Florine J. van Milligen

IgE epitopes on the cat (Felis domesticus) major allergen Fel d I: A study with overlapping synthetic peptides

BACKGROUND The major cat allergen Fel d I is composed of two disulfide-linked polypeptide chains, chain 1 (70 amino acid residues) and chain 2 (92 amino acid residues). Reduction and alkylation of Fel d I eliminates almost all antigenic and allergenic activity, and detection of linear epitopes with synthetic peptides is therefore not expected. METHODS We synthesized synthetic peptides of both chains of about 14 amino acid residues, overlapping by 7 residues. The peptides were coupled to Sepharose (Pharmacia, Uppsala, Sweden) and tested with sera of patients with cat allergy. RESULTS Three peptides showed specific binding of human IgE, residues 25-38 and 46-59 of chain 1 and residue 15-28 of chain 2. IgE binding was inhibited by Fel d I and the corresponding peptide. Of 61 patients with cat allergy tested, 65% showed IgE binding to at least one of the peptides; 46% showed IgE binding to peptide 25-38, 11% to peptide 46-59, and 28% to peptide 15-28. Each peptide was recognized by only one of the 78 patients with negative RAST results. By affinity chromatography with peptide-Sepharose anti-Fel d I antibodies were isolated, also confirming the specificity of IgE binding to the peptides. The percentage of IgE antibodies against Fel d I reactive with the peptides varied with the serum and the peptide-Sepharose used and ranged from 2% to 55%. CONCLUSIONS Because the affinity of IgE binding to the peptides was very low and only serum samples with high titers of Fel d I-specific IgE antibodies (RAST 4+/5+) showed significant binding, these peptides are not suitable for diagnostic purposes. However, the peptides are useful tools for comparing IgE and IgG responses and for studying the relationship to the T-cell epitopes on this molecule.

Presence of Felis domesticus allergen I in the cat's salivary and lacrimal glands

Most of the IgE response in persons allergic to cats is directed against the cat allergen Fel d I (Felis domesticus allergen I). Although the presence of Fel d I in saliva has been demonstrated, the source of Fel d I is not completely known. We measured Fel d I concentrations in the cats lacrimal and salivary glands and in the excretes lacrimal fluid, milk and saliva. The concentration of Fel d I, determined by a radioimmunoassay, in the lacrimal gland was 390-780 mU/g tissue and in the sublingual gland 610 mU/g tissue. In the parotid gland and especially the submandibular gland only low concentrations were found, respectively 70-210 and 40-50 mU/g tissue. In lacrimal fluid 6.8-14 U/ml Fel d I was detected, which is comparable to saliva, 4.2-7.0 U/ml, whereas in cats milk only 0.33 U/ml Fel d I was found. Immunohistochemical studies with monoclonal antibody directed against Fel d I (anti-Fel d I) showed the presence of Fel d I in the serous cells of the lacrimal gland. Thus, our results demonstrate that in particular the lacrimal gland might be a useful Fel d I source.

Epitope mapping of the cat (Felis domesticus) major allergen Fel d I by overlapping synthetic peptides and monoclonal antibodies against native and denatured Fel d I

The major cat allergen Fel d I is a homodimer of which each monomer consists of two disulfide‐linked polypeptide chains: chain 1 (70 amino acid residues) and chain 2 (92 amino acid residues). Twenty‐one synthetic peptides of 14 amino acid residues length, overlapping by seven residues and spanning the entire sequence of both chains, were synthesized. These peptides were coupled to CNBr‐activated Sepharose‐4B and used as solid‐phase antigens in epitope‐mapping studies with monoclonal antibodies against native and reduced/alkylated Fel d I.

IgE and IgG4 binding to synthetic peptides of the cat (Felis domesticus) major allergen Fel dI.

Specificities of IgE and IgG4 antibodies in 12 cat-allergic patients were compared with respect to their reactivity towards 3 IgE-binding synthetic peptides of Felis domesticus allergen 1 (Fel dI): peptides 25-38 and 46-59 of chain 1 and peptide 15-28 of chain 2. Peptides were coupled to Sepharose and anti-Fel dI antibodies were isolated by affinity chromatography. Fel dI-specific IgE- and IgG4 antibody activity in the peptide eluates was measured using Fel dI binding assays. Fel dI-specific IgE/IgG4 ratios in the eluates from peptide-Sepharose were determined and compared with the IgE/IgG4 ratios in the eluates from Fel dI-Sepharose. The mean ratio Fel dI-specific IgE/IgG4 in the peptide eluates (0.84; range 0.06-4.6) was significantly higher than the mean ratio in the eluates from Fel dI-Sepharose (0.31; range 0.13-1.1), demonstrating a higher reactivity of IgE antibodies with the peptides, compared to IgG4. These results indicate differences between the B cells producing IgE antibodies and the B cells producing IgG4 antibodies.

Differences between specificities of IgE and IgG4 antibodies: studies using recombinant chain 1 and chain 2 of the major cat allergen Felis domesticus (Fel d) I

IgE‐ and IgG4 antibodies were compared for reactivity with recombinant chain 1 and chain 2 of the cat allergen Felis domesticus (Fei d) I. Recombinanl chain 1 and chain 2 were coupled to sepharose and tested in IgE‐ and IgG4 radioallergosorbent test (RAST) experiments. Substantial IgE‐ and IgG4 binding was found. The fraction of Pel d I‐specific antibody that bound to the recombinant chains was calculated. For chain 1, the mean value of this fraction was 0.30 for IgE and 0.23 for lgG4 (P= 0.05). For chain 2, the mean value of this fraction was 0.19 for IgE and 0.13 for IgG4 (P= 0.02). These results indicate that differences in fine specificity exist between IgE and IgG4 antibodies. Moreover, these findings support our results with chemically prepared peptides derived from these two chains and suggest that the B cells producing IgE antibodies are more likely to recognize a less ‘native’ form of Pel d I, compared with IgG4.

IgE and IgG4 binding to the ocelot variant of the cat (Felis domesticus) major allergen Fel d l

The cross-reactivity of human IgE and IgG4 antibodies of 22 cat-allergic patients with the ocelot variant of Fel d I was studied. Throughout the Felidae species, this allergen, found in cat saliva, dander, sebaceous glands, and lacrimal glands (1-3, 5) is conserved, as shown in radioallergosorbent test (RAST) inhibition assays with affinity-purified Fel d I and hair extracts from various members of the Felidae family (6). The Felidae species we used in these experiments originate from different lineages (4). Because of the phylogenetic distance among these species, they are useful tools to study the specificities of IgE and IgG4 responses. Other feline species, e.g., tiger and lion, were found to be so closely related as to be almost immunologically identical, and therefore less suited for this type of investigation.