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1000
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Abstract/Summary
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OBJECTIVE: The aim of the present investigation was to determine the level of the active form of the endogenous protein Antisecretory Factor (actAF), and also the active form of AF immunoreactive molecules in the affinity purified egg yolk. DESIGN: Determination of AF in affinity purified egg yolk by means of in vivo and in vitro methods. SETTING: Sahlgrenska University Hospital and Chalmers University of Technology, both units located in Gothenburg, Sweden. The farm housing the egg-laying hens is situated some 10 miles west to Stockholm. SAMPLES: Egg yolk collected weekly from hens subjected to AF-stimulating feed for up to 18 weeks. METHODS AND MAIN OUTCOME MEASURES: The methods used were:
• A) The in vivo rat ligated jejunal loop assay.
• B) Two variations of ELISA, i.e.
o 1.
An indirect ELISA using a polyclonal antibody against AF-16 antibodies.
o 2.
A competitive enzyme immunoassay for detection of the peptide AF-16 and also related immunoreactive molecules.
• C) Matrix-Assisted Laser Desorption Ionization Mass Spectrometry, (MALDI-MS).
The numeric variables registered represents:
• A) The rat jejunal ligated loop assay demonstrates the influence of AF on the in vivo secretory response (mg/cm) to cholera toxin challenge. The ligated loop is some 12–15 cm long and placed on the mid part of jejunum, and the cholera toxin induced secretion is registered after a 5 h long challenge period.
• B) The indirect ELISA method demonstrates the relative concentration of immunogenic AF peptides/AF-16 peptides and also related immunoreactive compounds by means of absorbance values, while the values of the competitive immunoassay represent the concentration of AF-16 peptides including similar immunoreactive peptides in ng/ml.
• C) The MALDI-MS method provide information about the concentration of the AF-16 peptide down to nanogram per ml. levels after mass spectrometry analysis of the sample. RESULTS: All methods revealed similar results by demonstrating a continuous increase over time in the collected egg yolk samples. Thus, low AF activity was registered in egg yolk collected in the period of 1–10 weeks of AF-stimulated feeding, significantly higher AF values was registered in yolk collected between 10 and 15 weeks of feeding, while maximal AF concentration was determined after 15 weeks of feeding. Thus, in the period between 15 and 18 weeks of stimulated AF-feeding, no further increase of the endogenous AF activity could be registered despite continuous AF-stimulated feeding. CONCLUSION: During the period of AF-stimulated feeding of the egg laying hens the registration of AF concentration in the affinity purified egg yolk samples must be continuously registered over time. The various methods used for determination of AF concentration in the affinity purified egg yolk might all serve as tools in order to achieve the optimal concentration of active AF. Together, these methods will provide information about the optimal AF concentration in the final product consisting of spray dried egg yolk (Salovum®) used for disease treatment.
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