Abstract
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.
Highlights
A sensitive and specific immunoassay for mouse syndecan-4 is described
With polyclonal antibodies directed against the extracellular domain [14] or the cytoplasmic domain [26] of mouse syndecan-4, molecular weights of approximately 35,000, higher than the predicted 21,500, were reported
We have developed a sandwich ELISA for mouse syndecan-4, using a polyclonal antibody directed against the extracellular domain of the transmembrane protein
Summary
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 syndecan family of HSPGs contains four members (syndecan-1 to syndecan-4) with homologous transmembrane and cytoplasmic domains, and distinctive extracellular domains [1, 2] Their structure is characterized by hybrid glycosylation with long unbranched glycosaminoglycan (GAG) chains, containing heparan sulfate (HS) and chondroitin sulfate (CS) chains [3]. Because of the polyanionic nature of GAG chains, syndecans interact with numerous extracellular ligands, thereby affecting a large spectrum of biological activities in the cells [1, 2, 5]. Syndecan-4 mRNA expression has been studied during mouse embryogenesis [12] and measured in various tissues and cultured cells by Northern blot analysis [13,14,15], semiquantitative RT-PCR analysis [15,16,17] and in situ hybridization [10, 14].
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