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Whole-blood microassay for immunodetection of antigen specific cell mediated immunity using bromodeoxyuridine
L Smedman1, A Joki, P Perlmann
1Department of Immunology, Stockholm University, Sweden.
Insights
This study introduces a new bromodeoxyuridine (BrdU) assay using finger-prick blood for measuring T-cell proliferation in field settings. The novel assay shows high correlation with traditional methods, making it suitable for children
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Assessing in vitro proliferative responses is crucial for immunological studies.
- Traditional assays often require large blood volumes, limiting field applicability, especially in pediatric populations.
- Novel methods are needed for accessible immune response evaluation in resource-limited settings.
Purpose of the Study:
- To evaluate a modified bromodeoxyuridine (BrdU) incorporation assay for measuring in vitro proliferative responses using finger-prick blood.
- To adapt the assay for fieldwork, particularly for studies involving children in Africa.
- To compare the novel assay's performance against established proliferation assays.
Main Methods:
- Finger-prick blood samples were diluted and cultured with various stimulants (purified protein derivative, tetanus toxoid, concanavalin A).
- Bromodeoxyuridine (BrdU) was incorporated during the final 24 hours of culture.
- Cells were stained for activated T-cell markers (CD25) and BrdU incorporation using immunofluorescence and visualized via microscopy.
Main Results:
- The novel BrdU assay successfully detected proliferative responses in cultured white blood cells.
- Dual staining for CD25 and BrdU allowed for classification of cells as single- or double-positive.
- High rank order correlations (0.84 and 0.91) were observed between the BrdU assay and thymidine incorporation assays.
Conclusions:
- The developed bromodeoxyuridine (BrdU) assay is a viable method for assessing T-cell proliferation using small blood volumes.
- This assay is suitable for field-based immunological studies, especially in pediatric populations.
- The novel assay provides a reliable alternative to traditional proliferation assays, enhancing accessibility in diverse settings.
Abstract:
The study evaluates a novel version of the bromodeoxyuridine (BrdU) incorporation assay for in vitro proliferative responses, which works with finger-prick blood specimens. It was developed primarily for field-work involving children in Africa and elsewhere. Heparinized blood specimens from healthy volunteers were diluted 1:15 and cultured with purified protein derivative, tetanus toxoid, concanavalin A or medium alone for four-seven days and pulsed during the last 24 hours with bromodeoxyuridine. Thereafter, white and red cells were separated by Ficoll-Pacque centrifugation. The white cells were made to adhere to Multitest slides pre-treated with poly-L-lysin. After fixation with paraformaldehyde, the cells were incubated with mouse monoclonal antibodies to a surface marker for activated T cells (CD25, interleukin-2 receptor) and the reaction visualized with anti-mouse alkaline phosphatase conjugated antibodies and a Fast Red substrate. After treatment with cold acetone, the cells were covered with formamide and heated to 70 degrees C, without loss of surface staining. The incorporation of bromodeoxyuridine was visualized in the UV-microscope with mouse anti-bromodeoxyuridine monoclonal antibodies and a rabbit anti-mouse fluorescein conjugate. Both the surface staining and the nuclear fluorescence can be seen in UV-light and the cells be classified as single- or double-positive or negative. Parallel experiments on the same blood-sample from seven donors allowed for at least 42 paired comparisons between the proportion of BrdU positive cells and thymidine incorporation stimulation indices. The latter assay was carried out both in the conventional version and in a whole-blood micro-version. The rank order correlations were 0.84 and 0.91 respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
