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 0-4000 IU/l (3rd IS for hCG, 75/537) with an analytical sensitivity of 1.0 IU/l. The within and between batch coefficient of variation was < 7% for all doses. Cross-reactivity of < 1% with TSH, LH, FSH, hCG alpha, hCG beta 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 - > 500 000 IU/l 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 lectin-immunoassay were elevated in high risk Down's pregnancies (as defined by conventional screening tests between weeks 16-18 gestation, median multiple of median, 3.14; range 1.81-19.12, P < 0.001) 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 immunoassay had greater discriminatory power compared to conventional immunoassay of hCG and hCG beta between normal and both low and high risk Down's pregnancies. CONCLUSION This assay will allow analysis of serum samples for the investigation of sialylated variants of hCG glycoforms in various pathological and physiological situations.