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An enzyme-assessed microplate-assay for neutrophil adherence. I. IgA-induced adherence of human PMNs
W W Bullock1, M Rogers, W L Gabler
1Department of Oral Microbiology/Immunology, School of Dentistry, Oregon Health Sciences University, Portland 97201.
Abstract:
The binding of PMNs to extracellular matrix and cells is crucial to PMN host defense. Adherence mechanisms and the many families of molecules involved are major areas of study. We present here details of an enzyme-assessed microtiter plate assay for neutrophil adherence. This assay uses low numbers of cells (50,000/well) and permits analysis of several hundred wells in a short period of time, by using an ELISA reader. With this assay we observed 5- to 10-fold increases in the number of adherent human PMNs in response to nanogram amounts of LPS or as little as 5.0 micrograms/ml of aggregated IgA. Although fluoride blocked the LPS-induced adherence response, IgA-induced cell binding was largely unaffected.
Insights
A new enzyme-linked immunosorbent assay (ELISA) quantifies neutrophil adherence, revealing increased binding with lipopolysaccharide (LPS) and aggregated immunoglobulin A (IgA). LPS-induced adherence was blocked by fluoride, but IgA-induced binding was not.
Area of Science:
- Immunology
- Cellular Biology
- Assay Development
Background:
- Neutrophil adherence to extracellular matrix and cells is vital for host defense.
- Understanding adherence mechanisms and involved molecules is a key research area.
- Existing methods may require large cell numbers or extensive time.
Purpose of the Study:
- To develop and detail a novel enzyme-assessed microtiter plate assay for quantifying neutrophil adherence.
- To assess the assay's efficiency using low cell numbers and high throughput.
- To investigate the effects of lipopolysaccharide (LPS) and aggregated immunoglobulin A (IgA) on neutrophil adherence.
Main Methods:
- Development of an enzyme-assessed microtiter plate assay for neutrophil adherence.
- Utilized low cell numbers (50,000/well) for efficiency.
- Employed an ELISA reader for rapid analysis of multiple wells.
- Tested the impact of LPS and aggregated IgA on human neutrophil adherence.
- Investigated the effect of fluoride on LPS- and IgA-induced adherence.
Main Results:
- The assay allows for the analysis of several hundred wells rapidly.
- Observed 5- to 10-fold increases in adherent human neutrophils with nanogram amounts of LPS.
- Observed 5- to 10-fold increases in adherent human neutrophils with 5.0 µg/ml aggregated IgA.
- Fluoride significantly blocked the LPS-induced adherence response.
- IgA-induced neutrophil adherence was largely unaffected by fluoride.
Conclusions:
- The developed microtiter plate assay is a sensitive and efficient method for studying neutrophil adherence.
- Both LPS and aggregated IgA can significantly enhance neutrophil adherence.
- The mechanisms underlying LPS- and IgA-induced neutrophil adherence may differ, as suggested by the differential effect of fluoride.