Measurement of human latent transforming growth factor beta 1 using a latency associated protein reactive elisa
2012 (English)In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 379, no 1-2, 23-29 p.Article in journal (Refereed) Published
Human Transforming Growth Factor (TGF)-beta 1, one of three TGF-beta isoforms, is a pleotropic cytokine critical for many physiological and immunological processes. TGF-beta 1 is secreted in a latent form, linked to Latency Associated Protein (LAP). Analysis of Latent TGF-beta 1 by TGF-ELISA requires dissociation of TGF-beta 1 from LAP, e.g. by acidification of samples. The ELISA then measures total TGF-beta 1, equivalent to dissociated Latent TGF-beta 1 plus any free TGF-beta 1 present prior to acidification. Evolutionary conservation of TGF-beta 1 across mammals also renders TGF-beta 1 ELISAs reactive with TGF-beta 1 in bovine serum often used in human cell cultures. To enable a direct analysis of Latent TGF-beta 1, monoclonal antibodies were made against LAP from human latent TGF-beta 1 and used to develop a LAP ELISA detecting Latent TGF-beta 1. The ELISA did not react with LAP from human Latent TGF-beta 2 or 3, respectively, nor with Latent TGF-beta in bovine serum. EDTA-containing plasma from healthy subjects (n = 20) was analyzed by conventional TGF-beta 1 ELISA and LAP ELISA. By TGF-beta 1 ELISA, total TGF-beta 1 were detected in all samples (median 133 pM, range 34-348 pM); low levels of free TGF-beta 1 found in 8/20 non-addified samples showed that >98.5% of the total TGF-beta 1 derived from Latent TGF-beta 1. Latent TGF-beta 1 found in non-acidified samples by LAP ELISA (median 154 pM, range 48-403 pM) was comparable in molar levels to, and correlated with, total TGF-beta 1 (r(s) 0.96, p<0.0001). A similar agreement between the total TGF-beta 1 and the LAP ELISA was found with citrate- and heparin-containing plasma. The LAP ELISA facilitates analysis of Latent TGF-beta 1 without sample acidification and is not compromised by the presence of bovine serum in human cell supernatants.
Place, publisher, year, edition, pages
2012. Vol. 379, no 1-2, 23-29 p.
ELISA, Cytokine, TGF-beta 1, Latent TGF-beta 1, Total TGF-beta 1
Immunology Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:su:diva-79768DOI: 10.1016/j.jim.2012.02.016ISI: 000304019600003OAI: oai:DiVA.org:su-79768DiVA: diva2:551886