Technical Information

A Cost-Effective Antibody Cocktail for Broad Neutralization of Human Type I IFN Activity

A single anti-human IFN receptor 2 (IFNAR2) monoclonal antibody (cat. # 21385-1) has proven to be useful in neutralizing the activities of human Type I IFNs by blocking the Type I IFN receptor complex.1-3  However, a high amount of this antibody is sometimes required to effectively compete for the receptor complex in the presence of multiple Type I IFNs (e.g. IFN alpha, beta, and omega) that are present in certain samples.

 

Validation of a Highly Sensitive Immunoassay for the Quantitation of Interferon Beta in Autoimmune Sera

Authors: P.R. Conliff1, T.M. Stauffer2; S.F. Bungo1; M. Skawinski2; T. Lavoie2; I.S. DuBey1

Author Affiliation: 1. Smithers Avanza; 2. PBL Assay Science

 

 

Purpose:

The role of interferons (IFN) in autoimmune disease is crucial to understanding the etiology and treatment of these diseases.

A highly sensitive immunoassay was validated and utilized for the quantitation of interferon beta in autoimmune sera.

 

Flow Cytometry for IFNAR2 on Human PBMC

Protocol for Flow Cytometric Analysis of IFNAR2 and CD3 Surface Expression on Human Peripheral Blood Mononuclear Cells (PMBC)

 

Flow Cytometry for IFNAR2 on JURKAT cells

Protocol for Flow Cytometric Analysis of IFNAR2 Surface Expression on JURKAT Cells

 

Materials:

Phosphate buffered saline (PBS)

Wash buffer: PBS containing 0.5% BSA and 0.02% NaN3

 

General Procedure for Intra-Cellular Staining

  1. (a) Suspension cells: Transfer cells to a 15 ml conical tube and centrifuge at 1000-1200 rpm for 5 minutes. Discard media and wash cells 2X with 5 ml 1X dPBS. (b) Adherent cells: Trypsinize cells with Trypsin/0.25% EDTA. neutralize the trypsin with an equal volume of media once the cells are detached. Transfer cells to a 15 ml conical tube and centrifuge at 1000-1200 rpm for 5 minutes. Discard media and wash cells 2X with 5 ml 1X dPBS.

General Protocol for Human Interferon Alpha Cytopathic Effect (CPE) Assay

The antiviral-based Cytopathic effect (CPE) assay is amongst the most sensitive assays for interferon bioactivity, with often >100 fold more sensitivity than alternatives. Many combinations of cells and viruses can be used to measure interferon activity via CPE assay. Many researchers use the human lung carcinoma cell line A549 due to their ease of growth and relatively high sensitivity to all forms of human interfer­ons. The following is a summary of an A549/EMCV Cytopathic Effect Inhibition assay for Recombinant Human Interferon Alpha:

 

General Procedure for Cell Surface Staining

Materials:

  1. Appliable stimulated and un-stimulated cells
  2. 1X PBS pH 7.2
  3. Bovine Serum Albumin (BSA)
  4. Normal human serum
  5. 5 ml polystyrene tubes; BD Falcon Catalog No. 352052
  6. Isotype control: PE-Mouse IgG1 kappa; BD Pharmingen Catalog No. 554680

 

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