Do immunoassays differentially detect different acidity glycoforms of FSH?

The possibility of the carbohydrate residues of glycoproteins affecting their recognition in immunoassays is an important and unresolved issue. This study looked for evidence of differential recognition of FSH glycoform preparations, of variable isoelectric point (pI) and known molarity, using three routine assays employing different antibody configurations.

Seven glycoform preparations with differing pI bands (between 3.8 and 5.5) were produced by isoelectric focusing of recombinant human FSH and the molecular weights determined by mass spectroscopy. Three concentrations of each glycoform were assayed and the results expressed relative to unfractionated material. From the relative responses, recognition differences between the assay methods and between the glycoform preparations were investigated.

Three routine assays were employed: the commercially available Amerlite(R) enzyme immunoassay and Delfia(R) immunofluorometric assay, together with an in-house competitive two-site radioimmunoassay (RIA).

Overall, the three assays gave the same relative responses for equivalent glycoforms, with the only exceptions involving small differences between some assay pairs for the fractions at the extremes of the pI range investigated. Within each assay type, differences (P < 0.05) of up to 33% existed between glycoforms of different pI, however, these differences showed no patterns or trends across the entire acidity range examined.

Between the assay methods investigated in this study, few differences exist in the recognition of individual pI bands of FSH when expressed relative to a common unfractionated standard. Differences were apparent in the recognition of the different acidity glycoforms within each assay method, however, these were small and unlikely to be of clinical significance.