Sandwich immunoassay for the measurement of murine syndecan-4

A sensitive and specific immunoassay for mouse syndecan-4 is described. The assay is a non-competitive direct sandwich enzyme-linked immunosorbent assay based on the production of an affinity-purified polyclonal antibody directed against syndecan-4 extracellular domain, used both as a trapping and detecting antibody. The standard curve is constructed with recombinant 6-His-tagged syndecan-4 extracellular domain. The assay allowed quantification of syndecan-4 core protein in the partially purified proteoglycan fraction from adipocyte 3T3-F442A cells, as well as in the cellular whole and clarified lysates. Removal of glycosaminoglycan chains on syndecan-4 core protein is required for maximal epitope exposure. The standard curve ranged between 7 and 70 fmol per well. For the 14 standard curves run on different days, the absorbance at 490 nm for 35.5 fmol of recombinant syndecan-4 was 0.610 +/- 0.110 (n = 14) with a corresponding blank absorbance of 0.089 +/- 0.030. At low (5.7 fmol) and high (42.8 fmol) levels of whole cell syndecan-4, the intra-assay and inter-assay coefficients of variation were 4.9 and 15.3 percent, and 3.3 and 9.7 percent, respectively. A survey of the syndecan-4 expression in various mouse tissues shows that syndecan-4 is highly expressed in the kidney, brain, testes and liver, but can also be measured in several different adipose tissue sites.