Monoclonal antibodies to three distinct epitopes on human IgE: Their use for determination of allergen-specific IgE

Background: Lipid transfer proteins (LTPs) are relevant fruit allergens, recently proposed as model plant food allergens. No citrus fruit allergen has been characterized to date. We sought to identify and isolate citrus fruit LTPs and to explore their relevance in orange allergy. Methods: Twenty-seven patients, showing mainly oral allergy syndrome after orange ingestion, as well as positive prick responses and serum-specific IgE levels to orange, were selected. Natural orange and lemon LTPs, as well as a recombinant orange LTP isoform expressed in Pichia pastoris, were isolated by chromatographic methods and characterized by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionizaion mass spectrometry, and DNA sequencing of the corresponding cDNA in the case of the recombinant allergen. Specific IgE determination, immunodetection, ELISA-inhibition assays and in vivo skin prick tests (SPTs) were performed with all three purified allergens and with the major peach LTP allergen, Pru p 3. Results: The natural allergens purified from orange (nCit s 3) and lemon (nCit l 3) showed very similar N-terminal amino acid sequences (18 out of 20 identical residues), typical of LTPs, and molecular masses of 9,610 and 9,618 Da, respectively. The recombinant orange isoform (rCit s 3) expressed in P. pastoris (16 out of 20 residues identical to its natural counterpart in the N-terminal region) presented 92 amino acid residues and 9,463 Da, and 67% sequence identity with rPru p 3. Of the 27 sera analyzed, specific IgE to the purified allergens was found in 54% for nCit l 3, 48% for nCit s 3, 46% for rCit s 3 and 37% for rPru p 3. Positive SPT responses were obtained in 7 out of 26 patients tested for nCit s 3, 3 out of 8 for nCit l 3 and 10 out of 26 for nPru p 3. ELISA-inhibition assays showed an equivalent IgE-binding pattern for the natural and recombinant orange LTPs, and IgE cross-reactivity among the purified orange, lemon and peach LTP allergens. Conclusions: Members of the LTP allergen family are involved in allergy to oranges, displaying positive in vitro and in vivo reactions in 30–50% of the patients studied. Both orange and lemon allergens show cross-reactivity with the major peach allergen Pru p 3.

Section: Methodsmentioning

confidence: 99%

Lipid Transfer Proteins and Allergy to Oranges

Ahrazem¹,

Ibáñez

López‐Torrejón³

et al. 2005

“…The proteins separated by SDS-PAGE were transferred onto nitrocellulose membranes (0.4 × 7 cm) as described by Towbin et al [13]. After blocking with 1% (w/v) bovine serum albumin (BSA), 0.1% (v/v) Tween 20 in PBS (PBS-BSA-Tween), immunodetection of IgE-binding proteins was achieved by incubating with a 1/3 dilution of a pool of sera from 20 olive pollen-allergic patients, followed by sequential incubations with a 1/3,000 dilution of mouse anti-human IgE mAb HE-2 ascites fluid [14] and then with rabbit anti-mouse immunoglobulin antibodies conjugated with horseradish peroxidase (1/5,000 dilution, Dako, Glostrup, Denmark). IgE-binding proteins were detected by enhanced chemiluminescence following the manufacturer’s instructions (ECL, Amersham Biosciences).…”

Section: Methodsmentioning

confidence: 99%

Variability of Ole e 9 Allergen in Olive Pollen Extracts: Relevance of Minor Allergens in Immunotherapy Treatments

Duffort

Palomares

Lombardero

et al. 2006

Background: Clustered severe adverse reactions to immunotherapy with olive pollen extracts have been occasionally reported in areas where olive trees are extensively grown. Allergic patients from these areas, in addition to the major olive pollen allergen Ole e 1, frequently recognize a recently described allergen, Ole e 9. Objective: We aimed to develop an immunoassay to measure Ole e 9 concentration and to study the variability of this allergen in olive pollen extracts. Methods: Monoclonal antibodies (mAb) to Ole e 9 were produced from mice immunized with the pure allergen. One of these mAbs was used to develop a sandwich ELISA with an anti-olive pollen extract rabbit serum as the tracer. Olive pollen batches from several suppliers were analyzed using this method. These batches were also analyzed for Ole e 1 content and biological activity. Results: A 10-fold variation between the extreme values was found for the biological activity of the batches analyzed. Ole e 1 concentration showed a 25-fold variation. Variability of Ole e 9 concentration was extremely high, up to 161 times. The ratio Ole e 1/Ole e 9 varied in a range from 0.6 to 390.4. Conclusion: The availability of a mAb-based ELISA for Ole e 9 made it possible for us to detect an important source of variability in olive pollen batches. This variability may be the cause of outbreaks of adverse reactions in the course of immunotherapy treatments, which have sometimes been observed among olive-allergic patients living in areas with very high levels of airborne olive pollen.

“…For Western blot, separated proteins were electrotransferred to nitrocellulose membranes according to the method of Towbin et al [27]. Immunodetection of IgE-binding proteins using a serum pool from C. dactylon -allergic patients was accomplished with 125 I-anti-human IgE mAb HE2 [28]. …”

Section: Methodsmentioning

confidence: 99%

Monoclonal Antibody-Based ELISA to Quantify the Major Allergen of <i>Cynodon dactylon</i> (Bermuda Grass) Pollen, Cyn d 1

Duffort

Calabozo

González

et al. 2004

Background: Pollen of Bermuda grass (Cynodon dactylon) is an important cause of pollinosis in many areas of the world. Most patients show sensitivity to the major allergen Cyn d 1, a glycoprotein composed of a number of isoforms with a molecular mass of 31–32 kDa. The aim of this work was to develop a monoclonal antibody (mAb)-based ELISA to quantify Cyn d 1, and to assess the correlation of the allergen content with the biological activity of C. dactylon pollen extracts. Methods:After fusion of myeloma cells with spleen cells from a BALB/c mouse immunized with C. dactylon pollen extract, Cyn d 1-specific mAbs secreting hybridomas were selected, and the antibodies characterized. One of them (4.4.1) was used as the capture antibody in an ELISA method for Cyn d 1 quantitation. An anti-Cyn d 1 rabbit serum was used as the second antibody. Cyn d 1 was purified by immunoaffinity chromatography with mAb 4.4.1, characterized, and used as the standard in the assay. Results: The identity, purity and isoallergen composition of affinity-purified Cyn d 1 was confirmed by N-terminal amino acid sequencing, SDS-PAGE, Western blot and 2D electrophoresis. The Cyn d 1 ELISA is highly specific and sensitive, with a detection limit of 0.24 ng/ml and a linear range of 1.1–9.2 ng/ml. An excellent correlation was found when the content of Cyn d 1, measured in 16 different extracts, was compared with the allergenic activity of the same extracts determined by RAST inhibition. Conclusions: The results prove the usefulness of the Cyn d 1 ELISA for the standardization of C. dactylon-allergen products on the basis of major allergen content.