Chip and detecting reagent kit for detecting gastricism relevant indication marks object

A technology of markers and kits, applied in the biological field, can solve problems such as inconvenient clinical diagnosis and background interference

Inactive Publication Date: 2007-10-31
上海华冠生物芯片有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the current detection of these three factors is completed by three ELISA kits, and the requirements of the three ELISA kits for sample collection and processing (whether fasting or given due to too low Gastrin-17 content in serum) High protein agent stimulation), test sample pretreatment (56°C water bath or not), sample dilution degree (from 2 times to 200 times dilution), ELISA reaction time (from 2 hours to 4.5 hours), etc. are all different, And the background interference is very serious during detection, which brings inconvenience to clinical diagnosis
[0004] To sum up, at present, there are no rapid and sensitive detection products that can solve the above problems and can simultaneously detect multiple markers related to gastric disease at home and abroad.

Method used

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  • Chip and detecting reagent kit for detecting gastricism relevant indication marks object
  • Chip and detecting reagent kit for detecting gastricism relevant indication marks object
  • Chip and detecting reagent kit for detecting gastricism relevant indication marks object

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Chip preparation method (microsphere coating method)

[0131] (1) Take about 1.25×10 6Microspheres (purchased from Bio-rad, such as 171-506027, diameter 5.5 microns), oscillate and sonicate for 30 seconds → centrifuge at 14000×g for 4 minutes, discard the supernatant, resuspend in 100 μl of microsphere washing solution → oscillate at 1400 rpm, sonicate 30s→14000×g centrifugation for 4min, resuspend in 80μl activation solution→add 10μl of 50mg / ml EDC and S-NHS, shake at room temperature 1400rpm for 20min in the dark→add 150μl PBS buffer, shake at 1800rpm for 15s, centrifuge at 14000×g 4min→add 250μl of PBS buffer, shake at 1800rpm for 15s, centrifuge at 14000×g for 4min→resuspend in 100μl of PBS buffer→add 4μg of the corresponding capture antibody (anti-PGI primary antibody) and make up to 500μl with PBS buffer respectively; avoid light , 4°C, shake overnight at 1400rpm→centrifuge at 14000×g for 4min, wash the microspheres with 500μl of PBS buffer→centrifuge at 14000×g ...

Embodiment 2

[0136] Biotinylation of detection antibodies

[0137] (1) Dissolve BCA-SulfoNHS (Sigma) with 30 μl DMSO, and dilute to 0.5ml with 0.1mol / l sodium phosphate buffer solution, the final concentration is 10mg / ml→10mg / ml antibody in 1ml (for different gastritis markers , which are the secondary antibodies of PGI, PGII, and G-17) solution, add 38 μl of BCA-SulfoNHS solution → shake at room temperature in the dark for 30 minutes → separate and purify the biotinylated antibody through Sephadex G25 column → measure the recovery by BAC method amount of antibody protein.

[0138] (2) Detection of antibody biotinylation efficiency

[0139] Take 50 μl of biotin-labeled samples (labeled PGI, PGII, G-17 secondary antibody), add 5ul pronase protease, and bathe in water at 37°C for 90 minutes→mix 3.2ml of avidin (Avidin) with 100ul of 10mM Mix HABA[2-(4-hydroxyphenylazo)benzoic acid, 2-(4-Hydroxyphenylazo)benzoic acid] to obtain Avidin-HABA stock solution → mix 90ul of Avidin-HABA stock solu...

Embodiment 3

[0141] Preparation of detection kits

[0142] In this embodiment, a kit for detecting gastritis markers is prepared, and the kit includes the following components:

[0143] (a) A container a, and the microsphere mixture (prepared in Example 1) respectively coupled with PGI, PGII, and G-17 capture antibodies contained in the container.

[0144] (b) A container b, and a mixture of biotin-labeled PGI, PGII, and G-17 detection antibodies (prepared in Example 2) contained in the container.

[0145] (c) A container c, and 2× buffer A (10.1 mM Na 2 HPO 4 , 138mM NaCl, 1.76mM KH 2 PO 4 , 2.7mMKCl, 0.05% (V / V) Tween-20, pH7.4).

[0146] (d) A container d, and buffer B (10.1 mM Na 2 HPO 4 , 138mM NaCl, 1.76mM KH 2 PO 4 , 2.7mMKCl, 1% (m / V) BSA, 0.02% (m / V) Thimerosal, pH7.4).

[0147] (e) A container e, and streptavidin-phycoerythrin capable of binding to a biotin-labeled detection antibody contained in the container e.

[0148] (f) A set of 6 containers, and a series of mixe...

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Abstract

This invention discloses one protein chip, which comprises more than two kinds of micro ball fixed with different anti-gastritis label first antibody. This invention also discloses one method for test of the label. This invention also discloses one test agent case with protein chip. This invention protein chip has first antibody volume match design for accurate test.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a protein chip for detecting gastritis-related markers and a corresponding detection kit. Background technique [0002] Chronic gastritis is a common and frequently-occurring disease, generally there are two types: the inflammatory lesion is relatively superficial, limited to the surface layer of the gastric mucosa (no more than one-third), which is called chronic superficial gastritis; and the inflammatory lesion spreads to Full-thickness gastric mucosa, accompanied by gastric gland atrophy, is chronic atrophic gastritis. Gastroscopic surveys have confirmed that the incidence of chronic gastritis among the Chinese population is as high as 60%, and atrophic gastritis accounts for about 20% of them. Atrophic gastritis often develops from chronic superficial gastritis and is a chronic progressive lesion. If atrophic gastritis is not diagnosed and treated, it will cause dementia, neur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573G01N33/546
Inventor 赵芹何笑恬洪巍黄承
Owner 上海华冠生物芯片有限公司
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