In Vitro Assay Design: T Cell Function and Drug Efficacy in EAE Mice

This assignment focuses on designing in vitro assays to assess T cell function in mice with experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. The study involves mice induced with EAE using an immunogenic epitope (MOG) and treated with an experimental drug. The solution proposes two assays: ELISA (Enzyme-Linked ImmunoSorbent Assay) and Flow Cytometry (FCM). ELISA is used to detect cytokine release, which reflects T cell activity, by coating wells with antibodies and detecting cytokine-antibody binding. FCM is used to analyze memory T cells and detect cytokines through intracellular staining and analysis. Both assays aim to evaluate the drug's efficacy by measuring changes in T cell function and cytokine production in response to the drug. The assignment also provides detailed steps for each assay, including cell collection, fixation, permeabilization, and analysis, and references relevant research studies supporting the chosen methods.

Running head: HEALTHCARE
MEDICAL AND APPLIED IMMUNOLOGY
Name of the Student
Name of the University
Author Note

1HEALTHCARE
Assay 1: ELISA (Enzyme-Linked ImmunoSorbent Assay)
In the experiment, autoimmune disease (EAE equivalent) was introduced in mice strain
by administering an immunogenic epitope (MOG). Then it was suspended in Freund’s complete
adjuvant (CFA) before the immunization process. After a period of 9 to 14 days of introducing
with pertussis toxin, the mice were found to develop EAE (Experimental autoimmune
encephalomyelitis). One week after this experiment was performed, the mice were then
introduced with an experimental drug. ELISA can be used to check T cell functioning in
response to the above-stated drug. ELISA can be used for this test because of the fact that T cell
functioning is to be studied here. After the introduction of the drug, if there is an increase in the
efficacy of T cell functioning, it can be detected by ELISA. Body fluid from the mice needs to be
isolated and ELISA is to be performed with it. T cell activation is responsible for the release of
cytokines, which can be properly readout with ELISA. The ELISA wells need to be coated with
antibodies. Now, if T cell is properly functioning in the body of the mice, then it is bound to
release cytokines (Giavridis et al. 2018). These cytokines will bind to the antibody in the
microtitre well and are detected with the help of enzyme-substrate reaction. Thus, it can be stated
that if T cell functioning has increased in the mice after the introduction of the new drug, then
the cytokines will be detected in ELISA. Thus, it can be stated that ELISA can be used to assay
the functioning enhancement or deterioration of T cell after the introduction of a new drug. The
presence of adjuvant is also responsible for a slow release of antigen and thus makes the
detection process more accurate (Zhang et al. 2018). Various studies have been identified which
showed that ELISA can be used for T cell functioning in vitro assay procedures (Maj et al.
2017). Therefore, ELISA was chosen for the in vitro assay of T cell functioning of mice after the
introduction of a new drug for the prevention of EAE

2HEALTHCARE
Assay 2: Flow Cytometry (FCM)
FACs is defined as a specialized type of flow cytometry which is responsible for sorting a
heterogeneous mixture of cells into two or more containers. This analysis is responsible for the
analysis of memory T cells (Lugli et al. 2017). FCM can be used for the detection of cytokines,
in the same way as the previous assay does. In the experiment, autoimmune disease (EAE
equivalent) was introduced in mice strain by administering an immunogenic epitope (MOG).
Then it was suspended in Freund’s complete adjuvant (CFA) before the immunization process.
After a period of 9 to 14 days of introducing with pertussis toxin, the mice were found to develop
EAE (Experimental autoimmune encephalomyelitis). One week after this experiment was
performed, the mice were then introduced with an experimental drug. The steps needed to be
followed for this assay are a collection of mice immunological cells, fixation of the same
followed by its permeabilisation, blocking the cells, performing intracellular and analyzing the
same with FCM. This process provides a direct measurement of T cell functioning and also
diagnose whether it has enhanced after the incorporation of a new drug or not. In this assay, mice
lymphocytes will be collected both before the introduction of a new drug and after the
introduction of a new drug. Then the cells will be fixed on plates by applying chemical fixatives.
This will fix the cells at a place and will prevent them from moving. Permeabilisation will help
in the attachment of fluorophore-cytokine (EAE) complex designed against the antigens released
from the new drug. Combination of both will indirectly prove that T cell has been activated by
the introduction of a new drug. Since flow cytometry has been reported to be used in the
detection of cytokines, therefore it can be stated that the T cells can also be stated to be detected
indirectly with the detection of cytokines. These are the overall steps which can be followed in
this in vitro assays in order to analyze T cell functioning in the mice body after the introduction

3HEALTHCARE
of a new drug. FCM and FACs have been used by various research studies for the analysis of T
cell functioning in several immunological studies (Boland et al. 2018).

4HEALTHCARE
References
Boland, B., Oró, D., Veidal, S., Hansen, H.B., Jelsing, J., Vrang, N., Feigh, M., Fink, L. and Aps,
G., 2018, October. Flow Cytometry Reveals Distinct Changes in Hepatic Inflammatory Cell
Populations in a Diet-Induced Obese Mouse Model of Non-Alcoholic Steatohepatitis.
In HEPATOLOGY(Vol. 68, pp. 737A-738A). 111 RIVER ST, HOBOKEN 07030-5774, NJ
USA: WILEY.
Giavridis, T., van der Stegen, S.J., Eyquem, J., Hamieh, M., Piersigilli, A. and Sadelain, M.,
2018. CAR T cell–induced cytokine release syndrome is mediated by macrophages and abated
by IL-1 blockade. Nature medicine, 24(6), pp.731-738.
Lugli, E., Zanon, V., Mavilio, D. and Roberto, A., 2017. FACS analysis of memory T
lymphocytes. In T-Cell Differentiation(pp. 31-47). Humana Press, New York, NY.
Maj, T., Wang, W., Crespo, J., Zhang, H., Wang, W., Wei, S., Zhao, L., Vatan, L., Shao, I.,
Szeliga, W. and Lyssiotis, C., 2017. Oxidative stress controls regulatory T cell apoptosis and
suppressor activity and PD-L1-blockade resistance in tumor. Nature immunology, 18(12),
p.1332.
Zhang, J., Miao, J., Han, X., Lu, Y., Deng, B., Lv, F., Zhao, Y., Ding, C. and Hou, J., 2018.
Development of a novel oil-in-water emulsion and evaluation of its potential adjuvant function in
a swine influenza vaccine in mice. BMC veterinary research, 14(1), pp.1-11.