Product Protocol: Il2 antibody used to evaluate the cd8 t cell compartment plays a dominant role in the deficiency of brown-norway rats to mount a proper type 1 immune response (OASA08671)

Specificity: IL-2
Product Page: Il2 antibody (OASA08671)
Antibody Pair Available: OASA08671 Capture OASA08672 Detection
Experiment Type: Cytokine assays (by specific ELISA)

Protocol:
IFN-γ and IL-2 protein in the supernatant were measured by specific ELISA. Ninety-six-well plates were coated overnight at 4°C with 5 μg/ml of an anti-rat IFN-γ mAb (DB1) or 1 μg/ml rabbit anti-rat IL-2 Ab (BD PharMingen). Serial dilutions of tissue culture supernatant (100 μl/well), followed by biotinylated DB12, an anti-rat IFN-γ mAb, or biotinylated A38-3 (BD PharMingen), an anti-rat IL-2, were sequentially incubated for 2 h at room temperature, separated by three washes. The bound biotinylated Abs were revealed by an additional 60-min incubation with alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Avondale, PA). The assay was developed by adding the enzyme substrate 4-nitrophenylphosphate disodium (Sigma, St. Louis, MO) at 1 mg/ml in diethanolamine buffer, pH 9.6, for 90 min at room temperature. The absorbance was measured at 405 nm using an automated microplate ELISA reader (Emax, Molecular Devices, Sunnyvale, CA). For IFN-γ, values were expressed as units per ml derived from a standard curve constructed using rat recombinant IFN-γ. This cytokine and anti-rat IFN-γ mAbs were gifts from Dr. P. van der Meide (TNO, Rijswijk, The Netherlands). For IL-2, values were expressed as units per ml derived from a standard curve constructed using rat recombinant IL-2 (BD PharMingen). The IL-4 protein in the supernatant was analyzed by ELISA and biological assay based on the effect of IL-4 on MHC class II up-regulation on B cells. For quantification of IL-4 mRNA, total RNA was isolated from stimulated T cells using the Promega isolation kit (Promega, Madison, WI). The cDNA was prepared. Transcript levels of IL-4 and hypoxanthine phosphoribosyltransferase (HPRT) were quantified using real-time quantitative PCR and SYBR Green DNA dye (ABI PRISM 5700, Perkin-Elmer Applied Biosystems, Foster City, CA). Primer sequences were as follows: IL-4, 5′-CGGTGAACTGAGGAAACTCTGTAG-3′ (sense) and 5′-CACGGTGCAGCTTCTCAGTG-3′ (antisense); and HPRT, 5′-TGTTGGATACAGGCCAGACTTTGT-3′ (sense) and 5′-TCCACTTTCGCTGATGACACA-3′ (anti-sense). Results were expressed as the intrasample ratio of IL-4 to HPRT mRNA copy numbers.

Summary:
1. Tissue culture supernatants were assayed by capture ELISA for IL-2 and IFN-γ production after 36 (IL-2) and 60 h (IFN-γ) of stimulation. After 60 h of stimulation in the presence of BN APC the IL-2 production was slightly, but significantly, lower (p = 0.02) than that in the presence of LEW APC (data not shown). These data indicate that the APC are not involved in the defective IFN-γ response of BN rats. 2. In this APC-independent system, it first appeared that stimulation with anti-TCR Ab alone did not induce T cell proliferation or cytokine production, and second, that upon stimulation with anti-TCR and anti-CD28 mAbs, the peak of cytokine production was 24 and 48 h for IL-2 and IFN-γ, respectively. In this condition of stimulation, the production of IL-2 was not significantly different between T cells of both rat strains.

References:
1: Cautain B, Damoiseaux J, Bernard I, Xystrakis E, Fournié E, van Breda Vriesman P, Druet P, Saoudi A. The CD8 T cell compartment plays a dominant role in the deficiency of Brown-Norway rats to mount a proper type 1 immune response. J Immunol. 2002 Jan 1;168(1):162-70. PubMed PMID: 11751959.

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