Allergy Asthma Immunol Res.  2015 Jul;7(4):384-392. 10.4168/aair.2015.7.4.384.

IgE Reactivity of the Dog Lipocalin Allergen Can f 4 and the Development of a Sandwich ELISA for Its Quantification

• Rytkonen-Nissinen M
• Saarelainen S
• Randell J
• Hayrinen J
• Kalkkinen
• Virtanen T

• KMID: 2166686
• DOI: http://doi.org/10.4168/aair.2015.7.4.384

Abstract

PURPOSE
Divergent results on the IgE reactivity of dog-allergic subjects to Can f 4 have been reported. The aim of this study was to evaluate the significance of Can f 4 in dog allergy and to develop an immunochemical method for measuring Can f 4 content in environmental samples.
METHODS
We purified the natural dog allergen Can f 4 from a dog dander extract by monoclonal antibody-based affinity chromatography and generated its variant in a recombinant form. Sixty-three dog-allergic patients and 12 nonallergic control subjects were recruited in the study. The IgE-binding capacity of natural Can f 4 and its recombinant variant was assessed by ELISA, immunoblotting, and skin prick tests (SPT).
RESULTS
Eighty-one percent of the dog-allergic patients showed a positive result to the immunoaffinity-purified natural Can f 4 in IgE ELISA, but only 46% in IgE immunoblotting. Respective results with the recombinant Can f 4 variant were 54% and 49%. SPT results reflected those obtained in ELISA and immunoblotting. The overall IgE reactivity of the immunoaffinity-purified natural Can f 4 was found to depend strongly on the integrity of the allergen's conformation. A sandwich ELISA based on monoclonal antibodies was found to be functional for measuring Can f 4 in environmental samples.
CONCLUSIONS
Can f 4 is a major allergen of dog together with Can f 1 and Can f 5. In combination with other dog allergens, it improves the reliability of allergy tests in dog allergy.

Keyword

Allergen; Can f 4; Canis familiaris; dog; IgE; lipocalin

MeSH Terms

Allergens
Animals
Antibodies, Monoclonal
Chromatography, Affinity
Dander
Dogs*
Enzyme-Linked Immunosorbent Assay*
Humans
Hypersensitivity
Immunoblotting
Immunoglobulin E*
Lipocalins*
Skin
Allergens
Antibodies, Monoclonal
Immunoglobulin E
Lipocalins

Figure

• 

  Fig. 1 Monoclonal antibodies and a dog-allergic patient's serum IgE detect the Can f 4 allergen in the dog dander extract (ALK Abelló). Lane 1, mAb 41G; lane 2, mAb 26D; lane 3, mAb 48F; lane 4, patient serum (tested earlier to be Can f 1- and Can f 2-negative).

• 

  Fig. 2 IgE reactivity to the Can f 4. (A) Serum IgE reactivity of 63 dog-allergic patients to nCan f 4, rCan f 4 and murCan f 4 in ELISA. The prevalence of IgE reactivity to the preparations was 81%, 54% and 62%, respectively. There were no statistically significant differences in the IgE level between the allergen preparations (P>0.05, Two-tailed t-test). Horizontal lines represent the mean values and the dotted lines the cut-off values (mean OD value of healthy control subjects plus 3 SD) for each ELISA test. OD, optical density. (B) IgE prevalence to nCan f 4 and rCan f 4 among Finnish (gray and blue, respectively) and Spanish (orange and green bars, respectively) dog-allergic patients assayed with ELISA and Western blotting. The cut-off for a positive ELISA reaction was defined as the mean OD of the healthy control subjects plus 3 SD. n, natural; r, recombinant; mu, multimeric.

• 

  Fig. 3 ELISA inhibition analyses. Varying concentrations of natural Can f 4 (open symbols) or recombinant Can f 4 (closed symbols) were used to inhibit the binding of mAbs A or serum IgE B to the plate-bound counterpart allergen. (A) mAbs 26D (circles), 41G (triangles) and 48F (tetragons) were preincubated with the inhibitors and the residual antibody binding to the counterpart allergen was measured. (B) Three individual patient serum samples (circles, triangles, and tetragons) were preincubated with the inhibitors and the residual IgE binding to the counterpart allergen was measured. Data are expressed as percentages relative to the binding of an antibody without an inhibitor.

• 

  Fig. 4 ELISA inhibition of IgE binding to the natural Can f 4 allergen by lipocalin proteins. A pooled serum sample from six dog-allergic patients with IgE reactivity to nCan f 4 was preincubated with recombinant lipocalin allergens (rCan f 1, rCan f 2, rEqu c 1, rMus m 1, rBos d 2), or nCan f 4 as a positive inhibition control, or rTL and rPsoriasin as negative controls, and the residual IgE binding to the allergen was measured. n, natural; r, recombinant.

• 

  Fig. 5 Can f 4-specific mAbs and the sandwich ELISA for the quantification of Can f 4. (A) The epitope specificity of the three Can f 4-specific mAbs analyzed by competitive ELISA. Binding of the biotinylated mAbs to nCan f 4 was inhibited with the same mAbs which were not biotinylated. Percentages of inhibition are indicated. (B) The linear part of the recombinant Can f 4 standard curve is between 10 and 1,000 ng/mL in the sandwich ELISA. (C) Can f 4 concentrations of experimental home dust samples measured by the mAb-based sandwich ELISA. Horizontal lines represent the mean values.

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