Sandwich ELISA for the Quantification of Soluble Human Semaphorin 4D, a factor promoting skeletal metastases
05 March 2019 (online)
Semaphorin 4D (SEMA4D, CD100) is a type I integral membrane glycoprotein that regulates key cellular functions. It is over-expressed in a wide variety of cancers including malignancies of the prostate and the breast. The extracellular region of Sema4D can be proteolytically cleaved to generate a soluble molecule retaining its biological activity (sSema4D). The type 1 matrix metalloproteinases mediating this cleavage are upregulated in many malignant cells. Sema4D inhibits osteoblast function via its receptor Plexin-B1 and has been shown to promote the invasion of bone in tumor mouse models. To further investigate the potential role of Sema4D as a biomarker in physiological and pathological conditions, there is a need for a highly specific and sensitive assay for the quantification of Sema4D in peripheral blood.
We developed a sandwich ELISA that enables the accurate measurement of sSEMA4D in plasma samples. The assay utilizes two monoclonal anti-human Semaphorin 4D antibodies, both recognizing conformational epitopes. The epitopes have been mapped by overlapping constrained peptides and shown to involve amino acids AA30-AA34 and amino acids AA238-AA241, respectively.
We demonstrate that sSEMA4D can be reliably measured in various plasma preparations (EDTA, citrate, heparin) with a mean coefficient of variation of < 8% between these matrices. Serum measurement of sSEMA4D showed in average a 3 fold higher concentration than plasma, indicating that the measurement of sSEAMA4D in blood samples may be interfered by the in vitro release from platelets. Hence, the assay was optimized for human plasma samples only. The assay covers a wide calibration range between 0 to 2000 pmol/l and sSEMA4D EDTA-plasma concentrations in apparently healthy individuals are 239 +/- 59 pmol/l (n = 44). Assay characteristics, such as precision, dilution linearity and spike/recovery as well as sample stability have been analyzed and meet the international standards of acceptance.
Our ELISA provides a reliable and accurate tool for the quantitative determination of soluble, biologically active Semaphorin4D in human samples and could help to gain further insight into the role of Sema4D in cancer progression, prognosis, and therapy.