title: Comparison of two direct neutralizing assay formats using recombinant follicle-stimulating hormone as agonist.
abstract: Characterizing anti-drug antibodies for neutralizing activity is commonly part of the immunogenicity testing package for most therapeutic proteins. Cell-based neutralization assays can generally be categorized as direct- or indirect assays depending on whether they are associated with therapeutics with agonistic- or antagonistic properties. This paper's aim is a comparison of the two direct neutralization assay formats; the variable- and fixed concentration assay format, using recombinant follicle-stimulating hormone as drug agonist. Essential validation- and performance parameters, such as sample through-put, cut-point, precision, sensitivity and drug tolerance, were compared. The fixed concentration assay format offers superior sample through-put (40 versus 6 samples), precision (coefficient of variation of ≤14% versus 34%) and almost 6 times better sensitivity and is generally recommended as the better option particularly for quasi-quantitative assessments of neutralizing antibodies.
authors.affiliation: Ferring Pharmaceuticals A/S, Kaj Fiskers Plads 11, DK-2300 Copenhagen, Denmark. Electronic address: jrd@ferring.com.
authors.forename: J
authors.initials: J
authors.lastname: Ryding
authors.forename: E
authors.initials: E
authors.lastname: Hjertberg
authors.forename: B B
authors.initials: BB
authors.lastname: Rasmussen
mesh_terms: D000818:Animals; D019138:Antibodies, Blocking / Q000378:metabolism; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D009500:Neutralization Tests / Q000379:methods*; D015588:Observer Variation; D011962:Receptors, FSH / Q000819:agonists* / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity; D019076:Transgenes / Q000235:genetics
chemical_list: D019138:Antibodies, Blocking; D011962:Receptors, FSH; D005640:Follicle Stimulating Hormone
keywords: Antibodies; Bioassay; Cell-based; EC50; FSH; Format; NAb; NC; Neutralizing; PC LOW; RLU; cAMP; cyclic adenosine monophosphate; effective concentration at 50%.; human follicle-stimulating hormone; low positive control; negative control; recombinant human follicle-stimulating hormone; relative light units; rhFSH