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Related Experiment Videos

A method for specifically detecting internal immunoglobulin by immunofluorescence

G Mayrhofer

    Journal of Immunological Methods
    |January 1, 1979
    PubMed
    Summary

    Stripping surface immunoglobulin from lymphoid cells using pronase improves internal immunoglobulin detection. This method enhances accuracy by reducing background staining from membrane-bound immunoglobulin, allowing clearer identification of internal immunoglobulin levels.

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    Area of Science:

    • Immunology
    • Cell Biology

    Background:

    • Detection of internal immunoglobulin in lymphoid cells is crucial for understanding immune function.
    • Staining with fluorescein-conjugated antisera can be obscured by non-specific binding to membrane-bound immunoglobulin.
    • Distinguishing low internal immunoglobulin levels from membrane-bound immunoglobulin signal is challenging.

    Purpose of the Study:

    • To develop a method for accurately detecting internal immunoglobulin in lymphoid cells.
    • To overcome the limitations of non-specific binding in immunofluorescence staining.
    • To improve the reliability of quantifying internal immunoglobulin.

    Main Methods:

    • Lymphoid cells (tonsil cells) were treated with pronase to strip surface immunoglobulin.
    • Fixed cell preparations were stained with fluorescein-conjugated antisera.

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  • Comparison of staining patterns between pronase-treated and untreated cell preparations.

    • Main Results:

    • Pronase treatment effectively removed membrane-bound immunoglobulin without significant loss of cell viability.
    • Pronase-stripped smears showed reduced non-cellular debris and background staining.
    • A significant decrease in faintly stained small lymphocytes was observed in pronase-treated preparations.
    • Accurate scoring of cells specifically containing internal immunoglobulin was facilitated.

    Conclusions:

    • Pronase treatment is an effective method to enhance the specificity of internal immunoglobulin detection in lymphoid cells.
    • This technique improves the reliability of immunofluorescence assays for internal immunoglobulin.
    • The method allows for clearer differentiation between low internal immunoglobulin and surface immunoglobulin staining.