Product Protocol: GM-CSF antibody used to evaluate the effect of segmental bronchoprovocation with allergen on airway lymphocyte function (OASA04708)

Specificity: GM-CSF
Product Page: GM-CSF antibody (OASA04708)
Antibody Pair Available: OASA04706 Capture OASA04708 Detection
Experiment Type: Cytokine ELISAs

A sensitive two-step sandwich-type enzyme-linked immunosorbent assay (ELISA) was established to measure cytokines in culture supernatant fluids. ELISA plates (Easy Wash, Catalogue No. 25805-96; Corning, Costar Corp., Cambridge, MA) were coated overnight with a predetermined optimal concentration of purified monoclonal anticytokine antibody. Nonspecific binding sites on the plate were blocked with 10% dialyzed newborn calf serum. BALFs were concentrated by 20× with a low-protein-binding concentrator (Centriprep; Amicon, Beverly, MA) with a molecular-weight cutoff limit of 3 kDa. Cell-culture supernatant fluids were diluted to optimal concentrations. Test samples were incubated on antibody-coated plates for 2 h, and cytokines were detected with biotinylated anticytokine antibodies followed by addition of a streptavidin polymer (POLY-HRP-40; Research Diagnostics Inc., Flanders, NJ). A one-component substrate, 3,3′,5,5′-tetramethylbenzidine (TMB) (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD), was used for color development, and the reaction was stopped by addition of 0.18 M sulfuric acid. Optical density (O.D.) at 450 nm was determined with a Dynatech MR5000 microplate reader, and data were analyzed with Biolink Software (Dynatech Laboratories, Inc., Chantilly, VA). The concentration of cytokines in supernatant fluids was calculated by comparison with a standard curve generated with known amounts of recombinant human cytokines. The sensitivity for each cytokine assay was < 3 pg/ml. Summary:
1. The cells obtained 48 h after allergen SBP showed a large increase in the capacity to produce IL-5, and smaller but statistically significant increases in the production of IL-4, IL-10, and GM-CSF. The ability of cells to produce IFN-γ or IL-2 was not significantly altered after Ag SBP. 2. Furthermore, the levels of IL-4 and GM-CSF present in vivo in BALF were at or below the level of detection in all subjects (data not shown).

1: Kelly EA, Rodriguez RR, Busse WW, Jarjour NN. The effect of segmental bronchoprovocation with allergen on airway lymphocyte function. Am J Respir Crit Care Med. 1997 Nov;156(5):1421-8. PubMed PMID: 9372655.

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