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Human Serum Albumin Removal Kit

A technology of human serum albumin and kits, which is applied in the direction of biological testing, preparation of test samples, material inspection products, etc. It can solve the problems that protein components cannot be detected, limit the application of biological mass spectrometry, and albumin occupies analysis capacity, etc. , to achieve the effects of stable properties, wide range of applications, and easy preparation

Active Publication Date: 2020-09-18
山东民康生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A large amount of albumin occupies the analysis capacity of the mass spectrometer, resulting in many low-content protein components that cannot be detected, thus limiting the application of biological mass spectrometry in the detection of protein biomarkers in peripheral blood

Method used

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  • Human Serum Albumin Removal Kit
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  • Human Serum Albumin Removal Kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1, the preparation of anti-human serum albumin nanobody

[0018] (1) Synthesize the nucleic acid molecule encoding the above-mentioned anti-human serum albumin nanobody according to the sequence shown in the above-mentioned SEQ ID NO.2. Simultaneously, at the 5' end of the nucleic acid molecule, an Nde I restriction site (CATATG) is added, and a Not I restriction site (GCGGCCGC) is added at the 3' end.

[0019] (2) Use the pET30a(+) prokaryotic expression plasmid as the nanobody expression vector. The expression vector and the nucleic acid molecular fragments of the above-mentioned nanobodies were digested with Not I and Nde I enzymes. The digestion conditions were: 1 μg of DNA, 5 μL of 10X buffer, 1 μL of Not I and Nde I enzymes, added water to 50 μL, and 37°C After 20 minutes in water bath, inactivate at 80°C for 10 minutes, and recover the fragments after running the gel.

[0020] (3) Carrier and fragment ligation: 50 μg of carrier, 40 μg of fragment, 1 ...

Embodiment 2

[0027] Embodiment 2, the preparation of immunoaffinity material

[0028] (1) Take 2 mg of purified anti-human serum albumin nanobody, and make it into 1 mg / mL with cross-linking buffer;

[0029] (2) Get 0.3 mg of cyanogen bromide-activated agarose and soak it in 0.1M hydrochloric acid for 20 minutes to make cyanogen bromide-activated agarose microspheres;

[0030] (3) Wash the cyanogen bromide-activated agarose gel microspheres 3 times with cross-linking buffer, mix the nanobody solution with the gel microspheres, and incubate with shaking at room temperature for 2 hours;

[0031] (4) Alternately washing the cyanogen bromide-activated sepharose microspheres with cross-linking buffer and low pH acetic acid solution for 3 times;

[0032] (5) Resuspend the cyanogen bromide-activated sepharose microspheres in 1 mL of PBS and store at 4°C for later use.

Embodiment 3

[0033] Embodiment 3, the mensuration of immunoaffinity material adsorption capacity

[0034] (1) Dilute 25 μg of human serum albumin into 50 μg of PBS buffer, and prepare 5 copies in total;

[0035] (2) Take 12.5 μL, 25 μL, 37.5 μL, 50 μL, and 62.5 μL of cyanogen bromide-activated agarose gel microspheres, mix them with human serum albumin diluent, and incubate with shaking at room temperature for 20 minutes;

[0036] (3) Centrifuge at 100g room temperature for 2 minutes, and take the supernatant;

[0037] (4) Take 4 μL supernatant and 1 μL agarose gel microspheres, run SDS-PAGE gel electrophoresis, the result is as follows figure 2 As shown in the figure, the loading volume of the gel microspheres is 0.5uL, and the loading volume of the supernatant is 4uL;

[0038] (5) Calculate the capacity of 1 μL of cyanogen bromide-activated agarose gel microspheres to adsorb human serum albumin as 2 μg.

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Abstract

The invention discloses a human serum albumin removal kit, which is composed of an immunoaffinity material, a buffer solution, a washing solution, disposable consumables and a human serum albumin standard substance, wherein the immunoaffinity material comprises a carrier matrix and a nano antibody. Since the human serum albumin removal kit provided by the invention has the specific nano antibody and the carrier matrix of a special structure, human serum albumin can be specifically recognized and adsorbed, albumin in human plasma or serum can be removed, and the detection capability of the biomass spectrum on peripheral blood low-abundance protein indexes is improved, so that the application of the biomass spectrum in peripheral blood proteomics detection is promoted. In addition, the nanoantibody in the human serum albumin removal kit has the advantages of high affinity, stable properties, acid and alkali resistance, high salt resistance, easiness in preparation and the like, so thatthe human serum albumin removal kit is wide in application range.

Description

technical field [0001] The invention belongs to the technical field of immunology and biological mass spectrometry, and relates to a nanobody, an immunoaffinity material based on the nanobody, and in particular to a human serum albumin removal kit, which uses the immunoaffinity material for human serum albumin Removal kit for the removal of albumin from human plasma or serum. Background technique [0002] With the introduction of the concept of precision medicine and the development of precision medicine, proteomics based on biological mass spectrometry plays an increasingly important role in clinical diagnosis and precision treatment. High performance liquid chromatography tandem mass spectrometry (LC-MS / MS), as an emerging detection platform, has been widely used in the field of biomedical research. With the continuous innovation of technology, LC-MS / MS has gradually turned to clinical laboratory testing to provide more reliable test results for the diagnosis and treatmen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N1/34
CPCG01N1/34G01N33/6848G01N2560/00
Inventor 赵冠华靳昌忠
Owner 山东民康生物科技有限公司
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