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A novel sandwich enzyme-linked immunosorbent assay with covalently bound monoclonal antibody and gold probe for sensitive and rapid detection of bovine β-lactoglobulin


Bovine milk is a recognized allergenic food source with β-lactoglobulin (BLG) as its major allergen. Reliable detection of BLG epitopes can, therefore, be a useful marker for the presence of milk in processed food products, and for potential allergenicity. At the present, enzyme-linked immunosorbent assays (ELISA) for the detection of BLG are time-consuming and generally not specific to BLG IgE epitopes. In this study, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-activated anti-BLG IgE epitope monoclonal antibody (mAb 1G9) was covalently bound onto the KOH-treated microtiter plate surface. Using this mAb-bound plate in sandwich combination with biotinylated anti-BLG polyclonal antibody-labeled gold nanoparticles, a linear dynamic range between 31.25 and 64 × 103 ng mL−1 with a limit of detection for BLG of 0.49 ng mL−1 was obtained, which is 32 times wider and 16 times more sensitive than conventional sandwich ELISA (sELISA). Total recovery of BLG in spiked food samples was found, without matrix effects. Also in partially hydrolyzed infant formulas, the allergenic BLG residues were detected quantitatively. Compared with conventional and commercial BLG detection sELISAs, our sELISA is reliable, highly BLG epitope-specific, user-friendly, and time-saving and allows accurate detection of potentially allergenic residues in different types of processed foods. This improved sELISA protocol can be easily extended to detect other well-identified and characterized food allergens.

IgE epitope mAb-bound plate in sandwich combination with gold probe for sensitive and rapid detection of bovine β-lactoglobulin and its potentially allergenic residues

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Bovine α-lactalbumin




Colloidal gold nanoparticles


Bovine β-lactoglobulin


Bovine casein




Monoclonal antibody


2-(N-Morpholino)ethanesulfonic acid


Molecular weight


Rabbit anti-β-lactoglobulin polyclonal antibody


Polyacrylamide gel electrophoresis


Phosphate-buffered saline


PBS containing 0.1% Tween-20


Sandwich enzyme-linked immunosorbent assays


Ultra heat treated-milk


  1. Luyt D, Ball H, Makwana N, Green MR, Bravin K, Nasser SM, et al. BSACI guideline for the diagnosis and management of cow’s milk allergy. Clin Exp Allergy. 2014;44:642–72.

    Article  CAS  PubMed  Google Scholar 

  2. Spies J. Milk allergy. J Milk Food Technol. 1973;36:225–31.

    Article  Google Scholar 

  3. Hochwallner H, Schulmeister U, Swoboda I, Spitzauer S, Valenta R. Cow’s milk allergy: from allergens to new forms of diagnosis, therapy and prevention. Methods. 2014;66:22–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Taylor SL, Baumert JL, Kruizinga AG, Remington BC, Crevel RW, Brooke-Taylor S, et al. Establishment of reference doses for residues of allergenic foods: report of the VITAL Expert Panel. Food Chem Toxicol. 2014;63:9–17.

    Article  CAS  PubMed  Google Scholar 

  5. Trendelenburg V, Enzian N, Bellach J, Schnadt S, Niggemann B, Beyer K. Detection of relevant amounts of cow’s milk protein in non-pre-packed bakery products sold as cow’s milk-free. Allergy. 2015;70:591–7.

    Article  CAS  PubMed  Google Scholar 

  6. Cucu T, Jacxsens L, De Meulenaer B. Analysis to support allergen risk management: which way to go? J Agric Food Chem. 2013;61:5624–33.

    Article  CAS  PubMed  Google Scholar 

  7. Moreno Mde L, Muñoz-Suano A, López-Casado MÁ, Torres MI, Sousa C, Cebolla Á. Selective capture of most celiac immunogenic peptides from hydrolyzed gluten proteins. Food Chem. 2016;205:36–42.

    Article  CAS  PubMed  Google Scholar 

  8. Zhang H, Lu Y, Ushio H, Shiomi K. Development of sandwich ELISA for detection and quantification of invertebrate major allergen tropomyosin by a monoclonal antibody. Food Chem. 2014;150:151–7.

    Article  CAS  PubMed  Google Scholar 

  9. He SF, Li X, Gao JY, Tong P, Chen HB. Development of a H2O2-sensitive quantum dots-based fluorescent sandwich ELISA for sensitive detection of bovine β-lactoglobulin by monoclonal antibody. J Sci Food Agric. 2018;98:519–26.

    Article  CAS  PubMed  Google Scholar 

  10. He SF, Li X, Gao JY, Tong P, Chen HB. Development of sandwich ELISA for testing bovine beta-lactoglobulin allergenic residues by specific polyclonal antibody against human IgE binding epitopes. Food Chem. 2017;227:33–40.

    Article  CAS  PubMed  Google Scholar 

  11. Vashist SK, Marion Schneider E, Lam E, Hrapovic S, Luong JH. One-step antibody immobilization-based rapid and highly-sensitive sandwich ELISA procedure for potential in vitro diagnostics. Sci Rep. 2014;4:4407.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Vashist SK, Schneider EM, Luong JH. Rapid sandwich ELISA-based in vitro diagnostic procedure for the highly-sensitive detection of human fetuin a. Biosens Bioelectron. 2015;67:73–8.

    Article  CAS  PubMed  Google Scholar 

  13. Li YS, Meng XY, Zhou Y, Zhang YY, Meng XM, Yang L, et al. Magnetic bead and gold nanoparticle probes based immunoassay for beta-casein detection in bovine milk samples. Biosens Bioelectron. 2015;66:559–64.

    Article  CAS  PubMed  Google Scholar 

  14. Ciaurriz P, Fernández F, Tellechea E, Moran JF, Asensio AC. Comparison of four functionalization methods of gold nanoparticles for enhancing the enzyme-linked immunosorbent assay (ELISA). Beilstein J Nanotechnol. 2017;8:244–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Järvinen KM, Chatchatee P, Bardina L, Beyer K, Sampson HA. IgE and IgG binding epitopes on α-lactalbumin and β-lactoglobulin in cow’s milk allergy. Int Arch Allergy Immunol. 2001;126:111–8.

    Article  PubMed  Google Scholar 

  16. de Bievre P, Böttger D, Eastwood C, Hlavay J, Holmgren M, Horwitz W, et al. Eurachem Guide. Thefitness for purpose of analytical methods. A laboratory guide to method validation and related topics: second edition. 2014; (accessed July 14, 2017).

  17. Schägger H. Tricine-SDS-PAGE. Nat Protoc. 2006;1:16–22.

    Article  CAS  PubMed  Google Scholar 

  18. de Luis R, Lavilla M, Sanchez L, Calvo M, Perez MD. Development and evaluation of two ELISA formats for the detection of beta-lactoglobulin in model processed and commercial foods. Food Control. 2009;20:643–7.

    Article  CAS  Google Scholar 

  19. Badran AA, Morais S, Maquieira Á. Simultaneous determination of four food allergens using compact disc immunoassaying technology. Anal Bioanal Chem. 2017;409:2261–8.

    Article  CAS  PubMed  Google Scholar 

  20. Wu XL, He WY, Ji KM, Wan WP, Hu DS, Wu H, et al. A simple and fast detection method for bovine milk residues in foods: a 2-site monoclonal antibody immunochromatography assay. J Food Sci. 2013;78:M452–7.

    Article  CAS  Google Scholar 

  21. Ruiz-Valdepeñas Montiel V, Campuzano S, Conzuelo F, Torrente-Rodríguez RM, Gamella M, Reviejo AJ, et al. Electrochemical magnetoimmunosensing platform for determination of the milk allergen β-lactoglobulin. Talanta. 2015;131:156–62.

    Article  CAS  PubMed  Google Scholar 

  22. Eissa S, Tlili C, L’Hocine L, Zourob M. Electrochemical immunosensor for the milk allergen β-lactoglobulin based on electrografting of organic film on graphene modified screen-printed carbon electrodes. Biosens Bioelectron. 2012;38:308–13.

    Article  CAS  PubMed  Google Scholar 

  23. Wu XL, Li Y, Liu B, Feng Y, He WY, Liu ZG, et al. Two-site antibody immunoanalytical detection of food allergens by surface plasmon resonance. Food Anal Methods. 2016;9:582–8.

    Article  Google Scholar 

  24. Bonfatti V, Grigoletto L, Cecchinato A, Gallo L, Carnier P. Validation of a new reversed-phase high-performance liquid chromatography method for separation and quantification of bovine milk protein genetic variants. J Chromatogr A. 2008;1195:101–6.

    Article  CAS  PubMed  Google Scholar 

  25. Ji J, Zhu P, Pi FW, Sun C, Sun JD, Jia M. Development of a liquid chromatography-tandem mass spectrometry method for simultaneous detection of the main milk allergens. Food Control. 2017;74:79–88.

    Article  CAS  Google Scholar 

  26. Ansari P, Stoppacher N, Rudolf J, Schuhmacher R, Baumgartner S. Selection of possible marker peptides for the detection of major ruminant milk proteins in food by liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem. 2011;399:1105–15.

    Article  CAS  PubMed  Google Scholar 

  27. Chung CS, Yamini S, Trumbo PR. FDA’s health claim review: whey-protein partially hydrolyzed infant formula and atopic dermatitis. Pediatrics. 2012;130:e408–14.

    Article  PubMed  Google Scholar 

  28. Pescuma M, Hébert EM, Rabesona H, Drouet M, Choiset Y, Haertlé T, et al. Proteolytic action of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 reduces antigenic response to bovine β-lactoglobulin. Food Chem. 2011;127:487–92.

    Article  CAS  PubMed  Google Scholar 

  29. Vashist SK, Lam E, Hrapovic S, Male KB, Luong JH. Immobilization of antibodies and enzymes on 3-aminopropyltriethoxysilane-functionalized bioanalytical platforms for biosensors and diagnostics. Chem Rev. 2014;114:11083–130.

    Article  CAS  PubMed  Google Scholar 

  30. Park SJ, Jung WY. Effect of KOH activation on the formation of oxygen structure in activated carbons synthesized from polymeric precursor. J Colloid Interface Sci. 2002;250:93–8.

    Article  CAS  PubMed  Google Scholar 

  31. Ashley J, Piekarska M, Segers C, Trinh L, Rodgers T, Willey R, et al. An SPR based sensor for allergens detection. Biosens Bioelectron. 2017;88:109–13.

    Article  CAS  PubMed  Google Scholar 

  32. Curciarello R, Lareu JF, Fossati CA, Docena GH, Petruccelli S. Immunochemical characterization of Glycine max L. Merr. var Raiden, as a possible hypoallergenic substitute for cow’s milk-allergic patients. Clin Exp Allergy. 2008;38:1559–65.

    Article  CAS  PubMed  Google Scholar 

  33. Candreva ÁM, Smaldini PL, Curciarello R, Fossati CA, Docena GH, Petruccelli S. The major soybean allergen Gly m Bd 28K induces hypersensitivity reactions in mice sensitized to cow’s milk proteins. J Agric Food Chem. 2016;64:1590–9.

    Article  CAS  PubMed  Google Scholar 

  34. Mariager B, S⊘lve M, Eriksen H, Brogren CH. Bovine β-lactoglobulin in hypoallergenic and ordinary infant formulas measured by an indirect competitive ELISA using monoclonal and polyclonal antibodies. Food Agric Immunol. 1994;6:73–83.

    Article  CAS  Google Scholar 

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The work was supported by Research Project of State Key Laboratory of Food Science and Technology (No. SKLF-ZZA-201612, SKLF-ZZB-201712)

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Correspondence to Jinyan Gao or Hongbing Chen.

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All academic authors declare that they have no conflict of interest. Mr. Shandong Wu declares that he is the director of the Hangzhou Zheda Dixum Biological Gene Engineering Co., Ltd., but this company was not financially involved in the present study.

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He, S., Li, X., Wu, Y. et al. A novel sandwich enzyme-linked immunosorbent assay with covalently bound monoclonal antibody and gold probe for sensitive and rapid detection of bovine β-lactoglobulin. Anal Bioanal Chem 410, 3693–3703 (2018).

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