MICA-EK-200

Human soluble MICA ELISA Kit

MICA-EK-200 AMO+BAMO3+sMICA sMICA 200 reactions 1 + 1 + 1 450 Euro

Antigen: Soluble human MICA (MHC-class I-related chain A)

Catalog Number: MICA-EK-1000

Applications: Detection of soluble MICA in serum and cell culture supernatants

Capture antibody (1 vial):

Clone: AMO1 (mouse IgG1, kappa)

Concentration: 1 mg/ml

Formulation: 0.5 mg in 0.5 ml phosphate-buffered saline, pH 7.4, no sodium azide

Storage: Store at 4°C. For long-term storage freezing at -20°C is recommended.

Detection antibody (1 vial):

Clone: BAMO3 (mouse IgG2a, kappa)

Concentration: 0.2 mg/ml

Formulation: 0.1 mg in 0.5 ml phosphate-buffered saline, pH 7.4, no sodium azide

Storage: Store at 4°C. For long-term storage freezing at -20°C is recommended.

Standard (5 vials): recombinant soluble MICA*04 (purified from E.coli)

Concentration: 100 ng/ml

Formulation: 20 ng in 0.2 ml phosphate-buffered saline, 5% bovine serum albumin, pH 7.4

Storage: Store at -20°C. Use aliquots after thawing within 1-2 weeks.

Usage: See attached ELISA instruction sheet.

Description: MICA (MHC class I-related chain A) is a polymorphic, human MHC-encoded cell surface glycoprotein and ligand of the activating C-type lectin-like immunoreceptor NKG2D [1-5]. NKG2D engagement of MICA activates NK cells and costimulates CD8 T cells [3,6]. MICA is expressed on gastrointestinal epithelium and inducible by cell stress, viral and bacterial infection [2,6-8]. MICA is also expressed by malignant epithelial and haematopoietic cells, and MICA expression has been shown to enhance tumor rejection in vivo [9-12]. Tumor cells shed soluble MICA which is detectable in sera of patients with epithelial and haematopoietic malignancies and may counteract tumor immunosurveillance [10,13-15].

Conditions: For research use only. Not for use in diagnostic or therapeutic procedures. BAMOMAB is not responsible for any patent infringements caused by the use of this product.

Country of Origin: Germany

Literature: 1. Bahram S et al. Proc Natl Acad Sci USA 91, 6259-6263 (1994).

2. Groh V et al. Proc Natl Acad Sci USA 93, 12445-12450 (1996).

3. Bauer S et al. Science 285, 727-729 (1999).

4. Steinle A et al. Immunogenetics 53, 279-287 (2001).

5. Li P et al. Nat Immunol 2, 443-451 (2001).

6. Groh V et al. Nat Immunol 2, 255-260 (2001).

7. Spies T Proc Natl Acad Sci USA 99, 2584-2586 (2002).

8. Welte S et al. Eur J Immunol 33, 194-203 (2003).

9. Groh V et al. Proc Natl Acad Sci USA 96, 6879-6884 (1999).

10. Salih HR et al. Blood 102, 1389-1396 (2003).

11. Friese MA et al. Cancer Res 63, 8996-9006 (2003).

12. Wiemann K et al. J Immunol 175, 720-729 (2005).

13. Salih HR et al. J Immunol 169, 4098-4102 (2002).

14. Groh V et al. Nature 419, 734-738 (2002).

15. Holdenrieder S et al. Int J Cancer 118, 684-687 (2006).

Protocol MICA-Sandwich ELISA

1. COATING

􀂾 Incubate capture anti-MICA mAb (AMO1) at 5μg/ml (dilute stock solution 1:200 in PBS) overnight at 4°C in an ELISA plate (100μl/well).

o BAMOMAB recommends Nunc-Immuno 96 MicroWell Plates/MaxiSorp (from Nunc, Cat-Nr. 442404).

2. BLOCKING

􀂾 Add 15% BSA-PBS (100μl/well) to AMO1 and incubate for 1h at 37°C.

􀂾 After 1h incubation: Wash plate 4x with TWPBS (0.05%TWEEN-20 in PBS).

3. SAMPLING

􀂾 Add samples/standard diluted in 7.5% BSA-PBS (100μl/well) for 2h at 37°C.

o Standard: Use rMICA*04 at 20 ng/ml (dilute stock 1:5 in 7.5% BSA-PBS and titrate (in 1:2 dilutions) in 12 steps to 10pg/ml).

o Serum samples: Dilute 1:3 (1Vol. Serum + 2 Vol. 7.5% BSA-PBS) and spin 15 min with max. rcf in desk centrifuge. Take supernatant for analysis.

􀂾 After 2h sample incubation: Wash plate 4x with TWPS.

4. SANDWICHING

􀂾 Add purified anti-MICA/B mAb BAMO3 at 1μg/ml (dilute stock solution 1:200 in 7.5% BSA-PBS) (100μl/well) and incubate for 2h 37°C.

􀂾 After 2h BAMO3 incubation: Wash plate 4x with TWPS.

5. DETECTION

􀂾 Add HRP-conjugated anti-mouse IgG2a Ab (dilute 1:10.000 in 3.75% BSA-PBS) (100μl/well) for 1h at 37°C.

o BAMOMAB recommends HRP-conjugated goat anti-mouse IgG2a (from Southern Biotechnologies Cat. Nr. 1080-05).

􀂾 After 1h incubation: Wash plate 6x with 0.05%TWPS.

6. SUBSTRATE

􀂾 Develop with HRP substrate

o BAMOMAB recommends TMB 2-component microwell peroxidase substrate kit (from KPL, Cat. Nr. 50-76-00).

(mix equal volumes of TMB Peroxidase Substrate and Peroxidase Sol. B and add 100μl of the mixture/well. Incubate at RT 5-60 min)

7. STOP AND MEASURE

􀂾 Add 100μl 1M phosphoric acid and read results at 450 nm wavelength.

Attention: Do not use AZIDE-containing solutions after step 4. SANDWICHING !