Determination of soluble tumor necrosis factor receptor 2 produced by alternative splicing.
Soluble cytokine receptors have proven to be very useful biomarkers in a large variety of diseases, including cancer, infections, and chronic inflammatory diseases. These soluble receptors are produced by proteolytic cleavage or alternative splicing. Several cytokine receptors including tumor necrosis factor receptor 2 (TNFR2) can be generated by both mechanisms. However, the conventional ELISA systems do not differentiate between these two types of soluble receptors. We describe a sandwich ELISA to specifically quantify soluble TNFR2 protein generated by alternative splicing. This method requires the use of a capturing monoclonal antibody (mAb) specific of an epitope present in the soluble TNFR2 generated by alternatively splicing but absent in the proteolytically generated isoform. Here we present a detailed protocol for the production and validation of such a mAb. This method has the potential to be applied for measuring other soluble cell surface molecules generated by alternative splicing.
Immunology Unit, Department of Cell Biology, Immunology and Neurosciences, Medical School, University of Barcelona and IDIBAPS, C/Casanova 143, Barcelona, 08036, Spain.
Xavier
X
Romero
Juan D
JD
Cañete
Pablo
P
Engel
D017398:Alternative Splicing; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D009101:Multiple Myeloma / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D010446:Peptide Fragments / Q000008:administration & dosage / Q000276:immunology; D020033:Protein Isoforms; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000032:analysis* / Q000235:genetics*
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D020033:Protein Isoforms; D047889:Receptors, Tumor Necrosis Factor, Type II