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Chicken IgY- standard curve producing method and detection method using rabbit anti-chicken IgY- nanogold immune scattering probe

A standard curve and nano-gold technology, which is applied in the field of detection of chicken yolk immunoglobulin, can solve the problems of difficult and accurate quantitative detection by enzyme-linked immunoassay, cumbersome operation, expensive instruments, etc., and is conducive to popularization and application. The effect of high accuracy

Inactive Publication Date: 2015-12-09
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, ELISA is difficult to achieve accurate quantitative detection (Song Hongxin et al. 2004); high performance liquid chromatography is complicated to operate and the instruments used are relatively expensive (Chen Weifeng et al. 2012); electrochemical methods involve electrode preparation and the immobilization of detection substances on the electrode surface , the operation is more cumbersome (Bottarietal2014)

Method used

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  • Chicken IgY- standard curve producing method and detection method using rabbit anti-chicken IgY- nanogold immune scattering probe
  • Chicken IgY- standard curve producing method and detection method using rabbit anti-chicken IgY- nanogold immune scattering probe
  • Chicken IgY- standard curve producing method and detection method using rabbit anti-chicken IgY- nanogold immune scattering probe

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Experimental program
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Effect test

Embodiment 1

[0037] The preparation of embodiment 1 nano gold

[0038] Using trisodium citrate as a reducing agent, nano colloidal gold with uniform particles and uniform distribution was prepared. All glassware used must be in aqua regia (HNO 3 ︰HCl=1︰3,v / v) for 48 hours, take it out, wash it with plenty of tap water first, then wash it with double distilled water and dry it for later use.

[0039] The synthesis of gold nanoparticles is carried out according to the published methods of Kim et al.:

[0040] 1) Take 100mL of 0.1mg / mL chloroauric acid aqueous solution and heat it to 95°C until it boils. Similarly, heat 5mL of 10mg / mL sodium citrate solution to 95°C until boiling;

[0041] 2) Under the magnetic stirrer stirring at 120rpm, the above-mentioned sodium citrate solution was added dropwise into the aqueous chloroauric acid solution, and continued to boil for 10 minutes. Nanogold will form within 2-3 minutes. The color of the mixed solution changed from dark blue to bright red;...

Embodiment 2

[0043] Example 2 Preparation of AuNPs-Anti-IgY Immunoscattering Probe

[0044] The gold nanoparticles were combined with rabbit anti-chicken IgY antibodies to form IgY-specific immunoscattering probes. Specific steps are as follows:

[0045] 1 mL of 0.002 mg / mL Anti-IgY was added to 10 mL of 47.8 μg / mL AuNPs solution with appropriate pH under the action of magnetic stirring. After stirring for 10 minutes, 200 μL of polyethylene glycol 20000 (PEG20000) with a mass fraction of 3% was added as a stabilizer, and after stirring was continued for 15 minutes, it was stored at 4°C until use.

Embodiment 3

[0046] Embodiment 3 establishes the standard curve of rabbit anti-chicken IgY-nanogold (Anti-IgY-AuNPs) immunoscattering probe to detect IgY

[0047] Mix 0.5mL of Anti-IgY-AuNPs solution, IgY and 2.5mL of 0.02mol / L PBS buffer with a pH of 4.9, and then dilute to 5mL with double distilled water to obtain eight mixed solutions, among which, the mixed solution, IgY The concentrations were 4ng / mL, 10ng / mL, 20ng / mL, 40ng / mL, 80ng / mL, 120ng / mL, 160ng / mL and 200ng / mL.

[0048] The scattering spectra were measured with a RF-5301 fluorescence spectrophotometer from Shimadzu, Japan. Set synchronous scan △λ=0(λ em =λ ex ), the excitation wavelength is set to 225nm, the emission wavelength is set to 225-800nm, the slit width of both excitation and emission light is 5nm, and the response time is set to 0.25s. The obtained product was characterized by scattering spectroscopy ( figure 1 ).

[0049] The results show that the maximum scattering wavelength of AuNPs is at 564nm. The RRS in...

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Abstract

The present invention discloses a chicken IgY- standard curve producing method and detection method using a rabbit anti-chicken IgY- nanogold immune scattering probe. The method is as follows: preparing water-soluble well dispersed nanogold, the nanogold and a rabbit anti-chicken IgY antibody are conjugated into a specific immune probe used directly for IgY detection, and a new method for rapid and sensitive quantitative detection of IgY is established. The technology provides a new method for detecting IgY, has the advantages of high sensitivity, high accuracy, low detection limit, strong anti-interference ability, easy operation, and low equipment requirements, and is conducive to the promotion and application. The method can be applied to rapid and sensitive quantitative detection of IgY in egg products, health food, and other related products, and has broad application prospects.

Description

technical field [0001] The invention relates to a method for detecting chicken yolk immunoglobulin, in particular to a method and a detection method for detecting chicken IgY-standard curve by using rabbit anti-chicken IgY-nano gold immune scattering probe. Background technique [0002] Chicken yolk immunoglobulin (IgY) comes from oviparous animals, and most of the antigens studied at this stage originate from mammals. Therefore, compared with mammals, oviparous animals have a higher degree of reactivity to mammalian antigens and a higher rate of special antibody formation. Therefore, IgY can be used as a source of better antibody production and has advantages in immunology. In addition, the development of IgY health food has also been studied in recent years. Antibody IgY can combine with bacterial antigens in the body, thereby inhibiting the chance of contact between bacteria and intestinal wall cells, providing the body with passive immunity to resist the harm of antigen...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N21/64
CPCG01N33/6854G01N21/6402
Inventor 蔡朝霞王琪马美湖
Owner HUAZHONG AGRI UNIV
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