Test for Immune Status

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Test for Immune Status Cell mediated Immunity
Watchara Kasinrerk
Department of Clinical Immunology Faculty of
Associated Medical Sciences Chiang Mai University
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Test for CMI
1. In vivo test Skin Test
2. In vitro test - Determination of
lymphocyte - Lymphocyte Transformation Test
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4. Cytotoxicity Assay
CD8
Function of cytotoxic T cells
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Cytotoxicity Assay
Cytotoxic cells
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Lysis
AIDS with vaccine
Vaccine
Vaccine
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0
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2
4
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5. Assay for cytokine production
Interleukin
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Cytokine (interleukin) Assay
1. Bioassay Activity
2. Quantitative Assay Level, Amount
3. ELISPOT Cytokine secreting cells
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Bioassay for cytokines (Interleukine)
Interleukin 1 (IL1)
Interleukin 2 (IL2)
Interleukin 4 (IL4)
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1. Interleukin 1 (IL1) Assay
Monocytes
CD4
APC
Lymphokines
Interleukin 1
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Interleukin 1 Assay 2 steps
Step 1 IL1 production
Step 2 IL1 assay
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Step 1 IL1 production
Ficoll-Hypque Centrifugation
Haparinized blood
PBMC Lymph. Monocytes
PBMC
??????? plastic plate ???????? monocytes ????????
Incubate 37oC ??? 2-4 ???????
???? non-adherent cells ??? ??? adherent cells
(monocytes)
???? lipopolysaccharine (LPS) Inc. 37oC ??? 4
??????? ?? CO2 incubator
???? culture supernatant ??????????? IL1
??????????????
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Step 2 IL1 Assay
???? thymocytes ?????? Balb/c
culture supernatant ????????? IL1
?? 96 well plate
Add sub-optimal dose PHA Incubate 37oC 72 hours
Add 3H-thymidine
Incubate 37oC 2 hours
Harvest cells ?? glass fiber filter ???
3H-thymidine incorporation ???? Beta counter
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2. Interleukin 2 (IL2) Assay
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Interleukin 2 Assay 2 steps
Step 1 IL2 production
Step 2 IL2 assay
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Step 1 IL2 production
Ficoll-Hypque Centrifugation
Haparinized blood
PBMC Lymph. Monocytes
PBMC

• ???? specific antigen ???? mitogen (PHA or ConA)
• Incubate 37oC
• 18 hours ?????? Mitogen
• 48 hours ?????? Antigen

???????? culture supernatant ????????????? IL2
??????????????
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Step 2 IL2 Assay
- CTLL-2 - HT-2
culture supernatant ????????? IL2
IL-2 dependent cell line
?? 96 well plate
Incubate 37oC 72 hours
Add 3H-thymidine
Incubate 37oC 2 hours
Harvest cells ?? glass fiber filter ???
3H-thymidine incorporation ???? Beta counter
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3. Interleukin 4 (IL4) Assay
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Interleukin 4 Assay 2 steps
Step 1 IL4 production
Step 2 IL4 assay
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Step 1 IL4 production
Ficoll-Hypque Centrifugation
Haparinized blood
PBMC Lymph. Monocytes
PBMC

• ???? specific antigen ???? mitogen (PHA or ConA)
• Incubate 37oC
• 18 hours ?????? Mitogen
• 48 hours ?????? Antigen

???????? culture supernatant ????????????? IL4
??????????????
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Step 1 IL4 Assay
Culture supernatant ????????? IL4
Jijoye cell line (B lymphoma)
?? 96 well plate
Incubate 37oC 48-72 hours
????
????????????? anti-CD23 mAb-FITC ?????????????
CD23 expression ???? flow cytometry
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Bioassay Test for activity of cytokines
Disadvantage
IL2 assay
Sample
CTLL-2 IL2 dependent cell line
IL2low IL4high
Proliferation
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Quantitative Assay for cytokines (Interleukin)
Quantitative test
Antibody specific to cytokine
ELISA for measuring cytokine
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Quantitative Assay for IL2
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OD
Interleukin pg/ml
Conc.
Disadvantage Level of cytokines Activity
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Cytokine (interleukin) Assay
Bioassay Activity
Quantitative Assay Level, Amount
3. ELISPOT Cytokine secreting cells
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3. ELISPOT Cytokine secreting cells
T
T
Antigen
T
T
T
T
How many cells secret IL2?
Lymphokines
IL4
IFN
IL2
IL10
IL3
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Test for CMI status