Polypeptide nicks cause erroneous results in assays of human chorionic gonadotropin free beta-subunit.
Pregnancy and trophoblastic disease testing laboratories measure human chorionic gonadotropin (hCG; human choriogonadotropin) free beta-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 beta-subunit reported for persistent trophoblastic disease, for instance, range from 2.6% to 37% of total hCG. On hCG and its free beta-subunit, peptide bonds can be missing between beta-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 beta-subunit was due to differences in recognition of nicked molecules by different antibodies, we generated 0% and 100% nicked beta-subunit standards and investigated their recognition in three separate immunoassays of free beta-subunit. The immunoassay with antibody 1E5 did not recognize nicked beta-subunit (4% cross-reactivity with nicked beta-subunit) and thus detected only intact beta-subunit. The immunoassay with antibody FBT11 gave similar results with nicked and nonnicked thus standards (96% cross-reactivity with nicked beta-subunit), as did the assay with antibody B204 (73% cross-reactivity with nicked beta-subunit). These two immunoassays thus measured total (nicked + nonnicked) beta-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 beta-subunit molecules. The results obtained resembled the studies with nicked beta-subunit standards. The results from the FBT11 and B204 assays (total beta-subunit) were highest, results from the 1E5 assay (intact molecules only) being as much as 10 times lower. We conclude that nicks in beta-subunit and the extent of recognition of nicked molecules by different antibodies affect the concentrations reported for free beta-subunit.
Kardana
A
A
Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT 06510.
Cole
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LA
D000595:Amino Acid Sequence; D006063:Chorionic Gonadotropin / Q000097:blood* / Q000737:chemistry*; D018997:Chorionic Gonadotropin, beta Subunit, Human; D004314:Down Syndrome / Q000097:blood; D005260:Female; D006801:Humans; D006828:Hydatidiform Mole / Q000097:blood; D007118:Immunoassay / Q000592:standards; D019272:Leukocyte Elastase; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000097:blood* / Q000737:chemistry*; D011247:Pregnancy; D014328:Trophoblastic Neoplasms / Q000097:blood; D014594:Uterine Neoplasms / Q000097:blood
D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D010446:Peptide Fragments; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase