Detection of alkaline phosphatase by competitive indirect ELISA using immunoglobulin in yolk (IgY) specific against bovine milk alkaline phosphatase

Chao Cheng Chen, Yu Chang Tai, Szu-Chuan Shen, Yann Ying Tu, Ming Chang Wu, Hung Min Chang

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Immunoglobulin in yolk (IgY) (with a titer of 1.3 × 106) specific against bovine milk (BM) alkaline phosphatase (ALP) was obtained by intramuscularly immunizing hens on the thigh and was used as the primary antibody to conduct competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) to determine BMALP in ALPs from BM and Escherichia coli sources. A relationship between the ELISA value and the BMALP level (0.01-10 μg/mL) in whole milk (R2 = 0.9019) or in skimmed milk (R2 = 0.9402) was observed. The maximal inhibition (%) of BMALP on the microtiter plate by free BMALP at 10 μg/mL whole milk (3.89 mU/μg BMALP) was about 50%, while no inhibition (%) of BMALP by free E. coli ALP at concentrations between 0.01 to 10 μg/mL (60 mU/μg E. coli ALP) was determined. At BMALP levels higher than 0.1 μg/mL, CI-ELISA was proved to be effective in differentiating between BMALP and E. coli ALP and quantifying BMALP in whole milk or skimmed milk in the presence of E. coli ALP with an activity of 0.6 U/mL. Higher inhibition (about 70%) of BMALP on the microtiter plate by free BMALP in diluted (101-104 fold) milk samples was observed. The optimal conditions for CI-ELISA in determining BMALP (0.1-10 μg/mL) from ALPs in milk samples were using 103-fold diluted crude IgY specific against BMALP as primary antibody and 103-fold diluted goat anti-chicken IgG-ALP conjugate as the secondary antibody.

Original languageEnglish
Pages (from-to)213-220
Number of pages8
JournalFood Chemistry
Volume95
Issue number2
DOIs
Publication statusPublished - 2006 Mar 1

Fingerprint

immunoglobulins
Alkaline Phosphatase
alkaline phosphatase
Immunoglobulins
Milk
Enzyme-Linked Immunosorbent Assay
enzyme-linked immunosorbent assay
milk
Escherichia coli
whole milk
Immunosorbents
antibodies
Assays
Antibodies
Enzymes
thighs
hens
goats
chickens
Thigh

Keywords

  • Alkaline phosphatase
  • Bovine milk
  • Competitive ELISA
  • Escherichia coli
  • Immunoglobulin in yolk

ASJC Scopus subject areas

  • Analytical Chemistry
  • Food Science

Cite this

Detection of alkaline phosphatase by competitive indirect ELISA using immunoglobulin in yolk (IgY) specific against bovine milk alkaline phosphatase. / Chen, Chao Cheng; Tai, Yu Chang; Shen, Szu-Chuan; Tu, Yann Ying; Wu, Ming Chang; Chang, Hung Min.

In: Food Chemistry, Vol. 95, No. 2, 01.03.2006, p. 213-220.

Research output: Contribution to journalArticle

Chen, Chao Cheng ; Tai, Yu Chang ; Shen, Szu-Chuan ; Tu, Yann Ying ; Wu, Ming Chang ; Chang, Hung Min. / Detection of alkaline phosphatase by competitive indirect ELISA using immunoglobulin in yolk (IgY) specific against bovine milk alkaline phosphatase. In: Food Chemistry. 2006 ; Vol. 95, No. 2. pp. 213-220.
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abstract = "Immunoglobulin in yolk (IgY) (with a titer of 1.3 × 106) specific against bovine milk (BM) alkaline phosphatase (ALP) was obtained by intramuscularly immunizing hens on the thigh and was used as the primary antibody to conduct competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) to determine BMALP in ALPs from BM and Escherichia coli sources. A relationship between the ELISA value and the BMALP level (0.01-10 μg/mL) in whole milk (R2 = 0.9019) or in skimmed milk (R2 = 0.9402) was observed. The maximal inhibition ({\%}) of BMALP on the microtiter plate by free BMALP at 10 μg/mL whole milk (3.89 mU/μg BMALP) was about 50{\%}, while no inhibition ({\%}) of BMALP by free E. coli ALP at concentrations between 0.01 to 10 μg/mL (60 mU/μg E. coli ALP) was determined. At BMALP levels higher than 0.1 μg/mL, CI-ELISA was proved to be effective in differentiating between BMALP and E. coli ALP and quantifying BMALP in whole milk or skimmed milk in the presence of E. coli ALP with an activity of 0.6 U/mL. Higher inhibition (about 70{\%}) of BMALP on the microtiter plate by free BMALP in diluted (101-104 fold) milk samples was observed. The optimal conditions for CI-ELISA in determining BMALP (0.1-10 μg/mL) from ALPs in milk samples were using 103-fold diluted crude IgY specific against BMALP as primary antibody and 103-fold diluted goat anti-chicken IgG-ALP conjugate as the secondary antibody.",
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AU - Chen, Chao Cheng

AU - Tai, Yu Chang

AU - Shen, Szu-Chuan

AU - Tu, Yann Ying

AU - Wu, Ming Chang

AU - Chang, Hung Min

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AB - Immunoglobulin in yolk (IgY) (with a titer of 1.3 × 106) specific against bovine milk (BM) alkaline phosphatase (ALP) was obtained by intramuscularly immunizing hens on the thigh and was used as the primary antibody to conduct competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) to determine BMALP in ALPs from BM and Escherichia coli sources. A relationship between the ELISA value and the BMALP level (0.01-10 μg/mL) in whole milk (R2 = 0.9019) or in skimmed milk (R2 = 0.9402) was observed. The maximal inhibition (%) of BMALP on the microtiter plate by free BMALP at 10 μg/mL whole milk (3.89 mU/μg BMALP) was about 50%, while no inhibition (%) of BMALP by free E. coli ALP at concentrations between 0.01 to 10 μg/mL (60 mU/μg E. coli ALP) was determined. At BMALP levels higher than 0.1 μg/mL, CI-ELISA was proved to be effective in differentiating between BMALP and E. coli ALP and quantifying BMALP in whole milk or skimmed milk in the presence of E. coli ALP with an activity of 0.6 U/mL. Higher inhibition (about 70%) of BMALP on the microtiter plate by free BMALP in diluted (101-104 fold) milk samples was observed. The optimal conditions for CI-ELISA in determining BMALP (0.1-10 μg/mL) from ALPs in milk samples were using 103-fold diluted crude IgY specific against BMALP as primary antibody and 103-fold diluted goat anti-chicken IgG-ALP conjugate as the secondary antibody.

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KW - Competitive ELISA

KW - Escherichia coli

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