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Standardisation of a procedure for quantifying surface antigens by indirect immunofluorescence
1Institute for Animal Health, Compton, Newbury, Berkshire, UK. kyrlie.smith@bbsrc.ac.uk
Journal of Immunological Methods
|November 11, 1999
Summary
Standardizing quantitative indirect immunofluorescence (QIIF) for bovine MHC class I molecules requires optimizing cell washes and cell numbers. Accurate antibody titration and live cell gating are crucial for reliable quantification of cell surface antigens.
Area of Science:
- Immunology
- Flow Cytometry
- Veterinary Science
Background:
- Quantitative indirect immunofluorescence (QIIF) methods for measuring cell surface antigens yield inconsistent results.
- Accurate quantification of bovine classical major histocompatibility complex (MHC) class I molecules is essential for immunological studies.
Purpose of the Study:
- To standardize a flow cytometric method using the QIFIkit for quantifying surface-expressed bovine MHC class I molecules.
- To investigate critical parameters beyond antibody titration and live cell gating, including cell washes and cell numbers.
Main Methods:
- Utilized the QIFIkit (Dako, Denmark) for quantitative indirect immunofluorescence.
- Employed flow cytometry for cell surface molecule quantification.
- Focused on optimizing cell washing steps and evaluating the impact of varying cell numbers.
Main Results:
- Established the necessity of optimizing cell washes to remove excess antibodies.
- Demonstrated the influence of cell numbers on assay accuracy.
- Highlighted the requirement for understanding antibody binding properties for precise measurements.
Conclusions:
- Accurate numerical assessment of cell surface-expressed molecules using QIIF requires careful establishment of multiple critical parameters.
- Optimizing cell washes and cell numbers, alongside antibody titration and live cell gating, is vital for reliable QIIF results in bovine immunology.