IL8 Antibody (OASA04372)

Catalog#: OASA04372

• Size: 0.25mg

• Description of Target: MOUSE ANTI SHEEP INTERLEUKIN-8
• Gene Symbol: IL8
• Alias Symbols: N/A
• Protein Accession#: NP_001009401.1
• Swissprot Id: P36925
• Host: Mouse
• Protein Size (# AA): 101
• Specificity: IL-8
• Application: ELISA, FC, WB
• Clone: 8M6
• Immunogen: The immunogen for anti-IL8 antibody: recombinant ovine IL-8
• Product Format: Purified IgG - liquid
• Isotype: IgG2a
• Applications Info: ELISA : This antibody may be used in combination with OASA08778 in sandwich ELISA assays for ovine IL-8.
  Flow Cytometry: *
  Use 10ul of the suggested working dilution to label 10⁶ cells in 100ul.Method sheets are available
  Preservative Stabilisers: 0.09% - Sodium Azide
  Approx Protein Conc: IgG concentration 1.0 mg/ml
  
  Buffer Solutions: Phosphate buffered saline
• Reconstitution and Storage: Store at +4^oC or at -20^oC if preferred.
  
  This product should be stored undiluted.
  
  Storage in frost free freezers is not recommended. Avoid repeated freezing and thawing as this may denature the antibody. Should this product contain a precipitate we recommend microcentrifugation before use.
• Key Reference: 1. Caswell, J. L. et al. (1998) Expression of the Neutrophil Chemoattractant Interleukin-8 in the lesions of Bovine Pneumonic Pasteurellosis. Vet. Pathol. 35: 124 - 131.
  2. Pedersen, L. G. et al. (2002) Identification of monoclonal antibodies that cross react with cytokines from different animal species. Vet. Immunol. Immunopathol. 88: 111 - 122.
  3. Aasted, B. et al. (2002) Cytokine profiles in peripheral blood mononuclear cells and lymph node cells from piglets infected in utero with porcine reproductive and respiratory syndrome virus. Clin. Diagn. Lab. Immunol. 9: 1229 - 1234.
  4. Herndon, C.N. et al. (2010) Differential expression of interleukin-8 by polymorphonuclear leukocytes of two closely related species, Ovis canadensis and Ovis aries, in response to Mannheimia haemolytica infection. Infect Immun. 78: 3578-84.

Product Protocol: IL8 antibody used to evaluate cytokine profiles in peripheral blood mononuclear cells and lymph node cells from piglets infected in utero with porcine reproductive and respiratory syndrome virus (OASA04372)

Specificity: IL-8
Product Page: IL8 antibody (OASA04372)
Antibody Pair Available: OASA04372 Capture OASA08778 Detection
Experiment Type: Cytokine detection by flow cytometry

Protocol:
On the day of testing the cells were washed twice (5 min at 925 × g) with PBS and 0.1% (wt/vol) saponin (Sigma) (PBS-S) supplemented with 1% plasma from the animal under investigation. These fixed and saponin-treated cells were then transferred to U-bottom microtiter plates (0.2 × 106 to 1 × 106 cells in a volume of 50 μl), and the plates were incubated for 1 h at room temperature with 500 ng of MAbs. B. Aasted et al. used the following four MAbs: a cross-reactive anti-ovine IL-8-specific MAb (Serotec MCA 1660; immunoglobulin G2a [IgG2a] isotype), a cross-reactive anti-bovine IL-4-specific MAb (Serotec MCA 1820; IgG2a isotype), a cross-reactive anti-bovine γ-IFN-specific MAb (Serotec MCA 1783; IgG1 isotype), and an anti-porcine TNF-α-specific MAb (Endogen MP-390; IgG1 isotype). Nonreactive MAbs of the IgG1 isotype (DAK-G01; Dako) and the IgG2a isotype (DAK-G05; Dako) were used to stain the isotype controls. The cells were then washed twice with PBS-S and incubated with 500 ng of a fluorescein isothiocyanate-conjugated F(ab′)2 fragment of a rabbit anti-mouse immunoglobulin (F313; Dako). The cells were then washed once in PBS-S and once in FACS sheath fluid (J. T. Baker, Deventer, The Netherlands) and analyzed with a Calibur flow cytometer (Becton Dickinson, San Jose, Calif.). The results presented in this study were based on lymphocyte gating on a forward scatter-versus-side scatter diagram. All cells outside the lymphocyte gate (region 1) are named and regarded as monocytes (named macrophages in the lymph node preparations). Fluorescent histogram markers were defined and quantitated positively from negatively stained cells. The percentage of cytokine-positive cells was calculated after subtraction of eventual positive signals from the isotype control immunoglobulin preparations. The values for all animals in a group were plotted and were found to be normally distributed. Student's t test was used for statistical evaluations.

Summary:
1. Single-cell bronchial lymph node preparations exhibited very much the same cytokine profiles as peripheral blood mononuclear cells except for a lack of IL-8 production. 2. It should also be noted that the IL-8-positive cells almost exclusively belong to the monocyte cell population, while cells positive for γ-IFN and TNF-α fall in both gated cell populations. 3. The PMA and ionomycin treatment had a slight suppressive effect on IL-8 production by monocytes.

References:
1: Aasted B, Bach P, Nielsen J, Lind P. Cytokine profiles in peripheral blood mononuclear cells and lymph node cells from piglets infected in utero with porcine reproductive and respiratory syndrome virus. Clin Diagn Lab Immunol. 2002 Nov;9(6):1229-34. PubMed PMID: 12414754; PubMed Central PMCID: PMC130102.