Related Experiment Videos
Superoxide assay-leukocyte adherence inhibition test and a soluble factor which stimulates the adherence of
Abstract:
A modified method for the leukocyte adherence inhibition test is described. Peritoneal cells from immune guinea pigs or peripheral mononuclear cells from cancer patients were incubated with immunizing antigen or tumor extract in a 4-mm-wide glass microcell for spectrophotometer analysis. Instead of visual cell counting, the cells adherent to the bottom of the microcell were stimulated with cytochalasin E and wheat germ agglutinin, and the amount of the superoxide (O2.-) generated from the adherent macrophages or monocytes was assayed. Antigen-specific adherence inhibition of peritoneal cells of the immunized guinea pigs was detected 2 weeks after immunization. In contrast, cell adherence was stimulated after 3 weeks. It was shown that a soluble factor was responsible for the adherence stimulation and that nonadherent cells were necessary for its production. Peripheral mononuclear cells of 70% of the tumor-bearing lung cancer patients reacted to the lung tumor extract (9 adherence inhibitions and 7 adherence stimulations per 23 patients). Twenty-five % (3 of 12) of tumor-free patients showed positive reactions, all with adherence stimulation. Of the 12 healthy donors, 3 cases of pneumonia, one case of angiosarcoma of the left flank, one case of hemangiopericytoma of the mediastinum, and 8 cases of breast cancer, non reacted with the lung tumor extract, and only one of 7 lung tuberculosis patients showed positive adherence stimulation.
Insights
A modified leukocyte adherence inhibition test uses superoxide generation instead of cell counting. This assay detects antigen-specific immune responses in cancer patients and immunized animals.
Area of Science:
- Immunology
- Cellular Immunology
- Cancer Immunology
Background:
- The leukocyte adherence inhibition test is a valuable tool for assessing cell-mediated immunity.
- Traditional methods rely on visual cell counting, which can be subjective and time-consuming.
- A more objective and sensitive assay is needed to improve the reliability of the leukocyte adherence inhibition test.
Purpose of the Study:
- To develop and validate a modified leukocyte adherence inhibition test using superoxide anion (O2.-) generation as a readout.
- To assess the utility of this modified assay in detecting antigen-specific immune responses in cancer patients and immunized animals.
Main Methods:
- A modified leukocyte adherence inhibition test was developed using peritoneal cells from immune guinea pigs or peripheral mononuclear cells from cancer patients.
- Cells were incubated with antigen or tumor extract in microcells, and adherent cells were stimulated with cytochalasin E and wheat germ agglutinin.
- Superoxide (O2.-) generation from adherent macrophages or monocytes was measured spectrophotometrically, replacing visual cell counting.
Main Results:
- Antigen-specific adherence inhibition was detected in immunized guinea pigs 2 weeks post-immunization, with adherence stimulation observed after 3 weeks.
- A soluble factor produced by nonadherent cells was identified as responsible for adherence stimulation.
- Peripheral mononuclear cells from 70% of lung cancer patients showed reactivity to lung tumor extract, with both inhibition and stimulation observed.
- A low percentage of tumor-free patients and healthy donors showed positive reactions, primarily adherence stimulation.
Conclusions:
- The modified leukocyte adherence inhibition test, utilizing superoxide generation, provides an objective and sensitive method for detecting cell-mediated immunity.
- This assay shows promise for diagnosing and monitoring immune responses in cancer patients.
- Further research is warranted to explore the clinical applications of this modified assay in various immunological conditions.