Csf2 Antibody (OASA08403)

Catalog#: OASA08403

• Size: 0.1mg

• Description of Target: RABBIT ANTI MOUSE GM-CSF
• Gene Symbol: Csf2
• Alias Symbols: Csfgm; Gm-CSf; MGI-IGM; MGC151255; MGC151257; Csf2
• Protein Accession#: NP_034099.2
• Swissprot Id: P01587
• Host: Rabbit
• Protein Size (# AA): 141
• Specificity: GM-CSF
• Application: ELISA, WB
• Immunogen: The immunogen for anti-Csf2 antibody: recombinant mouse GM-CSF
• Product Format: Purified IgG - lyophilised
• Isotype: Polyclonal IgG
• Predicted Homology Based on Immunogen Sequence: Mouse
• Applications Info: ELISA : This product may be used in an indirect ELISA or as the capture reagent in a sandwich ELISA with OASA08404 as the detection antibody and OPSA11136 as the standard.
  Preservative Stabilisers: None present
  
  Antiserum Preparation: Antisera to mouse GM-CSF were raised by repeated immunisation of rabbits with highly purified antigen. Purified IgG was prepared by affinity chromatography.
  Approx Protein Conc: IgG concentration 1.0 mg/ml after reconstitution.
  
  Buffer Solutions: Phosphate buffered saline pH7.4
• Reconstitution and Storage: Reconstitution: Reconstitute with 0.1ml distilled water.
  Care should be taken during reconstitution as the protein may appear as a film at the bottom of the vial. Aviva Systems Biology recommend that the vial is gently mixed after reconstitution.
  

Product Protocol: Csf2 antibody used to evaluate incorporation of glycosylphosphatidylinositol-anchored granulocyte- macrophage colony-stimulating factor or cd40 ligand enhances immunogenicity of chimeric simian immunodeficiency virus-like particles (OASA08403)

Specificity: GM-CSF
Product Page: Csf2 antibody (OASA08403)
Antibody Pair Available: OASA08403 Capture OASA08404 Detection
Experiment Type: Quantitative ELISA for Env, Gag, GM-CSF, and CD40L, Production of VLPs

Protocol:
1. Quantitative ELISA for Env, Gag, GM-CSF, and CD40L:

To estimate the percentage of Env incorporation in VLPs, Ioanna et al. used a sandwich ELISA; 96-well Nunc Maxisorb flat-bottom plates were coated overnight with a 1:1,000 dilution of SIVmac251 gp120 monoclonal antibody (KK46) (NIH AIDS Research and Reference Reagent Program). The VLPs were pretreated with 0.1% radioimmunoprecipitation assay buffer (1 M Tris buffer [pH 8.0], 5 M NaCl, 10% Triton, 10% sodium deoxycholate, 10% SDS) and added at a concentration of 400 ng/well. Goat anti-SIV gp120 (1:4,000) and rabbit anti-goat IgG horseradish peroxidase (HRP) (1:4,000) diluted in PBS plus 0.05% Tween 20 supplementedwith 2% bovine serum albumin were used as primary and secondary antibodies. Purified SIVmac239 gp130 was used to construct the standard curve to detect the Env concentration and was provided from the NIH AIDS Research and Reference Reagent Program (National Institutes of Health, Rockville, MD).

To estimate the percentage of incorporation of growth factors into the chimeric VLPs, they used a direct ELISA. VLPs were used to coat each well (5 μg per well) of a 96-well Nunc Maxisorb flat-bottom plate. For the quantitative determination of GM-CSF, rabbit anti-mouse GM-CSF (1:2,000) and goat anti-rabbit IgG coupled to HRP (1:2,000) were used. For the quantitation of CD40L, goat anti-mouse CD40L (1:3,000) and rabbit anti-goat IgG HRP (1:5,000) were used. For the standard curves, they used soluble recombinant murine GM-CSF and CD40L (Peprotech, Inc, Rocky Hill, NJ). O-Phenylenediamine (OPD) substrate tablets (Zymed, San Francisco, CA) dissolved in citrate buffer, pH 5.0, were used to develop color in all aforementioned assays. Optical density was read at 450 nm. 2. Production of VLPs:

SIV VLPs were produced using a modification. For Gag VLPs, Sf9 insect cells were infected with rBV expressing SIVmac239 Gag at a MOI of 2 and incubated at 27°C for 72 h. SIV VLPs containing SIV Env and Gag were produced from Sf9 cells coinfected with rBVs expressing SIV Gag and SIV Env (SIVmac239) at MOI ratios of 1:4. Chimeric SIV VLPs were produced from Sf9 cells coinfected with rBV expressing SIV Gag, SIV Env, and CD40L or GM-CSF at MOI ratios of 1:4:4. Three days postinfection, the culture supernatants were collected and centrifuged at 1,500 × g for 20 min and filtered through a 0.45-μm-pore size filter, and the VLPs were pelleted at 100,000 × g for 1 h at 4°C in a Beckman SW28 rotor. The pellets were resuspended in PBS at 4°C overnight and VLPs were further purified through a 20-35-60% discontinuous sucrose gradient at 28,000 rpm for 1 h at 4°C. The VLP bands were collected, washed with PBS, pelleted, and resuspended overnight in PBS. To quantitate the yield of purified VLPs, the protein concentration of each sample was estimated with the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). For protein analysis, all samples were normalized to 1 μg/ml and loaded onto sodium dodecyl sulfate (SDS) polyacrylamide gels at the same concentration (5 or 10 μg), and SIV Gag and Env proteins, GM-CSF, and CD40L were probed using monkey anti-SIVmac239 serum (kindly provided by Silvija Staprans, Emory Vaccine Center), rabbit anti-mouse GM-CSF (Peprotech), and goat anti-mouse CD40L (Peprotech).

Summary:
1. In the chimeric VLPs, GM-CSF accounted for approximately 0.1% of the total VLP proteins. However, based upon quantitative ELISA analysis of SIV Env and GM-CSF incorporated into VLPs, the molar ratio of SIV Env trimers to GM-CSF is approximately 1:1, suggesting that GM-CSF is incorporated into VLPs as efficiently as SIV Env. 2. SIV Env proteins were found to be present at similar levels among various SIV VLP preparations, which were estimated to be 1.5 ± 0.2% of total VLP proteins by quantitative ELISA. Ioanna et al. measured the incorporation of immunostimulatory molecules by Western blot analysis of purified VLPs using antibodies specific to GM-CSF or CD40L GM-CSFCD59 and GM-CSFLFA3 were found to be incorporated into SIV VLPs at similar levels.

References:
1: Skountzou I, Quan FS, Gangadhara S, Ye L, Vzorov A, Selvaraj P, Jacob J, Compans RW, Kang SM. Incorporation of glycosylphosphatidylinositol-anchored granulocyte- macrophage colony-stimulating factor or CD40 ligand enhances immunogenicity of chimeric simian immunodeficiency virus-like particles. J Virol. 2007 Feb;81(3):1083-94. Epub 2006 Nov 15. PubMed PMID: 17108046; PubMed Central PMCID: PMC1797543.