The development of a sialic acid specific lectin‐immunoassay for the measurement of human chorionic gonadotrophin glycoforms in serum and its application in normal and Down's syndrome pregnancies

OBJECTIVE We have developed and validated a lectin‐immunoassay for the recognition of sialic acid residues on hCG glycoforms in serum. DESIGN This assay employs a hCG specific capture antibody and a sialic acid specific lectin (Wheat Germ Agglutinin) labelled with horse radish peroxidase. RESULTS The standard curve covered hCG concentrations of 0‐4000lU/I (3rd IS for hCG, 7W537) with an analytical sensitivity of 1.0 IUA. The within and between batch coefficient of variation was<7% for all doses. Cross‐reactivity of <1% with TSH, LH, FSH, hCGa, hCGβ and desialylated hCG confirmed assay specificity. Dilutions of serum of <10% final concentration were parallel to the standard curve (within and between batch CV,<6%). The assay working range was 100 – >500000 IUh and the recovery of hCG from serum was in the range of 94.5% to 115.4%, with a mean value of 102.1%. The assay detected a time dependent change in hCG sialylation during normal pregnancy with the relative abundance of sialylated hCG declining after week 9 and increasing after week 15 of gestation. In addition preliminary studies showed that maternal serum hCG concentrations measured with the lectln‐immunoassay were elevated in high risk Down's pregnancies (as defined by conventional screening tests between weeks 16‐18 gestatlon, medlan multiple of median, 3.14; range 1‐81–19.12, P< 001) and low risk (1.57, 0.49–6.14, P=0.034) compared to normal (1.00,0–32‐3.20) pregnancies. Furthermore, the lectin immunoas‐say had greater discriminatory power compared to conventional immunoassay of hCG and hCGB between normal and both low and high risk Down's pregnancies.

[1]  R. Cooke,et al.  Acidic isoforms of chorionic gonadotrophin in European and Samoan women are associated with hyperemesis gravidarum and may be thyrotrophic. , 1999, Clinical endocrinology.

[2]  R. Cooke,et al.  Acidic isoforms of chorionic gonadotrophin in European and Samoan women are associated with hyperemesis gravidarum and may be thyrotrophic , 1999 .

[3]  L. Cole,et al.  Serum hyperglycosylated hCG: a potential screening test for fetal Down syndrome , 1999, Prenatal diagnosis.

[4]  L. Cole,et al.  Urinary screening tests for fetal Down syndrome: II. Hyperglycosylated hCG , 1999, Prenatal diagnosis.

[5]  S. Birken,et al.  Early pregnancy human chorionic gonadotropin (hCG) isoforms measured by an immunometric assay for choriocarcinoma-like hCG. , 1999, The Journal of endocrinology.

[6]  L. Cole,et al.  Dissociation of Human Chorionic Gonadotrophin into its Free Subunits is Dependent on Naturally Occurring Molecular Structural Variation, Sample Matrix and Storage Conditions , 1998, Annals of clinical biochemistry.

[7]  L. Cole,et al.  Hyperglycosylated hcg, a potential alternative to hcg in Down syndrome screening , 1998, Prenatal diagnosis.

[8]  W. Robertson,et al.  Gonadotrophin heterogeneity and biopotency: implications for assisted reproduction. , 1998, Molecular human reproduction.

[9]  L. Cole,et al.  Immunoassay of human chorionic gonadotropin, its free subunits, and metabolites. , 1997, Clinical chemistry.

[10]  Roger Ekins,et al.  Point On the meaning of “sensitivity” , 1997 .

[11]  R Ekins,et al.  On the meaning of "sensitivity". , 1997, Clinical chemistry.

[12]  L. Díaz-Cueto,et al.  More in-vitro bioactive, shorter-lived human chorionic gonadotrophin charge isoforms increase at the end of the first and during the third trimesters of gestation. , 1996, Molecular human reproduction.

[13]  W. Olijve,et al.  Molecular biology and biochemistry of human recombinant follicle stimulating hormone (Puregon). , 1996, Molecular human reproduction.

[14]  R. de Leeuw,et al.  Structure-function relationship of recombinant follicle stimulating hormone (Puregon). , 1996, Molecular human reproduction.

[15]  B. Verrier,et al.  Biological and immunochemical characterization of recombinant human thyrotrophin. , 1995, Glycobiology.

[16]  M. Hearn,et al.  Application of a sensitive HPLC-based fluorometric assay to determine the sialic acid content of human gonadotropin isoforms. , 1995, Journal of biochemical and biophysical methods.

[17]  J. Y. Lee,et al.  A change in the isoforms of human chorionic gonadotropin occurs around the 13th week of gestation. , 1994, The Journal of clinical endocrinology and metabolism.

[18]  J. Hershman,et al.  Thyrotropic activity of basic isoelectric forms of human chorionic gonadotropin extracted from hydatidiform mole tissues. , 1994, The Journal of clinical endocrinology and metabolism.

[19]  M. Shao The use of streptavidin-biotinylglycans as a tool for characterization of oligosaccharide-binding specificity of lectin. , 1992, Analytical biochemistry.

[20]  R. Karlsson,et al.  Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. , 1991, Journal of immunological methods.

[21]  S. Chappel,et al.  Reevaluation of the roles of luteinizing hormone and follicle-stimulating hormone in the ovulatory process. , 1991, Human reproduction.

[22]  L. Cole,et al.  The heterogeneity of human chorionic gonadotropin (hCG). III. The occurrence and biological and immunological activities of nicked hCG. , 1991, Endocrinology.

[23]  A. J. Chapman,et al.  Gonadotrophin glycosylation and function. , 1990, The Journal of endocrinology.

[24]  H. Asakura,et al.  A Lectin-Based Monoclonal Enzyme Immunoassay to Distinguish Fucosylated and Non-Fucosylated α-Fetoprotein Molecular Variants , 1990, Annals of clinical biochemistry.

[25]  J. Parkkinen,et al.  A lectin-immunofluorometric assay using an immobilized Bandeiraea simplicifolia II lectin for the determination of galactosylation variants of glycoproteins. , 1989, Analytical biochemistry.

[26]  N. Taniguchi,et al.  Alpha-fetoprotein antibody-lectin enzyme immunoassay to characterize sugar chains for the study of liver diseases. , 1989, Clinica chimica acta; international journal of clinical chemistry.

[27]  N. Sharon,et al.  Occurrence and isolation , 1989 .

[28]  L. Cole,et al.  Characterization of antisera distinguishing carbohydrate structures in the beta-carboxyl-terminal region of human chorionic gonadotropin. , 1988, Endocrinology.

[29]  E. Köttgen,et al.  Demonstration of glycosylation variants of human fibrinogen, using the new technique of glycoprotein lectin immunosorbent assay (GLIA). , 1988, Biological chemistry Hoppe-Seyler.

[30]  B. Leijnse,et al.  Lectin-enzyme immunoassay of transferrin sialovariants using immobilized antitransferrin and enzyme-labeled galactose-binding lectin from Ricinus communis. , 1987, Analytical biochemistry.

[31]  L. Cole,et al.  Detection of desialylated forms of human chorionic gonadotropin. , 1987, Clinica chimica acta; international journal of clinical chemistry.

[32]  S. Imamura [Lectin immunoradiometric assay versus improved radioimmunoassay: a comparison of methods for determination of desialylated forms of human chorionic gonadotropin]. , 1985, Nihon Sanka Fujinka Gakkai zasshi.

[33]  A. Kobata,et al.  Detection of incompletely sialylated human chorionic gonadotropin by peanut agglutinin in choriocarcinoma. , 1985, The Japanese journal of experimental medicine.

[34]  R. Canfield,et al.  Immunochemical determinants unique to human chorionic gonadotropin: importance of sialic acid for antisera generated to the human chorionic gonadotropin beta-subunit COOH-terminal peptide. , 1980, Endocrinology.

[35]  M. Monsigny,et al.  Sugar-lectin interactions: how does wheat-germ agglutinin bind sialoglycoconjugates? , 1980, European journal of biochemistry.

[36]  V. Bhavanandan,et al.  The interaction of wheat germ agglutinin with sialoglycoproteins. The role of sialic acid. , 1979, The Journal of biological chemistry.