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Method for detecting specific immune globulin E of eriocheir sinensis

A Chinese mitten crab and immunoglobulin technology, which is applied in the cross-field of materials and biomedicine, can solve the problems of unstandardized sensitization efficiency, interference of allergy test results, high detection limit, etc.

Active Publication Date: 2018-01-30
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But it is also one of the eight types of highly allergenic foods, with an incidence rate of 15.6%-35%, leading to the occurrence of allergic asthma, anaphylactic shock and other diseases
[0003] At present, my country is in its infancy in the field of allergy-related research, and there are no relevant diagnostic reagents for shrimp and crab food allergies
Clinical detection reagents mainly rely on imports, such as the Immuno CAP system automatic detection system certified by the US FDA, its recognized detection limit is 0.24ng / mL, and the detection cost is relatively expensive, most allergic patients cannot afford it; The wide range of ELISA detection kits has a high detection limit and cannot accurately identify whether they are allergic patients
Moreover, the allergens used in allergy testing are crude extraction products of biological materials. The crude extraction products contain allergens, non-specific allergens, and suspected allergens that have not yet been characterized, which interfere with the results of allergy testing, and their sensitization efficiency cannot be standardized.

Method used

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  • Method for detecting specific immune globulin E of eriocheir sinensis
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  • Method for detecting specific immune globulin E of eriocheir sinensis

Examples

Experimental program
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Embodiment 1

[0027] Example 1: Preparation of fluorescent probes with different particle sizes FHMNs@PDDA@PAA

[0028] Dissolve cetyltrimethylammonium bromide (CTAB) powder in a mixture of 29mL deionized water, 12mL ethanol and 1mL ammonia water, then add polystyrene microsphere dispersion (dispersed in 10ml deionized ionized water), after magnetically stirring at room temperature for 60 min, tetraethyl orthosilicate (TEOS) was added dropwise, maintaining a constant stirring speed of 150 rpm / min, and continuing to stir at room temperature for 48 h. After the reaction was completed, the reaction solution was vacuum filtered and washed several times, and then dried in a vacuum oven at 65°C for 12 hours to obtain core-shell PS / SiO 2 Composite microsphere powder. Calcined at 550°C for 8h to remove template polystyrene to obtain hollow silica. Disperse 0.05g of HMNs and 0.012g of 5(6)-fluorescein isothiocyanate (FITC) in 5ml of deionized water, stir at room temperature for 48h, centrifuge the...

Embodiment 2

[0030] Example 2: Magnetic probe Fe 3 o 4 @SiO 2 Preparation of @PAA Nanoparticles

[0031] Weigh 300mgFe 3 o 4 Disperse in a mixture of 40mL ethanol and 4mL deionized water, after ultrasonication for 15min, maintain a certain stirring speed, add 5mL ammonia water, slowly add 2mLTEOS, react at room temperature for 12h, and use 0.1moL / L hydrochloric acid solution and deionized water after magnetic separation Wash each for 3 times, and dry at 40° C. for 12 hours to obtain a reddish-brown precipitate, which is ferric oxide / silicon dioxide core-shell nanoparticles. Fe will be produced 3 o 4 @SiO 2 Dispersed in dimethylamide (DMF), mixed with 33ml DMF dissolved in 2g polyacrylic acid (PAA), ultrasonicated for 30min, the mixture was vigorously stirred and heated to 110°C, added 3.3mL DMF dissolved in 4-dimethylaminopyridine and Dissolve N,N'-dicyclohexylcarbodiimide (DCC) in 6.6mL DMF, keep the mixture at 110°C and stir for 12h, then magnetically separate the product, wash w...

Embodiment 3

[0033] Embodiment 3: the establishment of allergy detection system fluorescent sensing platform (EFSP)

[0034] (1) Take 50 mg of prepared FHMNs@PDDA@PAA and activate PAA with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) / N-hydroxysuccinimide (NHS) In the carboxyl group, take an appropriate amount of activated FHMNs@PDDA@PAA and add a certain concentration of anti-IgE antibody (Ab 2 ), incubated at 37°C for 2h, centrifuged to remove the supernatant, washed 3 times with PBS, added blocking solution at 37°C for 1h, centrifuged to remove the supernatant, and collected the prepared FHMNs@PDDA@PAA-(Ab 2 ) were dispersed in PBS buffer and stored at 4°C for later use.

[0035] (2) the prepared Fe 3 o 4 @SiO 2 @PAA nanoparticles, the carboxyl group in PAA was activated by EDC / NHS, the purified hemocyanin, the main allergen of Chinese mitten crab, was added, incubated at 37°C for 120min, the supernatant was discarded by magnetic separation, washed 3 times with PBS buffer, added...

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Abstract

The invention discloses a method for detecting specific immune globulin E of eriocheir sinensis and belongs to the crossing field of material and biomedicine. The method includes: adopting fluorescenthollow mesoporous silicon dioxide FHMNs as a carrier resistant to IgE antibody and Fe3O4@SiO2 prepared by coating silicon dioxide with ferroferric oxide as an allergen carrier; allowing the two materials to capture a target-the specific IgE of eriocheir sinensis at the same time; utilizing antigen-antibody specific reaction to gather and separate the target-sIgE; utilizing fluorescent molecular signals in FHMNs to amplify signals of low-concentration target sIgE; correlating fluorescent intensity with IgE concentration to quickly and sensitively detect specific IgE of eriocheir sinensis in human blood. By the method, IgE detection limit is lowered effectively, and detection time is shortened.

Description

technical field [0001] The invention relates to a method for detecting the specific immunoglobulin E of Chinese mitten crab, which belongs to the cross field of materials and biomedicine. Background technique [0002] Shrimp and crab are the most common crustaceans in my country's aquatic product market, and they are high-protein and high-nutrition foods. But it is also one of the eight types of highly allergenic foods, with an incidence rate of 15.6%-35%, leading to the occurrence of allergic asthma, anaphylactic shock and other diseases. [0003] At present, my country is in its infancy in the field of allergy-related research, and there are no relevant diagnostic reagents for shrimp and crab food allergy. Clinical detection reagents mainly rely on imports, such as the Immuno CAP system automatic detection system certified by the US FDA, its recognized detection limit is 0.24ng / mL, and the detection cost is relatively expensive, most allergic patients cannot afford it; T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N21/64
Inventor 吴静云建荣姚瑞
Owner JIANGNAN UNIV
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