Polypeptide nicks cause erroneous results in assays of human chorionic gonadotropin free β-subunit

Abstract

Pregnancy and trophoblastic disease testing laboratories measure human chorionic gonadotropin (hCG; human choriogonadotropin) free β-subunit to screen for Down syndrome and to diagnose persistent trophoblastic disease (invasive mole and choriocarcinoma). The results from various laboratories, however, vary widely for similar groups of patients. Average concentrations of free β-subunit reported for persistent trophoblastic disease, for instance, range from 2.6% to 37% of total hCG. On hCG and its free β-subunit, peptide bonds can be missing between β-subunit residues 44 and 45 or between residues 47 and 48 (nicked molecules). To explore the possibility that the disparity in the reported concentrations of free β-subunit was due to differences in recognition of nicked molecules by different antibodies, we generated 0% and 100% nicked β-subunit standards and investigated their recognition in three separate immunoassays of free β-subunit. The immunoassay with antibody 1E5 did not recognize nicked β-subunit (4% cross-reactivity with nicked β-subunit) and thus detected only intact β-subunit. The immunoassay with antibody FBT11 gave similar results with nicked and nonnicked standards (96% cross-reactivity with nicked β-subunit), as did the assay with antibody B204 (73% cross-reactivity with nicked β-subunit). These two immunoassays thus measured total (nicked + nonnicked) β-subunit. We used the three immunoassays to examine sera from normal pregnancy, Down syndrome pregnancy, hydatidiform mole, and persistent trophoblastic disease, all of which contain nicked and nonnicked β-subunit molecules. The results obtained resembled the studies with nicked β-subunit standards. The results from the FBT11 and B204 assays (total β-subunit) were highest, results from the 1E5 assay (intact molecules only) being as much as 10 times lower. We conclude that nicks in β-subunit and the extent of recognition of nicked molecules by different antibodies affect the concentrations reported for free β-subunit.