Allergy Asthma Immunol Res.  2017 Sep;9(5):417-422. 10.4168/aair.2017.9.5.417.

Monoclonal Antibodies to Recombinant Fag e 3 Buckwheat Allergen and Development of a Two-site ELISA for Its Quantification

• Jeong KY
• Park KH
• Lee JH
• Park JW

• KMID: 2383989
• DOI: http://doi.org/10.4168/aair.2017.9.5.417

Abstract

PURPOSE
Buckwheat is a major cause of anaphylaxis, and Fag e 3 is the key major allergen in buckwheat. However, an immunoassay system for the quantification of Fag e 3 has yet to be developed.
METHODS
We developed a 2-site enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (mAbs) produced against recombinant Fag e 3. We applied this ELISA to quantify native Fag e 3 in total buckwheat extract.
RESULTS
Four clones of mAbs were produced, and all recognized vicilin allergens not only from buckwheat, but also from peanut and walnut. However, the ELISA using these antibodies was only able to quantify Fag e 3 in the total extract after addition of 1% sodium dodecyl sulphate (SDS) and heating, which facilitated dissociation of the allergen. The detection limit of the developed 2-site ELISA was 0.8 µg/mL. The measurement of Fag e 3 in the total extract of buckwheat showed that approximately 12% of protein in total buckwheat extract was Fag e 3.
CONCLUSIONS
We have developed an ELISA system for the quantification of the group 3 buckwheat allergen, Fag e 3, specifically. This assay will be useful for standardization of buckwheat allergens and monitoring of buckwheat contamination in foods.

Keyword

Buckwheat; 2-site ELISA; Fag e 3; vicilin

MeSH Terms

Allergens
Anaphylaxis
Antibodies
Antibodies, Monoclonal*
Arachis
Clone Cells
Enzyme-Linked Immunosorbent Assay*
Fagopyrum*
Heating
Hot Temperature
Immunoassay
Juglans
Limit of Detection
Sodium
Allergens
Antibodies
Antibodies, Monoclonal
Sodium

Figure

• 

  Fig. 1 SDS-PAGE analysis of recombinant Fag e 3 (5 µg) and antibody responses to buckwheat extract. Recombinant protein was separated onto a 15% polyacrylamide gel under reducing and non-reducing conditions (A). We examined reactivity of mAbs produced against recombinant Fag e 3 to buckwheat extract by ELISA using hybridoma culture supernatants (B). SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; mAbs, monoclonal antibodies; ELISA, enzyme-linked immunosorbent assay.

• 

  Fig. 2 Recognition of native vicilin-like allergens by mAbs. We probed Fag e 3 and its homologous allergens with mAbs from extracts (10 µg) that were transferred onto a PVDF membrane after running on 15% gels that were run under reducing (A) and non-reducing (B) conditions. mAbs used to detect Fag e 3 are denoted beneath each membrane. M, molecular mass standard; B, buckwheat extract; P, peanut extract; W, walnut extract; mAbs, monoclonal antibodies; PVDF, polyvinylidene difluoride.

• 

  Fig. 3 Dose-dependent curve of recombinant Fag e 3 by a 2-site ELISA. The 2-site ELISA was quantified with 4-fold dilution of recombinant Fag e 3 from 25 µg/mL. The detection limit was 0.1 µg/mL. ELISA, enzyme-linked immunosorbent assay.

• 

  Fig. 4 Application of detergents in total buckwheat extract to facilitate native Fag e 3 quantification. One percent detergent was added to the extract (A) and to recombinant Fag e 3.

• 

  Fig. 5 Quantification of native Fag e 3 in total buckwheat extract. Addition of SDS decreased the sensitivity to recombinant Fag e 3 (A), but increased the sensitivity to native Fag e 3 in total buckwheat extract (B). The detection limit for recombinant Fag e 3 was 6 µg/mL (A). We created a dose-dependent curve of with 4-fold dilution of buckwheat extract from 1 mg/mL (B). SDS, sodium dodecyl sulphate.

• 

  Fig. 6 Heating after adding SDS to the buckwheat. Heating improves the sensitivity of the 2-site ELISA. The detection limit was 0.8 µg/mL of recombinant Fag e 3. SDS, sodium dodecyl sulphate; ELISA, enzyme-linked immunosorbent assay.

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