After the nucleic acids were extracted by the perchloric acid method, absorbance at 260 mµ was measured. Subtraction of results with a given dilution of normal rabbit serum from results with the same dilution of hyperimmune serum permitted evaluation of the cytotoxic activity of the antiserum. Results in tests performed with and without the several enaymes are presented to demonstrate the increase in sensitivity provided by using them. Absorbance data for several rabbit antisera are compared with results in neutralization tests in mice. The titers tend to parallel one another in the two tests. As another example of the use of the cell-leakage test to detect low levels of antibody, measurement of anti-V2x rabbit carcinoma antibody in rabbits bearing the V2x tumor and the alterations in the antibody level during treatment of the rabbits with a vasodilator are presented. A good correlation existed between the level of cell leakage induced by the rabbit serum and regression or lack of regression of the transplanted tumor. 1 Supported in part by USPHS Research Grant No. CA-08061 from the National Cancer Institute and in part by the Michigan State Legislature from an allocation to The University of Michigan Medical School for Cancer Research. 2 Present address: University of Edinburgh, Edinburgh, Scotland.