An event from European Scientific Conferences - Euroscicon "Specialising in communicating cutting edge technology & methodology in the Life Sciences"
After our successful ELISPOT
technology: The latest tricks event which took place in October 2009 we
are delighted to announce our follow up event, which will be our 5th event on
This event is discussion
workshop. We have invited 6 experts to discuss their work in an informal
lecture setting, discussion and demonstration groups, one2one sessions and
Meeting Chair: Prof.
Paul V. Lehmann - Case Western Reserve University Cleveland, USA
This event has CPD
On registration please
submit your questions to the panel that will be asked by the chair on the day
of the event
9:00 – 9:20
9:20 – 9:30
Introduction by the Chair: Prof. Paul V. Lehmann -
Case Western Reserve University
Talks by Invited Experts:
9:30 – 9:45 EliSPOT Standaridsation: How do we get the ELISPOT
into the Clinic?
College London, UK
ELISPOT assay is one of the most useful techniques for
immunological monitoring of trials and has gained increased application as a
measure of specific T cell activation. However there are still issues with
standardisation particularly across centres. Multi-centre clinical trials pose
the challenge of collecting, shipping and processing samples in a way that
ensures consistency and reproducibility hence it is critical to have validated
assays that ensure that data is reliable.
9:45 – 10:00
Developing Multicolor Isotype Revealing Antigen Specific B cell ELISPOT
University of Nebraska-Lincoln, USA
The B cell
response to vaccines, commensal bacteria, and pathogens reflect the T cells and
inflammatory environment that provide "Help" to differentiating B
cells. We are developing a multicolor ELISPOT assay that will measure both the
frequency and isotype of antigen specific B cells in the same wells using
florescent secondary antibodies and the appropriate filters. This will allow us
to measure 5-6 isotypes in the same wells, greatly increasing the number of
samples that can be analyzed and decreasing the number of B cells from tissue
or blood that will be required for the assay. We will describe the progress in
this development, using mouse splenic and lamina propria B cells, in the
context of oral vaccination of gnotobiotic mice colonized with microbes known
to shift the balance of the immune response from Th1, to Th2 T cells.
10:00 – 10:15
Future of ELISPOT Assays:
Lymphocytes and Beyond
ELISPOT assays are traditionally used to study cytokine secretion from
immune system cells. However, this type of cell-based assay is quite flexible
and can be used for many other cells types including neuronal and glial cells
in the central and peripheral nervous system, endocrine and exocrine cells as
well as stem cells to mention few. Using ELISPOT for cells other than
lymphocytes require adjusting assay conditions and detection chemistry which
will be addressed in this presentation.
10:15 – 10:30 Statical Analysis of ELISPOT Data
Professor Marcus Dittrich
University of Wurzburg, Germany
The principal goal
of most ELISPOT experiments is the reliable identification of a positive
antigen response. Different approaches are commonly used, mainly either
statistically tests or empirical rules of thumb (e.g. based on the mean spot
count difference or ratio between the antigen-containing the negative control wells).
Albeit enjoying some popularity empirical rules in general do not have a
theoretical justification and provide no measurement of confidence. Instead,
the application of solid statistical tests is highly recommended. First data
from cell transfected cell lines indicate that the standard t-test and related
statistics should be applicable in most cases.
10:30 – 10:45
Qualitative and Quantitative Analysis of HIV-1-Specific T-cell Responses
Dr Nesrina Imami
College London, UK
Mechanisms by which
immune-based therapies increase T-cell numbers and function in chronically
HIV-1-infected treated patients are not fully understood. Our experimental data
suggests that by utilising various immunotherapies we can affect T-cell
proliferation, survival, development and differentiation and/or maturation and
thymic output, all of which lead to enhancement of T cell function. This
implies that just as in vitro, in vivo HIV-1-specific T cell defects might be
corrected by administration of exogenous stimuli such as cytokines, hormones
and/or therapeutic immunisation. Understanding the precise biochemical,
molecular and cellular mechanisms involved will be crucial for the optimisation
and development of these and other modes of immune-based therapies. Our
research work is aimed at carrying out basic research and clinical
studies/trials aimed at development of novel immunotherapies. Utilisation of
new technologies to assess full functionality of anti-HIV-1 responses combined
with expression profiling will be essential in application to human health.
10:45 – 11:00 Fluorospot
for Dual and Triple Cytokine Analysis: Applications
Associate Prof. Bernt Axelsson
Cytokine ELISPOT has become a powerful routine tool for
the analysis of disease- as well as vaccine-induced T-cell responses. The metod
is limited, however, in that only one cytokine at a time is assessed.
Fluorospot is a development of the ELISPOT method that facilitates the analysis
of single cells secreting several cytokines, e. g. polyfunctional T cells, which are suggested to be of
protective importance in various infectious diseases. By detecting each
cytokine with a certain flurophore and analyzing two- or three-colored spots by
fluorophore-specific filter systems, spots derived from cells producing single
or multiple cytokines are identified. Fluorospot maintains the simplicity and
sensitivity of the ELISPOT while taking the analysis a step forward towards
11:05 – 11:30
11:30 – 12:30
Question and Answer Session
Working Lunch (in Exhibition Area)
Discussion Groups and One to One Sessions
· Round table discussion
groups will be throughout the afternoon
Delegates will rotate at 15 minute intervals so
that they may participate in all the discussion tables
All delegates will also be allocated an slot to
visit the exhibition stands
One to one sessions can also be held after lunch
(in parallel to the discussion groups)
Where appropriate delegates will be able to bring
their samples to the discussions
12:45 – 13:20 Discussion Groups (Sessions 1 & 2)
13:40 Effect of T-Cell XTend on the Performance of the TSPOT.TB Assay
Med Microbiology & Immunology Diakonessen
Hospital, The Netherlands
Vacutainer CPT tubes are commonly used for collection of
whole blood for the TSPOT.TB assay, but
require that blood samples are processed within 8 hours. In this study we
evaluated the feasibility of T-Cell XTend
for isolating peripheral blood mononuclear cells (PBMC). This procedure would
allow storage of blood samples for batched processing.
blood specimens from 59 individuals were collected in Vacutainer CPT tubes
(CPT) and lithium heparin (LH) tubes. CPT tubes were processed within 8 hours.
T-Cell XTend was added to LH tubes
after 24 or 48 hours. We measured total white blood cell counts (WBC) and
proportions of lymphocytes and granulocytes in the isolated PBMC’s. We also
evaluated the performance of T-Cell XTend
in the TSPOT.TB assay.
Results PBMC yields from T-Cell XTend treated LH samples did not differ
from PBMC yields from CPT tubes, but T-Cell XTend had a pronounced effect on the proportions of lymphocytes and
granulocytes. The mean lymphocyte percentage in PBMC’s isolated with CPT was
84.31 ± 1.14 %, but was decreased to 52.72 ± 3.34 % (p<0.05) in untreated LH
blood left to stand for 48 hours. This effect was neutralized by T-Cell XTend (85.44 ± 0.74 %). We observed a similar
but opposite effect on granulocytes: The mean proportion in untreated LH blood
was increased to 40.9 ± 3.67 % (p <0.001) compared to CPT blood (8,26 ± 0.89
%). Treatment of LH samples with T-Cell XTend
(48 hours) restored the proportion of granulocytes to 8.47 ± 0.61 %.
Enumeration and analysis of spots in the TSPOT.TB assay demonstrated good agreement between CPT and T-Cell XTend results, even after 48 hours.
Conclusion T-Cell XTend efficiently removes granulocytes
from PBMC suspensions and increases the proportion of lymphocytes. TSPOT.TB results from T-Cell XTend treated blood samples are at least
comparable to the results obtained from the current CPT method.
also observed increased spot counts in control wells of T-Cell XTend treated samples, less
indeterminate results and a possible increase of positive TSPOT.TB results.
of T-Cell XTend® can be a
feasible for ELISPOT purposes, but further research is warranted to investigate
the need of (re-)establishing specific cut-off levels for T-Cell XTend treated samples.
13:45 – 14:20 Discussion Groups (Sessions 3 & 4)
14:20 – 14:45 Afternoon Break
14:45 – 15:20 Discussion Groups (Sessions 5 & 6)
15:25 – 15:40 Talk by Associate Prof. Bernt Axelsson
for Dual and Triple Cytokine Analysis: Applications: Technical Aspects
Groups (Sessions 7 & 8)
Table 1: ELISPOT:
Challenges and Opportunities
Hosted by Prof. Paul Lehmann, who trained as a
T cell immunologist. He introduced and patented image analysis for ELISPOT
(United States Patent No 08/577,957) dedicating 40 of his 100 publications to
the basics of ELISPOT, including single cell resolution, per cell productivity,
cognate vs. bystander cytokine, T cell avidity measurements, determinant
mapping etc. In 1998, he founded CTL to assist scientists in ELISPOT analysis.
CTL offers GLP-compliant ELISPOT contract research, ELISPOT readers (visible
light and UV), PBMC libraries and reference.
Table 2: Quality Control
and Multi Centre Validation
Hosted by Dr Sefina Arif, who did
her PhD in Immunology at King’s College London focusing on the identification
of autoantigens in type 1 diabetes. Since then she has continued in this field
specifically working on the role of T cells in type 1 diabetes characterising
autoreactive T cells in both patients and healthy controls specifically T cells
producing interferon-g in patients and cells making IL-10 in healthy controls
Her recent studies focus on the role of Th17 cells in this type 1 diabetes.
Table 3: Developing
Multicolor Isotype Revealing Antigen Specific B cell ELISPOT Assay
Hosted by Prof. Daniel Peterson, who grew
up outside of Lincoln NE and attended the University of Nebraska, studying
Animal Science graduating in 1993. From there he went to Washington University
in St Louis to pursue a MD/PhD in Immunology with Emil Unanue. He then went to
Switzerland to study human immune responses to the Cancer Melanoma. Following
internship in Internal Medicine at Case-Western Reserve University Hospitals of
Cleveland, he returned to Wash U to complete a Residency in Clinical Pathology
and a postdoctoral Fellowship with Jeffrey Gordon. In 2008, he moved back to
Lincoln NE, and set up his laboratory focused on the specific immune response
to gut microbes. Since his arrival at UNL he has established a germ-free
gnotobiotic facility and immune monitoring core with Flow Cytometry, ELISA and
ELISPOT capabilities in his laboratory.
Table 4: Future of ELISPOT Assays:
Lymphocytes and Beyond
Hosted by Dr Alex Kalyuzhny, who gained a Ph.D. in embryology and histology in 1988, in 1998 set up
immunocytochemistry and ELISPOT assays department at R&D Systems, Inc. and
manage it till now. Developed more than 50 ELISPOT assays which are currently
manufactured and sold by R&D Systems, Inc. worldwide. A faculty at the
department of Neuroscience at the University of Minnesota. Author of 27
peer-reviewed publications, 16 chapters in textbooks, editor of 3 books
including both 1st (published in 2005) and 2nd (will be published in 2011)
editions of the Handbook of ELISPOT (Humana Press). Professional interest:
developing multiplex ELISPOT assays for veterinary and human diagnostics.
Table 5: Statical Analysis of ELISPOT Data
Hosted by Dr Marcus Dittrich, who studied Medicine at
University of Tübingen. From there he went to the Case Western Reserve
University in Cleveland (Ohio) where he did his MD thesis in the Lab of Prof.
Paul V. Lehmann. After internships at the University Hospital of Zürich he
joined the MD/PhD program in Würzburg where he graduated in bioinformatics
about novel systems biological approaches to the functional analysis of
cellular networks. Today he leads a bioinformatical research group at the
University of Würzburg and mainly works on the integrated statistical analysis
of multivariate high-throughput data (e.g. microarray, proteomics) in the
context of biological networks.
Table 6: Design of Assessment
of HIV-1-Specific T-cell Function in HIV-1 Infected Individuals and Vaccine
Recipient: Concomitant Assessment of Perforin, IFN-gamma, IL-2 and IL-4 Secretion
Hosted by Dr Dr Nesrina Imami, who is a Reader in Immunology and Fellow of the Royal
College of Pathologists, qualified in medicine, microbiology and immunology.
She further specialised in viral immunology, in particular HIV-1, and focused
on cell mediated immunity and immunotherapeutic development. She has published
primarily on the immunology of T cells and their responses to viral infection,
was awarded a University of London PhD in 1992 and after a subsequent period of
postdoctoral work was awarded a Wellcome Trust Fellowship. She embarked on a
long-term detailed programme of research to assess HIV-1-specific T-cell immune
responses, mechanisms of non-responsiveness/anergy and to evaluate the effects
of drugs, cytokines and other immunomodulators such as vaccines on these
responses. The Wellcome Trust Fellowship enabled her to establish her research
group at the forefront of international research into the immunopathogenesis of
HIV-1 infection and immune reconstitution in HIV-1 disease, and to continue her
work in an area that integrates basic biological science with clinical science,
with direct application to human health. She has completed and set up novel
immunotherapy and drug clinical trials for which she has been awarded the
prestigious MRC Experimental Medicine Award.
Table 7: Fluorospot
Hosted by Associate Prof. Bernt Axelsson, who
gained a PhD in immunology at Stockholm University (SU) 1984 under
late prof. Peter Perlmann. He continued as a lecturer and researcher in
immunology at SU to 1995, mainly engaged in mechanisms of T cell activation (35
peer reviewed articles). He then went to the swedish biotech company Biovitrum
AB as a manager engaged in immunological aspects of obesity and diabetes type
II. Since 2006 he has been at
Mabtech AB developing the Fluorospot method.
– 17:00 Expert Panel's
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