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

  • Shengfa He
  • Xin Li
  • Yong Wu
  • Shandong Wu
  • Zhihua Wu
  • Anshu Yang
  • Ping Tong
  • Juanli Yuan
  • Jinyan Gao
  • Hongbing Chen
Research Paper
  • 29 Downloads

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.

Graphical abstract

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

Keywords

Bovine β-lactoglobulin Sandwich ELISA Monoclonal antibody IgE epitope Covalent immobilization Gold nanoparticle 

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

Notes

Acknowledgments

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

Compliance with ethical standards

Conflict of interest

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.

Supplementary material

216_2018_1019_MOESM1_ESM.pdf (183 kb)
ESM 1 (PDF 183 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Shengfa He
    • 1
    • 2
    • 3
  • Xin Li
    • 1
    • 2
  • Yong Wu
    • 1
    • 3
  • Shandong Wu
    • 4
  • Zhihua Wu
    • 1
    • 3
  • Anshu Yang
    • 1
    • 3
  • Ping Tong
    • 1
  • Juanli Yuan
    • 1
    • 5
  • Jinyan Gao
    • 1
    • 2
  • Hongbing Chen
    • 1
    • 3
  1. 1.State Key Laboratory of Food Science and TechnologyNanchang UniversityNanchangChina
  2. 2.School of Food Science & TechnologyNanchang UniversityNanchangChina
  3. 3.Jiangxi-OAI Joint Research InstituteNanchang UniversityNanchangChina
  4. 4.Hangzhou Zheda Dixun Biological Gene Engineering Co., Ltd.HangzhouChina
  5. 5.School of Pharmaceutical ScienceNanchang UniversityNanchangChina

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