Abstract
A novel trophoblast cell surface antigen has been defined by a monoclonal antibody 5T4, raised following immunisation with wheat germ agglutinin (WGA) purified glycoproteins from deoxycholate (DOC) solubilised human syncytiotrophoblast plasma membrane (StMPM). The distribution of the antigen was determined by indirect immunoperoxidase staining of sections of normal organ and placental tissues as well as immunofluorescence and radiobinding assays with a wide variety of cell lines representing differing normal and tumour cell types. In frozen sections of normal full term placenta, 5T4 is strongly expressed only by the syncytiotrophoblast, some extravillous cytotrophoblast and the amniotic epithelium. The 5T4 antigen is apparently not expressed by any maternal component of the placenta nor is it detected in adult liver, lung, bronchus, heart, testis, ovary, brain, or muscle. The antigen is apparently expressed by several specialised epithelia. Immunoprecipitation of radiolabelled StMPM indicated that 5T4 molecules are glycoproteins of mol. wt of approximately 72 kD on SDS-PAGE. 5T4 antigen is selectively expressed by diverse tumour cell lines, including those of developmental origin. The molecular characteristics, relatively restricted normal tissue distribution and expression by certain tumour cell types make this antigen worthy of future study for use as a diagnostic marker of malignancy.
Highlights
The preliminary screen by immunodot showed that the antigen recognised was none of the following major proteins associated with the trophoblast; IgG, transferrin, PLAP, human placental lactogen (HPL), albumin, calmodulin, nor was it detectable in serum
On the basis of immunoperoxidase staining of frozen sections from normal tissue, 5T4 antigen is expressed by certain epithelial cell types
It should be noted that several 'trophoblast-characteristic' antigens, such as PLAP, are found in normal tissues at trace concentrations (McLaughlin, 1986)
Summary
StMPM was purified from full term human placentae, obtained within I h post partum, by the method of Smith et al (1974). The StMPM pellet was solubilised in 0.5% DOC in tris-buffered saline (TBS, 0.15 M NaCl, 25mM tris, pH 8.0) containing 0.1 mM phenylsulphonylmethyl fluoride (PMSF) and centrifuged at 14,000g for 10min. The WGA-reactive glycoproteins were purified by incubation of the supernatant with WGA-Sepharose (5mg ligand ml-1 Sepharose) for 1 h at room temperature. The beads were washed extensively in TBS/0.5% DOC, and the bound glycoproteins eluted in 5ml of 0.2M N-acetyl glucosamine (Sigma) in TBS. The eluted fraction was extensively dialysed against 30 mM ammonium bicarbonate (pH 7.9), and lyophilised. Received 24 September 1987; and in revised form 27 November 1987
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