1.
BT602: Seminar II
Course Instructor: Ms. Nighat Syed

2.
Name: Salma Zafar.
Student of BS Biotechnology.
Current Semester no: 8
VU Student ID: BC180405936
VU Mail ID:
BC180405936@vu.edu.pk

3.
Introduction.
Main Content.
Discussion.
Conclusion.
Future perspectives.
References

4.
• ELISPOT assay is the modified form of
ELISA assay.
• This assay proceeds with the
quantitative determination of the number
of cells in a population. Those cells are
capable of producing a number of
antibodies specified with some given
antigen.
• It allows the quantitative determination
of the number of the cells in a
population that are producing a number
of antibodies specifies at a given antigen
or an antigen for which one has a
specific antibodies.
Introduction
to
ELISpot
assay

5.
• T cells (also known as T lymphocytes and
thymocytes) are further developing cells of
stem cells originated from bone marrow.
• They get matured within thymus and then
released into the blood stream throughout the
body.
• They are the active members of immune
system, developing strength against infections
and severe autoimmune diseases including
cancer.
• Their roles include immune response
regulation, direct killing of the infected body
cells while activating new immune cells.
T-Cells

6.
T-cell ELISpot assay can be defined as quantitative method
for the measurement of relevant parameters of T-cells
activation in immune system at single cell level.
It is a most sensitive method allowing the detection of T-
cells having low-frequency and antigen specificity
secreting cytokines and effector molecules including
perforin and granzyme B.
The T-Cell ELISPOT ASSAY is used for the
characterization of T-cell subsets.
This is because the T-cell assay can use for the detection of
the production of cytokines including IL-2, IL-4, IL-5, IL-
13, TNF-alpha and IFN-y.
Three cytokines are produced by Th-1 cells while the other
three are produced by Th-2 cells.

7.
•The enzyme-linked immuno-spot (ELISpot)
assay is a highly sensitive immunoassay even
for the investigation of single cell, which allows
the measurement of cytokine-secreting cells
frequency.
•A specific capturing antibody taken, on which
cells are allowed to grow, to capture the
secreted proteins including cytokines. The
captured cells after a specific incubation period
are removed and the detection of secreted
molecule is done by using a detector antibody
through the ELISA employed procedure.

8.
Antibody coating
first of all the captured monoclonal antibodies are immobilized
through an ethanol treated membrane plate.
Incubation of cells
After antibody coating, the extracted cells are added to the wells
no matter activating stimuli is present or not at that time, and then
it put to the incubation till the secretion of cytokine obtained.
Cytokine capture
Membrane that surrounds the activated cells, possess antibodies
along-with, thus, the secreted cytokines captured through them.
Addition of detection antibodies
After the capturing of cytokines, and removal of cells from the
wells, antibodies are added to those wells.

9.
Addition of enzyme-conjugate complex
An enzyme and its conjugate complex is added to the wells for the
formation of spots on membrane.
Substrate addition
now in the wells, a substrate of calorimetric nature is added and after
its catalyzing by enzyme, the mixture will form an insoluble
precipitate indicating the releasing of cytokine in the mixture.
Analysis
The last step is the analyzing the obtained product by an automated
apparatus or through dissecting microscope to count the frequency of
secreted cells.

10.
 There are a few significant differences between ELISA and ELISpot assay
 One of the major is because of its functionality as the ELISpot assay is the
combination of both bioassay and immunoassay because cells are cultured in
the wells of ELISpot plate while the ELISA is only an immunoassay.
 An ELISA can be used for the detection of whole concentration from secreted
signaling proteins while an ELISpot assay can be used to measure the
frequency of secreted proteins on cellular level as well as the respective cell of
that cytokine secretion.
 So, the ELISpot assay should be use along with ELISA as an addition but not
as replacement. ELISpot assay is far sensitive as compared to conventional
ELISA as the capture of secretion done on the well directly before its addition
and dilution in the supernatant of culture media, capturing through receptors of
adjacent cells and degradation through proteases enzyme.

11.
 Elispot assay has a wide range of applications including cancer research, vaccine
development, macrophage characterization and characterization of dendritic cells
and monocytes, to treat the allergic diseases, analysis of apoli-proteins,
transportation of organs and for veterinary research.
 The assay has an important application in the analysis and diagnosis of
autoimmune diseases including diabetes and multiple sclerosis.
 For the measurement of tumor secretory products, immune cells and low
frequency tissues in body.
 In gene therapy it has an application to detect the transfection efficiency.
 In peripheral blood cell preparation, it can be used for the measurement of antigen
specific responses and post vaccination effects.
 ELISpot assay now has an application in the coronavirus research. It can be used
as a powerful method to analyze the immune response against viral infection.

12.
 The ELISPOT assay uses pairs of antibodies, directed against distinct epitopes of a
cytokine, which capture molecules secreted by individual activated T cells.
 Differently to ELISA, the cytokine captured by the immobilized antibody is detected in
situ using an insoluble peroxidase substrate.
 Thus, the cytokine secretion by individual cells is clearly visualized.
 This technique has shown to be particularly useful in the analysis of CD8q T cell
responses of very low-frequencies.
 In our hands, antigen-specific T cells at frequencies as low as 1–2 per 100,000 cells could
be reproducibly detected by the ELISPOT assay.
 The ELISPOT technique permits the evaluation of T cell responses in different
compartments, such as lymph nodes, peripheral blood, spleen or liver.
 As illustrated in this protocol, for spleen or liver cells, red blood cells RBC lysis is not
necessary before the assay because they are not present in sufficient quantity to interfere
with the formation of a cell monolayer to coated nitrocellulose membrane.
 However, for the analysis of peripheral blood mononuclear cells removal of RBC is
highly recommended.

13.
 Clearly, reliable measurements of several key T cell functions will be
required for a better understanding of these cells’ roles in diverse immune
processes, and for mediating different clinical outcomes.
 These parameters include the type of cytokine, chemokine, and other
mediators T cells produce, their cytolytic activity, migratory properties,
proliferative potential, and their functional avidity.
 It will take the thoughtful utilization and combination of several different
techniques to assess these functions.
 ELISPOT will continue to be the technique of choice for screening,
measurements of effector functions mediated by secretory products, fine
specificity, and avidity.
 Flow cytometry will continue to be indispensable for multiparameter
phenotypic analysis.
 Neither of the two, however,
 will obviate the need for a new generation of killer assays, or migration
assays. Each of these techniques excels in providing a specific type of
information and does not permit interpretations beyond what actually is
being measured.

14.
 mabtech.com/knowledge-center/assay-principles/elispot-assay-
principle/elispot-step-step
 J Simon Lunn, Stacey A Sakowski, Thais Federici, Jonathan D Glass,
Nicholas M Boulis2 & Eva L Feldman. (1 University of Michigan
Department of Neurology, 109 Zina Pitcher Place, 5017 BSRB, Ann
Arbor, MI 48109, USA 2 Department of Neurosurgery, Emory
University, Atlanta, GA, USA 3 Department of Neurology, Emory
University, Atlanta, GA, USA, Author for correspondence).
 Genevieve Gowing & Clive N. Svendsen (Published online: 9
September 2011)
 Irene Faravelli, Monica Bucchia, Paola Rinchetti, Monica
Nizzardo, Chiara Simone, Emanuele Frattini & Stefania Corti
(Published: 14 July 2014)