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ELISA fluorescence detection kit for detecting content of human Galectin-3

A fluorescence detection and kit technology, applied in the biological field, can solve problems such as large detection range, large error, and inability to obtain absorbance, and achieve the effect of eliminating reagent or operating errors and improving accuracy

Pending Publication Date: 2021-09-17
INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, some Galectin-3ELISA detection kits have been launched at home and abroad, but they all have certain shortcomings. For example, the detection concentration range of the kit k093758 is 1.4-94.8ng / ml, and the error is large when the detection is below 10ng / mL ; This detection range is relatively large. In most diseases, the blood concentration of Galectin-3 is between 5-15ng / mL, which is not suitable for detection
Another example: R&D Company and abcam Company have Galectin-3 detection kits with a detection range of 0.16-10ng / mL, but these kits use the absorbance detection method, and it is easy to obtain absorbance because the sample concentration exceeds 10ng / mL, so it is not suitable for Large-scale testing of human blood samples

Method used

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  • ELISA fluorescence detection kit for detecting content of human Galectin-3
  • ELISA fluorescence detection kit for detecting content of human Galectin-3
  • ELISA fluorescence detection kit for detecting content of human Galectin-3

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0063] 1.2 Preparation of biological samples

[0064] 1.2.1 Preparation of human serum samples

[0065] Centrifuge the serum sample under the conditions of 1000g centrifugal force and centrifugation for 15 minutes, and analyze the obtained serum sample immediately; if it cannot be used immediately, the sample can be frozen and stored at -80°C. It should be noted that all blood components should be handled as potential hazards while avoiding contamination of the samples in the vials.

[0066] 1.2.2 Preparation of cell lysate samples

[0067] (1) Cultivate cells, when the cells cover more than 85% of the culture surface, wash twice with PBS, and dry the PBS;

[0068] (2) Add cell extraction buffer (Cell Extraction Buffer), the amount of cell extraction buffer depends on the amount of cells, the ratio between the two is 1×10 7 Add 1mL of cell extraction buffer to each cell, collect the cells with a cell scraper, and transfer them to a microcentrifuge tube;

[0069] (3) After ...

experiment example 1

[0072] The method for detecting Galectin-3 content in a biological sample using the ELISA fluorescence detection kit described in the composition and configuration of the ELISA kit is specifically:

[0073] (1) Place the serum samples to be tested, standard samples, and positive control samples at room temperature and pre-balance the temperature, then dilute the serum samples by half with biological sample diluent (if you encounter a sample with a very high concentration, you can carry out four One-half and one-eighth dilutions); the standard was diluted to seven gradients of 20ng / mL, 10ng / mL, 5ng / mL, 2.5ng / mL, 1.25ng / mL, 0.625ng / mL, and 0.3125ng / mL; Directly select standard diluent as negative control;

[0074] (2) Add 100uL of biological sample diluent to the microplate, add the above-mentioned samples to the microwells of the microplate in turn, and repeat the experiment twice for each sample, 50uL per well. After incubation at 25°C for 3 hours, Galectin-3 will bind to the...

experiment example 2

[0080] The method of using the ELISA fluorescence detection kit of Experimental Example 1 to detect the content of Galectin-3 in biological samples, specifically measuring the stability of the samples in the ELISA detection plate of different sources of human Galectin-3 in serum samples, cell culture medium supernatants, and cell lysates Sex, and the stability of samples between ELISA detection plates of human Galectin-3 serum samples.

[0081] 3.1 Human Galectin-3 Serum Sample ELISA Detection Panel Sample Stability

[0082] By the ELISA method of Example 2, in the same 96-well plate, the samples were detected in parallel 20 times, and the stability of the serum sample detection in the plate was obtained by calculating the mean value, standard deviation, and CV of the 20 results (such as figure 2 and Table 1), the results showed that the stability of human Galectin-3 in different serum samples in the plate was high.

[0083] Table 1 Serum sample detection stability results i...

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Abstract

The invention belongs to the technical field of biology, and discloses an ELISA fluorescence detection kit for detecting the content of human Galectin-3.The kit comprises a Galectin-3 standard substance, a human serum Galectin-3 positive control, a reaction strip plate coated with a Galectin-3 capture antibody, an anti-Galectin-3 specific detection antibody, an enzyme-labeled secondary antibody, a biological sample diluent, a cleaning solution and a fluorescent light-emitting solution. The invention further discloses application of the kit in the aspect of detecting the content of Galectin-3 in a biological sample. A double-antibody sandwich method is adopted to carry out ELISA fluorescence detection on the content of human Galectin-3, and a human Galectin-3 specific monoclonal antibody is taken as an antibody for capturing Galectin-3, so that the specificity of a detection result is ensured; in addition, the kit can also reduce test errors and improve the accuracy of detection results.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to an ELISA fluorescence detection kit for detecting the content of human Galectin-3. Background technique [0002] Galectin-3 belongs to the galectin family. There are currently 14 members of this family reported, all of which have at least one conserved sugar recognition domain that binds galactose. Galectin-3 is a special one in this family, which consists of an N-terminal unstructured domain similar to collagen sequences and a C-terminal carbohydrate recognition domain (CRD). Galectin-3 is located on chromosome 14, and Galectin-3 can guide the polymerization of Galectin-3 monomers through the N-terminal domain, forming self-multimers, or forming a multimeric grid with other glycoproteins or glycolipids. The N-terminal domain can also assist Galectin-3 to enter the nucleus. Through the phosphorylation of 6-serine, the N-terminal domain can affect the binding of the CRD domain to su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/58G01N33/577G01N33/543
CPCG01N33/581G01N33/6893G01N33/577G01N33/54306G01N2333/4724
Inventor 李平平马春晓柳星峰崔冰姜茜孔丽娟侯少聪
Owner INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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