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Measurement of polyclonal immunoglobulin synthesis using the reverse plaque technique
1Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA.
Insights
The reverse hemolytic plaque assay quantifies immunoglobulin (Ig)-secreting cells in various cell populations. This method is crucial for immunology research, enabling precise measurement of antibody-producing cells.
Area of Science:
- Immunology
- Cell Biology
Background:
- Immunoglobulin (Ig)-secreting cells are vital for adaptive immunity.
- Accurate quantification of these cells is essential for immunological studies.
Purpose of the Study:
- To describe the reverse hemolytic plaque assay (RHPA) for measuring Ig-secreting cells.
- To provide a detailed protocol for conducting the RHPA.
Main Methods:
- The RHPA involves incubating test cell populations with protein A-sensitized sheep red blood cells (SRBC), complement, and anti-Ig antibody.
- Incubation is followed by scoring of resulting hemolytic plaques.
- A support protocol details the preparation of specialized plaquing chambers.
Main Results:
- The RHPA effectively measures the number of Ig-secreting cells in a population.
- The assay can be applied to diverse cell sources, including peripheral blood mononuclear cells and tonsil cells.
Conclusions:
- The reverse hemolytic plaque assay is a valuable and effective technique for enumerating Ig-secreting cells.
- This method facilitates the study of humoral immunity and antibody production in various biological samples.
Abstract:
This unit describes the reverse hemolytic plaque assay, an effective method for measuring the number of immunoglobulin (Ig)-secreting cells present in a cell population at any particular time. Cell populations that can be assayed using the technique include peripheral blood mononuclear cells or cells from tissues such as the tonsils. The basic protocol is divided into three stages. First, protein A-sensitized sheep red blood cells (SRBC), guinea pig complement, and anti-Ig antibody are prepared. Test samples are then combined and incubated with the SRBC, complement, and antibody in appropriate chambers. Finally, the resulting plaques are scored. A support protocol describes the preparation of plaquing chambers.
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