Urinary gonadotrophin peptide – isolation and purification, and its immunohistochemical distribution in normal and neoplastic tissues

Abstract

Summary A urinary gonadotrophin peptide (UGP) was isolated and purified from semi-purified human chorionic gonadotrophin (hCG), prepared from pregnancy urine. The peptide showed hCG-B subunit activity and no hCG-alpha subunit activity as demonstrated by binding studies with the relevant antibodies. It had a molecular weight significantly less than hCG-B subunit. The peptide was linked to thyroglobulin and this conjugate used to immunise rabbits and mice. A radioimmunoassay (RIA) using 1251-UGP and the rabbit antiserum (AK 12) was used to monitor chromatographed urine fractions from patients with ovarian carcinoma, seminoma and hydatidiform mole. UGP was also found in the urine extract of a healthy male, but at a much lower level. In each case the UGP detected had the same molecular weight as the pregnancy preparation and appeared to be the main gonadotrophin constituent in those urine samples. Initial immunohistochemical screening of normal and neoplastic tissues with the rabbit antibody (AK12) showed reactivity with some tumours including carcinomas of the lung, ovary, cervix and breast as well as trophoblastic and germ cell tumours. Reactions with non-neoplastic tissues were confined to some specialised epithelia and macrophage populations. A more comprehensive immunohistochemical study was made using a monoclonal antibody to UGP (2C2), with a monoclonal antibody to conformational hCG (INN 13) and another monoclonal antibody to free B subunit (1 E5) as controls. Similar patterns of reactivity were produced by the AK12 and 2C2 antibodies in both neoplastic and non-neoplastic tissues. Additional tissues were investigated with the three monoclonal antibodies. The 2C2 antibody reacted with 93% (77/83) of tumours examined; the INN 13 antibody reacted with only the syncytiotrophoblast cells of choriocarcinoma, hydatidiform mole, placental site trophoblastic tumour, and in one case of seminoma; the 1E5 reactivity was confined to only choriocarcinoma syncytiotrophoblast cells.

Characterisation of UGP and its relationship with beta-core fragment

“…It has also been detected in 93% (77/83) of tumours examined immunohistochemically (Kardana et al, 1988). The apparent molecular weight is 15,000.…”

“…A commercial preparation of hCG (Pregnyl) derived from pooled pregnancy urines was subjected to gel filtration on Sephadex G 100 and immunoadsorption with a mouse monoclonal P-directed antibody (W14) immobilised on cyanogen-bromide activated Sepharose 4B, the required material being eluted with 3 M ammonium thiocyanate followed by immediate desalting by G25 chromatography, according to procedures published earlier (Kardana et al, 1988). Aliquots of the immunoadsorbed material were further purified on FPLC using a Superose 12 column (Pharmacia FPLC systems) or re-subjected to gel-filtration using Sephadex G-100.…”

“…UGP from normal male urine or ovarian carcinoma urine was extracted using acetone precipitation (as described in Kardana et al, 1988), whilst urine from a patient with hydatidiform mole was extracted using either acetone precipitation (as above) or as an alternative kaolin adsorption was used. Two litres of urine were adjusted to pH=4.5 with glacial acetic acid and then 35 g of kaolin (a modification of the method of Albert, 1956, that does not include the final precipitation step) stirred into this solution.…”

“…Beta core-fragment on iso-electric focusing was found to have a pl > 9.5, peptide 3 showed two bands at pl = 3.5 and 3.8 whilst insufficient purified peptide 1 was available to determine its iso-electric point. Bioassay studies on UGP showed that any biological activity could be attributed to trace contamination with hCG.Urinary gonadotrophin peptide (UGP) has been purified from the urine of patients with trophoblastic and nontrophoblastic disease (Kardana et al, 1988). It has also been detected in 93% (77/83) of tumours examined immunohistochemically (Kardana et al, 1988).…”

“…Urinary gonadotrophin peptide (UGP) has been purified from the urine of patients with trophoblastic and nontrophoblastic disease (Kardana et al, 1988). It has also been detected in 93% (77/83) of tumours examined immunohistochemically (Kardana et al, 1988).…”