The Detection of expressed IL-32 in the Human Stomach Cancer using an ELISA and Immunostaining. Seo, Eun-Hee October 6, 2008 Lab of Cell Biol& ImmunoBiochem Department of Bioscience & Biotechnology Konkuk University, Seoul, Korea

Introduction of IL-32 Summary Material & Method C ontents Purpose Evaluation of IL-32 ELISA

Interleukin-32 Proinflammatory cytokine In activated T cells, NK-cells, monocytes, epithelial cells Human chromosome 16p13.3 Eight small exons & Six splice variants ( IL-32α / β / γ / δ / ε and ζ ) Inducer of TNF- α, IL-1β, MIP-2, IL-8 Trigger of the IκB/NF- κB cascade, p38 MAPK Autoimmunity Reviews 6 (2007) 131–137 Ann Rheum Dis 2006;65(Suppl III):iii61–iii64. IL-32 promotes a number of proinflammatory cytokines.

Purpose Preparation of monoclonal antibodies reactive to IL-32 Evaluation of sandwich ELISA base on the combination of various antibody pairs Determination of IL-32 level in the sera of cancer patients Detection of intact IL-32 level in mammalian cells

Antibody purification KU32-07 ; IL-32α KU32-52 ; IL-32α β γ δ KU32-56 ; IL-32α β KU32-09 ; IL-32α β γ δ Affinity column (Protein A/G agarose) “ Hybridoma cell ” Antibody B-cell Myeloma cell Stimulation of B-cell Hybridoma screening Material & Method

Antigen; IL-32α (yeast) [ Lane ] 1. Extract of yeast expressed IL KU32-52 Affinity bead (Ne control) 3. IL-32 bound to KU32-52 Affinity bead 4. Eluant 5. Eluted KU32-52 Affinity bead 72kD 55kD 40kD 33kD 24kD 17kD Extract of yeast expressed IL-32 KU32-52 Affinity bead IL-32α (20kDa)

Sandwich ELISA The ELISA (Enzyme-Linked ImmunoSorbent Assay) can be used both qualitatively and quantitatively to measure antigen-antibody binding. Depending on what variation you use, it will detect antigen (hormones, enzymes, microbial antigens) or antibody in body fluids or tissue culture supernatents. The Sandwich ELISA measures the amount of antigen between two layers of antibodies. Sandwich ELISAs are especially valuable when the concentration of antigens is low and/or they are contained in high concentrations of contaminating protein. Detection Antibody TMB Capture Antibody HRP HRP-linked Antibody

(A) Negative isotype control mouse IgG1 antibody (B) KU32-09 ( IgG1) (400X) Fig. 1] Representative immunohistochemical staining of IL-32 in stomach cancers. Result

Empty vector IL-32αIL-32ß IL-32 (pg/ml) 56 kD 40 kD 33 kD 24 kD 17 kD IL-32α IL-32β Empty vector K-562 (A) Fig. 2] Detection of expressed or secreted IL-32 from the stable K-562 transfectants expressing IL-32α or β. (B)

Dilution linearity of hIL-32α ELISA hIL-32α ELISA using PoAb and biotinylated KU32-52 Standard curve y = x IL-32 alpha (pg/ml) O.D A Observed Value IL-32 alpha (pg/ml) y = x R 2 = Theoretical Value IL-32 alpha (pg/ml) B Fig. 3] ELISA for IL-32 using a coating polyclonal Ab and a capturing biotinylated mAb KU32-52.

A) Run to run precision ControlNXSD%CVRecovery (%) IL-32α (pg/ml) Low (250 pg/ml) Medium (1000 pg/ml) High (2500 pg/ml) B) Within run precision ControlNXSD%CVRecovery (%) IL-32α (pg/ml) Low (250 pg/ml) Medium (1000 pg/ml) High (2500 pg/ml) C) Dilution linearity Dilution factorExpected (pg/ml)Observed (pg/ml)Recovery (%)

D) Cross-reaction of monoclonal antibody for hIL-32α and the related cytokines CytokinesCross-reaction (%) hIL-32α100 hIL-32β100 hIL-1α< 0.1 hIL-1β< 0.1 hIL-2< 0.1 hIL-6< 0.1 hIL-8< 0.1 hIL-10< 0.1 hIL-18< 0.1 hTNF-α< 0.1 Table 1] Evaluation of ELISA for hIL-32 using a polyclonal anti-hIL-32 antibody and a biotinylated mAb KU32-52 as a coating and a capturing antibody, respectively.

IL-32 ELISA NormalTumors IL-32 (pg/mL) X=186.3 %CV=55.5 X=119.9 %CV=18.8 n=10 n=16 Fig. 4] IL-32 protein levels in the plasma of stomach cancer patients and healthy individuals.

Summary A new cytokine, IL-32 usually exerts a role inside the cells and less amount of IL-32 would be secreted from the cells. The established sandwich ELISA can sensitively and specifically detect 100 pg/ml of IL-32 alpha. but not other cytokines. The mAb, KU32-52, can be effectively used in the functional study of IL-32 in the pathogenesis of cancer and inflammatory diseases. This immunoassay and immunohistochemistry can be used in the detection of expressed and secreted IL-32 in stomach cancer patients. The mAb, KU32-09, was shown to react strongly on immunohistochemistry in the tissues of stomach cancer.

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