A human monoclonal antibody based immunoenzymetric assay to measure Fel d 1 concentrations in cat hair and pelt allergenic extracts

Abstract

In the United States, 19 allergen extracts of different specificities are standardized, which means that their potencies are determined in comparison to a US reference standard. For cat allergen extracts, potency is determined by measuring Fel d 1 content expressed in in Fel d 1 units, and with a unitage that correlates with skin test reactions (bioequivalent allergy units or BAU). Currently, Fel d 1 content is measured with a radial immunodiffusion (RID) assay that uses polyclonal sheep antisera to detect the allergenic protein by producing a white precipitin line in agar gel. However, the RID is considered cumbersome, and the polyclonal sera may qualitatively vary among animals and may recognize epitopes irrelevant to human allergic disease. In this report, we describe a quantitative two-site immunoenzymetric assay (IEMA) for Fel d 1 that uses immobilized capture and soluble biotin-labeled detection Fel d 1-specific human IgE monoclonal antibodies (mAb) that have been class-switched to IgG4. Together, they sandwich Fel d 1 molecules from extracts. Using purified natural Fel d 1 as a calibrator, the historically reported ∼4 micrograms Fel d 1/Fel d 1 unit assignment was directly measured in this mAb-based IEMA at 3.12 ± 0.24 micrograms of Fel d 1 per Fel d 1 unit. This IEMA appears to be equivalent to RID in the measurement of biological potencies of commercial cat hair and cat pelt extracts marketed in the United States.

Keywords: Fel d 1; IEMA; IgE; IgG4; allergen extract; allergen immunotherapy; allergy; cat.

Figure 1

Affinity measurement and epitope binning of four human mAbs specific to Fel d 1. (A–D) Binding curves for each mAb against nFel d 1. Serial dilution of nFel d 1 used (blue to green): 20 nM, 4 nM, 0.8 nM, and 0.16 nM. pM = picomolar. (E) Pairwise epitope binning of nFel d 1. Blue = a pair of mAbs sandwiching nFel d 1; red = a ligand mAb blocks the binding of an analyte mAb to nFel d 1; dark red = a ligand mAb blocks itself as an analyte from binding nFel d 1 (see discussion of binning in the methods).

Figure 2

The Fel d 1 IEMA is reproducible and precise. (A) EC₅₀ of two reference cat hair extracts reflects their relative potency, which was tightly distributed over 142 measurements. (B) Distribution of E9 extract potency values over 142 measurements by RID, using the E8 reference extract value of 14.7 Fel d 1 Units/ml. (C) IEMA determination of mass (ug) per unit of Fel d 1; n = 30.

Figure 3

No correlation between potencies measured by RID and IEMA. The relationship between potency values as determined by RID and IEMA was not linear. All correlation coefficients in the figures were r²< 0.5. The top panel includes all extract data in a single graph while the lower panels show the trends broken down by company (panels 2 and 3) and source type (Hair—panel 4 vs. pelt—panel 5).