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Indomethacin increases the sensitivity of the monocyte migration inhibition assay
R Van der Gaag1, L Broersma, F D De Hon
1Department of Ophthalmo-Immunology, Netherlands Ophthalmic Research Institute, Amsterdam.
Insights
Indomethacin enhances the sensitivity of the monocyte migration inhibition (MIF) assay, enabling detection of immune responses with minimal cell samples. This method is crucial for ophthalmo-immunological studies requiring small biological fluid volumes.
Area of Science:
- Ophthalmology
- Immunology
- Pharmacology
Background:
- Ophthalmo-immunological studies require assays sensitive to small sample volumes and cell numbers.
- Indomethacin is known to augment immune responses in vitro and in vivo.
Purpose of the Study:
- To evaluate the effect of indomethacin on the monocyte migration inhibition (MIF) assay using low cell or antigen doses.
- To determine if indomethacin can increase the sensitivity of the MIF assay for detecting cellular immune responses.
Main Methods:
- The monocyte migration inhibition (MIF) assay was performed with varying low doses of antigen and mononuclear cells.
- Indomethacin was added during the initial step of the MIF assay.
- Titration experiments were conducted to assess sensitivity changes.
- Prostaglandin E2 and leukotriene B4 were added to evaluate their modulatory effects.
Main Results:
- Adding indomethacin significantly increased the sensitivity of the MIF assay by 10-50 fold.
- This enhanced sensitivity allows for the use of fewer cells and lower antigen doses.
- The increased response could be partially reversed by prostaglandin E2 and enhanced by leukotriene B4 within a specific concentration range.
- A challenge was noted regarding the activation of specific cells present at very low frequencies in non-immunized individuals.
Conclusions:
- Indomethacin significantly enhances the sensitivity of the MIF assay, making it highly suitable for ophthalmo-immunological investigations.
- The modified MIF assay allows for the detection of cellular immune responses in minimal biological fluid samples with very few antigen-specific lymphocytes.
- Further research may be needed to manage potential activation of low-frequency specific cells.
Abstract:
In ophthalmo-immunological investigations only small samples of ocular tissues and fluid are available and assays which are feasible with very small volumes or cell numbers are mandatory. Indomethacin, which is known to augment the immune response both in vivo and in vitro was therefore tested for its effect on the monocyte migration inhibition (MIF) assay using low cell or antigen doses. The sensitivity of the MIF assay may be greatly increased by adding indomethaci during the first step of the assay. Titration of either the antigen dose, the mononuclear cells number or both per assay, resulted in a 10-50-fold increase in sensitivity of the assay, with a broad inter-individual variability. Increasing the sensitivity of the MIF assay with indomethacin has clear advantages with regard to the number of cells required but also confronts us with a new problem: activation of specific cells that circulate at very low frequencies in non-immunized individuals. The enhanced response could be reversed to some extent by adding prostaglandin E2 together with indomethacin to the first step of the assay. Moreover, adding leukotriene B4 to the first step of the assay had an enhancing effect over a limited concentration range. We conclude that in the presence of indomethacin, the MIF assay provides a highly sensitive technique for the demonstration of cellular immune responses in small samples of biological fluids containing very small numbers of antigen-specific lymphocytes.
