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Method for measuring digestive enzyme activity of tissues of seahorse baby with enzyme linked immunosorbent assay

An enzyme labeling method and a technology of digesting enzymes, which are applied in the preparation of test samples, biological testing, color/spectral property measurement, etc. material resources and other issues, to achieve the effect of low price, low cost, and easy to learn methods

Inactive Publication Date: 2011-10-12
EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional digestive enzyme analysis method has the following problems: 1) it often requires larval tissues with a weight of several hundred milligrams, which requires the consumption of dozens or even hundreds of larvae
2) You need to configure the analysis reagents yourself or purchase a commercial kit
And the price of the test kit often reaches hundreds or even thousands of yuan.
3) For tissue homogenization before experimental testing, manual glass homogenizers or high-speed homogenizers are often used, but generally only one sample can be homogenized at a time
This not only requires high analysis costs, but also consumes a lot of manpower and material resources
And because the test process of a large number of samples takes more than ten or even dozens of days, this greatly increases the deviation caused by the long span of the test process

Method used

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  • Method for measuring digestive enzyme activity of tissues of seahorse baby with enzyme linked immunosorbent assay
  • Method for measuring digestive enzyme activity of tissues of seahorse baby with enzyme linked immunosorbent assay
  • Method for measuring digestive enzyme activity of tissues of seahorse baby with enzyme linked immunosorbent assay

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] 1) Put 10 mg of frozen hippocampal seedlings into a 1.5ml container, and then add 90 μl of homogenate medium (pH 7.4, 0.01mol / L Tris-HCl, 0.0001mol / L EDTA-2Na, 0.01mol / L sucrose, 0.8% g / mlNaCl solution), homogenate into a homogenate, and then in a TGL-16G refrigerated centrifuge at 4°C, centrifuge at 9000rpm for 10min, take 80ul of the supernatant, 20ul of which is used for the following detection, and another 60ul can be reserved for other use;

[0027] 2) Take 10 ul of the above supernatant and use the Coomassie Brilliant Blue G250 method to measure the total tissue protein concentration to be 0.34 mg / ml, wherein bovine serum albumin is the standard protein;

[0028] 3) Take two clean containers A and B, respectively add 50ul of the "substrate starch buffer" in the kit, and then add 10ul of the supernatant obtained in step 1) to B, and shake B with a vortex shaker to make Mix the reagents well; then heat A and B in a constant temperature water bath at 37°C for 7.5 m...

Embodiment 2

[0033] 1) Add 20 mg of frozen hippocampus seedlings to a centrifuge tube, and then add 180 μl of 2 in ice bath. Homogenize 0.7% g / ml NaCl physiological saline at around ℃ to form a homogenate, then centrifuge in a refrigerated centrifuge at 10000rpm for 10min at 4℃, take 160ul of the supernatant, of which 20ul is used for the following detection, and the other 140ul can be reserved for other purposes;

[0034] 2) Take 10 ul of the above supernatant and use the Coomassie Brilliant Blue G250 method to measure the total tissue protein concentration to be 0.30 mg / ml, wherein bovine serum albumin is the standard protein;

[0035] 3) Take two clean centrifuge tubes A and B, add 50ul of the "substrate starch buffer" in the kit respectively, then add 10ul of the supernatant obtained in step 1) to B, and shake tube B with a vortex shaker , to fully mix the reagents in the tubes; then heat A and B in a constant temperature water bath at 37°C for 7.5 minutes, take them out and add 50ul ...

Embodiment 3

[0040] The concentration of total tissue protein was determined by Coomassie Brilliant Blue G250 method:

[0041] 1) The preparation of homogenate supernatant and centrifugation process are the same as step 1) of Example 2;

[0042] 2) Take three clean centrifuge tubes (blank tube A, standard tube C, and measuring tube B), and then add 10ul of distilled water to the blank tube, 10ul of standard solution into the standard tube, and 10ul of sample homogenate supernatant into the measuring tube ;Add 300ul Coomassie Brilliant Blue chromogen to each tube respectively, then mix well, and let stand for 10min;

[0043] 3) Mix well, take 270ul of the solution and drip it into the microplate (96 wells with a volume of 405ul per well), adjust the wavelength of the microplate reader to 595nm, and measure the absorbance as blank tube A: 0.323, measuring tube B: 0.424, Standard tube C: 0.511; then calculate the total tissue protein concentration according to the formula to be 0.30mg / ml, of w...

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Abstract

The invention relates to a method for measuring the digestive enzyme activity of tissues of a seahorse baby with enzyme linked immunosorbent assay. The method comprises the following steps of: (1) putting a frozen seahorse baby into a container, adding normal saline of NAC1 with the concentration of 0.7 percent or a homogenizing medium in an ice bath, homogenizing by using a multifunctional sample homogenizer, centrifuging with a centrifuge and taking supernatant fluid for later use; (2) measuring the total protein concentration of the tissues with a part of the supernatant fluid; (3) adding a substrate starch buffer solution into a container A and a container B respectively, adding the supernatant fluid obtained in the step (1) into the container B and adding iodine reaction liquid and distilled water into the containers A and B respectively; (4) sucking the solutions in the containers A and B, dripping the solutions into two holes of an enzyme label plate, putting the enzyme label plate into an enzyme label reader and detecting the absorbency of the solutions in the two holes simultaneously; and (5) calculating the amylase activity of a sample. The method disclosed by the invention is easy to operate, has low cost and high efficiency and is environment-friendly.

Description

technical field [0001] The invention belongs to the field of measuring the activity of digestive enzymes, in particular to a method for measuring the activity of digestive enzymes in hippocampal seedling tissue by an enzyme labeling method. Background technique [0002] Hippocampus is a traditional precious Chinese medicine with high economic value. In recent years, seahorses have been regarded as advanced ornamental animals, making their value soar. However, due to the serious destruction of natural resources, the supply cannot meet the demand, and artificial breeding has become the only option. In seahorse seedling cultivation, whether the bait nutrition is sufficient and whether the environmental conditions are suitable have become important factors affecting the success of seahorse seedling cultivation. Digestive enzyme activity is an important index reflecting the ability of fish to utilize different nutrients in aquatic research. In addition to bait nutrition, envir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N21/31G01N1/38
Inventor 尹飞孙鹏彭士明施兆鴻
Owner EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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