Do immunoassays differentially detect different acidity glycoforms of FSH?

Area of Science:

• Biochemistry
• Immunology
• Analytical Chemistry

Background:

• Glycoprotein carbohydrate residues can affect recognition in immunoassays, a critical factor for accurate diagnostic testing.
• Understanding glycoform variability is essential for interpreting immunoassay results, particularly for hormones like follicle-stimulating hormone (FSH).

Purpose of the Study:

• To investigate the impact of varying isoelectric point (pI) on the recognition of follicle-stimulating hormone (FSH) glycoforms in routine immunoassays.
• To compare the performance of three different immunoassay platforms in recognizing FSH glycoform preparations.

Main Methods:

• Seven recombinant human FSH glycoform preparations with distinct pI values (3.8-5.5) were generated using isoelectric focusing.
• Molecular weights were confirmed by mass spectrometry, and glycoforms were assayed at known molarities in three different immunoassay formats.
• Results were analyzed relative to unfractionated FSH to identify differential recognition patterns.

Main Results:

• All three immunoassays (enzyme immunoassay, immunofluorometric assay, radioimmunoassay) showed largely consistent recognition of FSH glycoforms relative to a standard.
• Minor discrepancies were observed between assay pairs for glycoforms at the extreme ends of the pI range.
• Significant differences (up to 33%) in recognition were found within each assay type for different pI glycoforms, but without a clear trend across the acidity spectrum.

Conclusions:

• Few significant differences exist in FSH glycoform recognition across the investigated immunoassay methods when normalized to a common standard.
• While intra-assay differences in glycoform recognition were noted, they were generally small and unlikely to be clinically significant.
• The carbohydrate structure of FSH glycoforms has a limited impact on their recognition in routine immunoassays.