Method for quantitatively measuring Ngb (neuroglobin) with double-antibody sandwich method and application of method

A quantitative determination, double-antibody sandwich technology, applied in the field of protein analysis, can solve the problems of low concentration, unsatisfactory detection limit detection accuracy, unstable rhodopsin, etc., achieves low detection limit, improved specificity, high Effects of Specificity and Sensitivity

Inactive Publication Date: 2017-05-31
GUANGZHHOU HUAHONG BIOLOGICAL TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, rhodopsin has certain advantages as a photodamage biomarker. However, rhodopsin is very unstable and its concentration may be too low to make it an ideal photodamage marker. people are satisfied

Method used

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  • Method for quantitatively measuring Ngb (neuroglobin) with double-antibody sandwich method and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Preparation of Ngb monoclonal antibody

[0031] 1.1 Preparation of hybridoma cells

[0032]1ml of 0.25g / L Ngb protein was fully emulsified with an equal volume of Freund's complete adjuvant and injected into 5 Balb / c mice subcutaneously. The immunization was boosted once every 3 weeks, and Freund's incomplete adjuvant was used for the booster immunization, and the antigen weight and injection route remained unchanged. 7d after the second booster immunization, tail blood was taken to measure the titer. Three days before fusion, the same dose of antigen PBS solution was injected through the tail vein to boost the immunization once again. The spleen cells and NS-1 cells of the immunized mice were collected and fused at a ratio of about 1:5 under the action of 50% PEG according to conventional methods. The fused cell pellets were lightly suspended with 2.5ml of complete culture medium, added with 22.5ml of semi-solid medium Medium D, mixed, and then poured into...

Embodiment 2

[0034] Example 2 Preparation and purification of biotinylated Ngb polyclonal antibodies

[0035] 2.1 Preparation and purification of rabbit anti-Ngb protein polyclonal antibody: The purified Ngb protein was thoroughly mixed with complete Freund's adjuvant, and the Japanese rabbits were immunized subcutaneously at multiple points. After 2 weeks, it was mixed with incomplete Freund's adjuvant. Subcutaneous booster immunization, 1 week later, the serum was collected, and the antibody titer was detected by ELISA. The immune serum was precipitated by salting out saturated ammonium sulfate, 50% ammonium sulfate, and 33% ammonium sulfate solution. The immune serum purified by salting out was then purified by SephadexG25 desalting and purified by DEAE-SephadexG-100 column chromatography.

[0036] 2.2 The cross-linking and purification of Ngb protein polyclonal antibody and biotin were carried out with reference to references and reagent instructions. The detailed process is as follow...

Embodiment 3

[0037] Embodiment 3 A kind of method that utilizes double antibody sandwich method to quantitatively measure cerebroglobin

[0038] A method for quantitatively determining cerebroglobin by double-antibody sandwich method, the detection method is carried out according to the following steps:

[0039] 1) Coating: Dilute the Ngb monoclonal antibody with sodium carbonate buffer pH 9.6 to 10 μg / ml, add 100 μl to each well, incubate at 37°C for 4 h and wash as follows, discard the liquid in the well, and wash with PBST for 4 times, 2 minutes each time, shake off the liquid in the hole and pat dry on absorbent paper;

[0040] 2) Blocking: Add 200 μl of PBS containing 5% BSA blocking solution to each well to block, incubate at 37°C for 4 hours, wash as follows, wash twice with PBST for 2 minutes each time, shake off the liquid in the well and pat dry on absorbent paper ;

[0041] 3) Capture antigen: Dilute the antigen to an appropriate concentration, add 100 μl per well to the corre...

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Abstract

The invention discloses a method for quantitatively measuring Ngb (neuroglobin) with a double-antibody sandwich method and an application of the method and belongs to the technical field of protein analysis. The detection method comprises steps as follows: a coated Ngb monoclonal antibody is taken as a solid-phase antibody on an ELISA detection plate, and a biotin-labeled Ngb polyclonal antibody is taken as an enzyme-labeled antibody, so that HRP is combined with biotin on the polyclonal antibody through Avidin coupled with the HRP, the absorption value is measured in the position of 450 nm single wave length through TMB color development, and quantitative detection of the Ngb protein can be realized. The method adopts the double-antibody sandwich ABC method and has high specificity and high sensitivity. The content of Ngb in retina tissue can be accurately detected, so that the light injury degree of illumination of an LED light source to retinas is evaluated.

Description

technical field [0001] The invention relates to a method for quantitatively determining cerebroglobin by using a double-antibody sandwich method and its application, in particular to a method for quantitatively detecting changes in the expression level of retinal cerebroglobin based on an ELISA detection method and its use for evaluating the degree of retinal light damage caused by an LED light source The application belongs to the technical field of protein analysis. Background technique [0002] A light-emitting diode (LED) is a solid-state semiconductor that converts electrical energy directly into visible light. Unlike tungsten lamps, LED lamps use blue light to excite phosphors to produce white light, so they contain a very high visible spectrum of harmful components. Because the short wavelength spectrum (400-500nm) is especially dangerous, LED lighting is more likely than any other light source to cause photochemical damage to the retina. However, there are few stud...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 邹伟权
Owner GUANGZHHOU HUAHONG BIOLOGICAL TECH
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