世翔國際有限公司

(1)

Autoimmun Diagnostika GmbH

Ebingerstraße 4 D-72479 Straßberg Tel.: +49 (0) 7434/9364-0 [email protected]

The AID EliSpot/FluoroSpot Reader portfolio and their features

08/12/2010, Vetereans General Hospital, Taichung, Taiwan

Juergen Haeussler AID GmbH, Germany

世翔國際有限公司

(2)

Autoimmun Diagnostika GmbH

Part 1: How it works

Part 2: EliSpot in diagnostics

Part 3: Limitations of EliSpot and Development of FluoroSpot

Part 4: The AID EliSpot/FluoroSpot Reader portfolio

Part 5: Live-Demo of the AID iSpot Reader

(3)

Autoimmun Diagnostika GmbH

Part 1: How it works

(4)

Autoimmun Diagnostika GmbH

Measuring antigen specific T-cell reactions

Enzyme-linked ImmunoSpot assay: Method to detect cytokine release of T-cells on the single cell level. EliSpot

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Autoimmun Diagnostika GmbH

Lymphocyte-isolation (FICOLL)

Y Y

Y Y Y

Y Y

Usually Elispot Assays are performed in a 96well plate with a membrane bottom in each well. The membrane is precoated with capture antibody specific for a cytokine (e.g. IFN-γ)

Add cells together with antigen (mitogen) and incubate for 16-20h at 37°C.

Some cells will be stimulated to produce cytokine molecules, which are bound to the membrane by the capture antibody.

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Autoimmun Diagnostika GmbH

Y

Remove cells, add detection AB, conjugated with substrate-specific enzyme.

Add substate and perform enzyme-substrat reaction to visualize developing spots.

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Autoimmun Diagnostika GmbH

Extremely sensitive

Close to the in vivo-situation

Analyses secreted cytokines (post-translational regulation)

Easy to perform

Results within 16 hours

Allows monitoring of TH ₁ /TH ₂ balance

High throughput assays (96-well format)

Well suited for screening and standardization

Disadvantages

Only one cytokine can be analysed

Reader is needed

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Autoimmun Diagnostika GmbH

0 200 400 600 800 1000 1200 1400 1600

1983 - 1985 1986 - 1990 1991 - 1995 1996 - 2000 2001 - 2005 2006 - 2010

No.: of publications

Year of publication

Publications "EliSpot" NCBI

Availability of EliSpot Reader systems

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HIV 18%

TB 9%

Malaria 3%

Transplantation 10%

EBV 3%

CMV 3%

Cancer 24%

Allergy 5%

others 25%

Investigation of specific immune response in infections Mainly (>90% ) INF-γ is used

Standard tool in development vaccine canditates

Method of choice to monitor new (HIV) vaccine canditates

(10)

Autoimmun Diagnostika GmbH

Part 2: EliSpot in Diagnostics

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Lalvani et al. , Am J Respir Crit Care Med, 163, 824-828 (2001)

Major drawbacks of the commonly used tuberculin skin test (TST):

- low sensitivity and low specificity

- false-positive results due to BCG vaccination

- needs return visit

- administration and reading is highly operator dependent

12. Aug. 2004: First available diagnostic kit based on the EliSpot technology (Oxford Immunotec, UK).

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RD1 Gen-Region: ESAT-6: Early Secreted Antigenic Target (6kDa) CFP-10: Culture Filtrate Protein (10kDa)

Region of difference, RD1

CFP-10 ESAT-6

M. tuberculosis M. africanum, M. bovis

M. kansasii, M. marinum, M. szuigai

M. tuberculosis BCG

Most environmental strains Andersen P et al. Lancet 2000;356:1099

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Negative control Positive control

ESAT-6 CFP-10

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• Not impaired by BCG-Vaccination

• High sensitivity and specificity

• Responds early to infection

• Diagnoses active and latent TB

• Diagnoses extrapulmonary TB

• Single patient visit, fast procedure

• Data from more than 20.000 subjects available

• Worldwide accepted as diagnostic tool

• Also known as one of the “IGRA”, Interferon-γ Releasing Assay (& ELISA)

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Method TST EliSpot ELISA

BCG vaccination Affected Not affected Not affected

Specificity Low > 95% > 95%

Sensitivity Low (<70%) >95% >80%

HIV subjects Yes Yes (Lower sensitivity)

Elderly/children/IS Yes Yes (Lower sensitivity)

Readout system EliSpot (cell-counting) ELISA (OD)

Regulatory approvals Europe /Canada/FDA Europe/Canada/FDA

IGRA: Interferon-γ releasing assays

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Autoimmun Diagnostika GmbH

Part 3: Limitations of EliSpot

Development of FluoroSpot

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Autoimmun Diagnostika GmbH

Giuseppe Pantaleo and Alexandre Harari

NATURE REVIEWS | IMMUNOLOGY, vol 6 417-23, 2006

Levels of disease activity and viral replication correlate with distinct functional signatures of T-cell responses (e.g. IFN/IL-2)

Examples:

• HIV

• HCV

• CMV, EBV

• and others

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Autoimmun Diagnostika GmbH

Red signal

Blue signal

?

IL-2

IL-2/IFN

IFN

IFN ??

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Autoimmun Diagnostika GmbH

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Autoimmun Diagnostika GmbH

FITC

Overlay

Result Reset

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Autoimmun Diagnostika GmbH

Part 4: The AID EliSpot/FluoroSpot Reader portfolio

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Autoimmun Diagnostika GmbH

EliSpot Reader Systems

Basic EliSpot Reader for 96-well plates 5 Mpix firewire-driven camera

AID EliSpot Reader Systems „Classic“ iSpot Reader System

FluoroSpot/EliSpot Reader for 96-well plates

2 Mpix firewire-driven camera, fluorescent optimized 2-3 color assays

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Autoimmun Diagnostika GmbH

EliSpot Reader Systems

iSpot Spectrum

• High Resolution, multi-color FluoroSpot /EliSpot analysis

• 5 Megapixel firewire camera

• 7&1 filter wheel for 7 single banded fluorescent filters plus one LED

• Fully automated input / output module for 96-well plates

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Autoimmun Diagnostika GmbH

vSpot Reader System

• High resolution, multi-color FluoroSpot /EliSpot and Virus Plaque analysis

• 5 Megapixel firewire camera, optimized for fluorescent imaging

• 7 &1 filter wheel for 7 single banded fluorescent filters plus one LED

• 6,12, 24, 48 and 96 well layout EliSpot Reader Systems

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Autoimmun Diagnostika GmbH

MultiSpot Reader System

The MultiSpot Reader System

• Enzymatic and fluorescent EliSpot assays

• Cell counting and viability tests, autofocus microscope with 2.5x, 20x and 40x objective

• IF-based tissue assays

• HLA screening assays

• Tailor-made solutions on 96well plates, slides and Terasaki plates

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Autoimmun Diagnostika GmbH

600 users

 UK and Australia >95% of the market

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Autoimmun Diagnostika GmbH

National Institute of Health (NIH), US National Cancer Institute (NCI), US

Food and Drug Administration (FDA ), US

Center for Disease Control and Prevention (CDC), US CDC, China

Bayer Healthcare Merck & Co.

Merck Research Laboratories Sanofi Pasteur, Canada

Sanofi Pasteur, USA Centocor

Pfizer Novartis Baxter Roche Amgen Genzyme Celgene

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世翔國際有限公司

Autoimmun Diagnostika GmbH

The AID EliSpot/FluoroSpot Reader portfolio and their features

08/12/2010, Vetereans General Hospital, Taichung, Taiwan

Juergen Haeussler AID GmbH, Germany

Autoimmun Diagnostika GmbH

Part 1: How it works

Part 2: EliSpot in diagnostics

Part 3: Limitations of EliSpot and Development of FluoroSpot

Part 4: The AID EliSpot/FluoroSpot Reader portfolio

Part 5: Live-Demo of the AID iSpot Reader

Autoimmun Diagnostika GmbH

Part 1: How it works

Autoimmun Diagnostika GmbH

Measuring antigen specific T-cell reactions

Enzyme-linked ImmunoSpot assay: Method to detect cytokine release of T-cells on the single cell level. EliSpot

Autoimmun Diagnostika GmbH

Lymphocyte-isolation (FICOLL)

Usually Elispot Assays are performed in a 96well plate with a membrane bottom in each well. The membrane is precoated with capture antibody specific for a cytokine (e.g. IFN-γ)

Add cells together with antigen (mitogen) and incubate for 16-20h at 37°C.

Some cells will be stimulated to produce cytokine molecules, which are bound to the membrane by the capture antibody.

Autoimmun Diagnostika GmbH

Y

Remove cells, add detection AB, conjugated with substrate-specific enzyme.

Add substate and perform enzyme-substrat reaction to visualize developing spots.

Autoimmun Diagnostika GmbH

Extremely sensitive

Close to the in vivo-situation

Analyses secreted cytokines (post-translational regulation)

Easy to perform

Results within 16 hours

Allows monitoring of TH 1 /TH 2 balance

High throughput assays (96-well format)

Well suited for screening and standardization

Disadvantages

Only one cytokine can be analysed

Reader is needed

Autoimmun Diagnostika GmbH

Publications "EliSpot" NCBI

Availability of EliSpot Reader systems

Investigation of specific immune response in infections Mainly (>90% ) INF-γ is used

Standard tool in development vaccine canditates

Method of choice to monitor new (HIV) vaccine canditates

Autoimmun Diagnostika GmbH

Part 2: EliSpot in Diagnostics

Lalvani et al. , Am J Respir Crit Care Med, 163, 824-828 (2001)

Major drawbacks of the commonly used tuberculin skin test (TST):

- low sensitivity and low specificity

- false-positive results due to BCG vaccination

- needs return visit

- administration and reading is highly operator dependent

12. Aug. 2004: First available diagnostic kit based on the EliSpot technology (Oxford Immunotec, UK).

RD1 Gen-Region: ESAT-6: Early Secreted Antigenic Target (6kDa) CFP-10: Culture Filtrate Protein (10kDa)

Region of difference, RD1

M. tuberculosis M. africanum, M. bovis

M. kansasii, M. marinum, M. szuigai

M. tuberculosis BCG

Most environmental strains Andersen P et al. Lancet 2000;356:1099

Negative control Positive control

ESAT-6 CFP-10

• Not impaired by BCG-Vaccination

• High sensitivity and specificity

• Responds early to infection

• Diagnoses active and latent TB

• Diagnoses extrapulmonary TB

• Single patient visit, fast procedure

• Data from more than 20.000 subjects available

• Worldwide accepted as diagnostic tool

• Also known as one of the “IGRA”, Interferon-γ Releasing Assay (& ELISA)

Method TST EliSpot ELISA

BCG vaccination Affected Not affected Not affected

Specificity Low > 95% > 95%

Sensitivity Low (<70%) >95% >80%

HIV subjects Yes Yes (Lower sensitivity)

Elderly/children/IS Yes Yes (Lower sensitivity)

Readout system EliSpot (cell-counting) ELISA (OD)

Regulatory approvals Europe /Canada/FDA Europe/Canada/FDA

IGRA: Interferon-γ releasing assays

Autoimmun Diagnostika GmbH

Part 3: Limitations of EliSpot

Allows monitoring of TH ₁ /TH ₂ balance