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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

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Abstract

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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Abbreviations

ALA:

Bovine α-lactalbumin

APTES:

3-Aminopropyltriethoxysilane

AuNPs:

Colloidal gold nanoparticles

BLG:

Bovine β-lactoglobulin

CN:

Bovine casein

EDC:

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

mAb:

Monoclonal antibody

MES:

2-(N-Morpholino)ethanesulfonic acid

MW:

Molecular weight

pAb-BLG:

Rabbit anti-β-lactoglobulin polyclonal antibody

PAGE:

Polyacrylamide gel electrophoresis

PBS:

Phosphate-buffered saline

PBST:

PBS containing 0.1% Tween-20

sELISA:

Sandwich enzyme-linked immunosorbent assays

UHT-milk:

Ultra heat treated-milk

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Acknowledgments

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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Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, Jiangxi, China

    Shengfa He, Xin Li, Yong Wu, Zhihua Wu, Anshu Yang, Ping Tong, Juanli Yuan, Jinyan Gao & Hongbing Chen

  2. School of Food Science & Technology, Nanchang University, Nanchang, 330031, Jiangxi, China

    Shengfa He, Xin Li & Jinyan Gao

  3. Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang, 330047, Jiangxi, China

    Shengfa He, Yong Wu, Zhihua Wu, Anshu Yang & Hongbing Chen

  4. Hangzhou Zheda Dixun Biological Gene Engineering Co., Ltd., Hangzhou, 310052, Zhejiang, China

    Shandong Wu

  5. School of Pharmaceutical Science, Nanchang University, Nanchang, 330006, Jiangxi, China

    Juanli Yuan

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  1. Shengfa He
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  2. Xin Li
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  3. Yong Wu
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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). https://doi.org/10.1007/s00216-018-1019-5

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  • DOI: https://doi.org/10.1007/s00216-018-1019-5

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