Egg allergy antigen
an egg allergy and antigen technology, applied in the field of new antigens of egg allergies, can solve the problems of insufficient detection rate of patients by measuring allergen components, low diagnostic efficiency,
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example 1
[0226]Proteins contained in egg were investigated using a two-dimensional electrophoresis method described below.
[0227]Protein Extraction
[0228]Raw chicken eggs were separated into egg white and egg yolk.
[0229]The extraction and the purification of proteins contained in the egg white and the egg yolk were performed as follows. A solubilization agent was added to the egg white to extract proteins and then water or a urea buffer was added to obtain a protein extract. The composition of the urea buffer is as follows.
[0230]30 mM Tris
[0231]2 M Thiourea
[0232]7 M Urea
[0233]4% (w / v) CHAPS:
[0234]3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate
[0235]A proper quantity of dilute hydrochloric acid
Distilled water was added and the total volume was adjusted to 100 mL. PH was 8.5. Subsequently, 25 μg each in terms of protein weight was mixed to obtain an extract. A surfactant buffer (Mammalian Lysis Buffer (MCLI), SIGMA) was added as a solubilization agent to the egg yo...
example 2
ation of Antigens by Immunoblotting (1)
[0275]Identification of antigens by immunoblotting was carried out by taking all the steps up to the step of “Second-dimensional SDS-PAGE” as described above in Example 1, followed by the steps of “Transfer to membrane”, “Immunoblotting” and “Analysis” as described below.
[0276]Transfer to Membrane
[0277]Transfer to membrane was done using the following transfer system and transfer buffer.
Transfer system: XCell SureLock Mini-Cell and XCell II Blot Module (produced by Life Technologies)
Transfer buffer: NuPAGE Transfer Buffer (X20) (produced by Life Technologies), used in a form diluted 20-fold with milliQ water.
[0278]To be specific, proteins in the two-dimensional electrophoresis gels were transferred to a membrane (PVDF membrane) according to the following procedure.
[0279](1) The PVDF membrane was immersed in 100% methanol followed by milliQ water, and then moved into the transfer buffer to hydrophilize the PVDF membrane.
[0280](2) After sponge, f...
example 3
trometry and Identification of Antigens (1)
[0288]The amino acid sequences of the antigens that form the three protein spots of Example 2 were identified by mass spectroscopy.
[0289]To be specific, protein extraction and mass spectroscopy were done by the following procedure.
(1) Egg yolk of raw chicken egg was subjected to protein extraction, two-dimensional electrophoresis and transfer to membrane by following the procedures described in Examples 1 and 2, and the resulting membrane was stained by shaking in a solution of 0.008% Direct blue in 40% ethanol and 10% acetic acid.
(2) Then, the membrane was decolorized by repeating a 5-minute treatment with 40% ethanol and 10% acetic acid three times, washed with water for 5 minutes, and then dried by air.
(3) A protein spot of interest was cut out with a clean cutter blade and put into a centrifugal tube. The cut membrane was subjected to hydrophilization with 50 μL of methanol, followed by washing with 100 μL of water twice and then centri...
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