Interleukin-4 (IL-4)
Description
IL-4 has an important role in regulating antibody production, hematopoiesis and inflammation, and the development of effector Th2-cell responses. It is produced only by a subset of activated hematopoietic cells, including T cells and FceRl+ mast cells and basophils. IL-4 plays an important role in the development of certain immune disorders, particularly allergies and some autoimmune diseases (Brown and Hural 2017). There are no available assays for marmoset IL-4.
Alignment
Protein alignment for human, rhesus macaque and marmoset IL-4:
Protein alignment for marmoset, owl monkey, and squirrel monkey IL-4:
References
- Brown, M. A. and J. Hural (2017). "Functions of IL-4 and Control of Its Expression." Crit Rev Immunol 37(2-6): 181-212.
Status
Immunization of mice was performed with in-house HEK293-produced tagged MAR IL-4 (C-tag or C-tag plus immunogenicity tag). Serological responses were detected with soluble Twin-tagged antigen and good responses were also seen with coated antigen. A total of 10 clones were selected after a screening process that included ELISA, FluoroSpot (monoclonality) and Octet (affinity scout & epitope bin) assays. These clones, and 2 additional ones (IL-4I and IL-4II), were tested directly with samples generated by NWM. Further Octet-analysis showed that the IL-4 does bind to its soluble receptor, and that binding of the selected mAb clones are not affected by IL-4 binding to its soluble receptor.
A total of 12 monoclonal antibodies were prepared in pure and biotinylated form, and ELISPOT assays were performed to determine reactivity with NWM samples. Because IL-4 is generally released at low levels and consumed by cells in culture, ELISPOT assays, which can identify few cytokine-producing cells, were chosen as they are the most sensitive ones compared to ELISA and LMX.
An example of one of the several ELISPOT plates that were run appears below in Figure 1. Antibody clones are color coded based on the IL-4 epitope that they recognize.
In the Figure 1 example plate, wells A1 to 9 and B1 to 9 were coated with the mAb BR8. The wells A1 to 9 received unstimulated marmoset PBMC, whereas well B1 to 9 received PBMCs stimulated with the mitogens PMA/ionomycin at the concentrations described in section 1.a. After 48 hours of incubation plates were washed and BR20, BR26, BR9, BR16, BR19, BR30, BR1, BR29, and IL-4 (II) biotinylated antibodies were added to wells A1-B2, A2-B2, A3-B3, A4-B4, A5-B5, A6-B6, A7-B7, A8-B8, and A9-B9, respectively. Spots were visualized by the addition of Streptavidin-alkaline phosphatase, followed by the chromogenic substrate NBT/BCIP.
The outcome of the example ELISPOT plate is shown in Figure 2, which was able to detect IL-4-procuding cells for wells D11 (IL-4II and BR1), D12 (IL-4II and BR29), and H9 (BR1 and IL-4II).
Based on the outcome of the first ELISPOT assay, antibodies BR1, BR29, and IL-4(II) were selected for further testing. Another ELISPOT assay was designed with PBMCs from four different marmosets, in which BR29 was used as capture and IL-4(II) as detection, or IL-4(II) as capture and BR29 as detection (Figure 3). The results of this assay showed that either combination was able to detect IL-4-producing cells in the stimulated wells, but the combination BR29 capture with IL-4(II) detection produced clearer backgrounds. This assay also showed the individual variability of outbred marmosets, with one animal (Marmoset 2) barely producing any IL-4 after PMA/ionomycin stimulation (row D of Figure 3).
Having demonstrated that BR29 and IL-4(II) were able to recognize marmoset IL-4, we performed an additional ELISPOT assay using PBMCs obtained from owl monkeys (Figure 4). The same antibody pair detected owl monkey IL-4-producing cells. An ELISPOT assay for squirrel monkey IL-4 has not been performed yet.
In summary, BR29 and IL-4(II) monoclonal antibodies recognize marmoset and owl monkey IL-4 in an ELISPOT format. Production of these antibodies for distribution is underway.