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Kit and method for quantitatively detecting human lipopolysaccharide binding protein

A protein-binding and quantitative detection technology, applied in the field of immunoassay, can solve the problems of low precision, low sensitivity, short detection range, etc., and achieve the effects of high sensitivity, high detection sensitivity and low cost

Pending Publication Date: 2021-12-21
SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Existing immunological research solutions for detecting LBP, such as conventional enzyme-linked immunoassay (ELISA), have a short detection range, generally 0.8-50ng / mL and poor linearity; the sensitivity is not high, generally 2ng / mL About, and the blood sample needs to be pre-treated, and tested after 50-200 times dilution; at the same time, the standard deviation is about 15%, and the precision is not high

Method used

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  • Kit and method for quantitatively detecting human lipopolysaccharide binding protein

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Embodiment 1

[0038] The preparation of embodiment 1 kit

[0039] 1. Preparation of chemiluminescent plates coated with monoclonal or polyclonal antibodies to human lipopolysaccharide-binding protein (LBP)

[0040] Dilute human lipopolysaccharide-binding protein (LBP) monoclonal antibody or polyclonal antibody to 2-4 μg / mL with phosphate buffer or carbonate buffer, add 100 μl / well to the chemiluminescence plate, and place at 4 °C After 16 to 20 hours, take out the chemiluminescence plate, dry the residual liquid, and use bovine serum albumin, casein, gelatin, animal or human serum, etc. to block, add 250 μl / well (concentration 5-20 μg / mL) For samples, place at 37°C for 2 hours or at 4°C for 16-20 hours, shake off the residual liquid, dry and store at 4°C for later use.

[0041] 2. Preparation of biotin-labeled human lipopolysaccharide-binding protein (LBP) monoclonal antibody or human lipopolysaccharide-binding protein (LBP) polyclonal antibody

[0042] A 10 mM NHS-DPEG4-Biotin working so...

Embodiment 2

[0061] Example 2 The method and methodological evaluation of using the kit to detect human lipopolysaccharide-binding protein (LBP)

[0062] (1) The method for quantitatively detecting human lipopolysaccharide-binding protein with the kit described in Example 1 is as follows:

[0063] (1) Dilute the human blood sample by 50 times with the standard diluent, take the gradient concentration of the standard product and serum / plasma samples according to 50 μL / well, add them to the corresponding plate wells, and make a good label;

[0064] (2) Take biotin-labeled human lipopolysaccharide-binding protein (LBP) antibody working solution, and add 50 μL / well to the corresponding plate well;

[0065] (3) Incubate at 37°C for 1 hour, take out, wash 250 μL of working solution per well, wash 4 times, and pat dry;

[0066] (4) Take the streptavidin-labeled horseradish peroxidase working solution (concentration: 50mIU / mL), add 50μL / well to the corresponding reaction well, and place it at 37℃...

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Abstract

The invention relates to the technical field of immunoassay, and in particular relates to a chemical light kit for quantitatively detecting human lipopolysaccharide binding protein (LBP) and a detection method. The kit comprises a pore plate coated with a human lipopolysaccharide binding protein antibody. The detection method comprises the following steps: (1) adding a sample to be detected into the pore plate coated with the human lipopolysaccharide binding protein antibody; (2) adding a biotin-labeled human lipopolysaccharide binding protein antibody; (3) carrying out incubating, washing and drying; (4) adding horseradish peroxidase labeled by streptomycin and avidin or alkaline phosphatase labeled by streptomycin and avidin for reaction, washing and drying; and (5) adding an enzyme luminescence substrate, and reading a luminescence value. The method is higher in detection sensitivity and stronger in specificity; blood sample treatment is not needed, and operation is easy and convenient; and the method has no high requirements on required equipment, can realize large-flux detection, is low in cost, can realize semi-automatic or full-automatic batch operation, and has a good effect and a good application prospect.

Description

technical field [0001] The invention relates to the technical field of immune analysis, in particular to a chemical light kit for quantitatively detecting human lipopolysaccharide binding protein (LBP). Background technique [0002] Lipopolysaccharide-binding protein (LBP) is a glycoprotein with a molecular weight of about 58KDa, which is synthesized by liver cells and belongs to the lipid-binding protein family. Its family also includes BPI, PLTP, CETP. LBP binds to the lipid A part of lipopolysaccharide (LPS), the cell wall component of Gram-negative bacteria, promotes the monomerization of LPS, catalyzes the combination of LPS monomer and cytokine CD14, and induces an immune response that promotes LPS. This indicator can reflect Body endotoxin load level. [0003] Existing immunological research solutions for detecting LBP, such as conventional enzyme-linked immunoassay (ELISA), have a short detection range, generally 0.8-50ng / mL and poor linearity; the sensitivity is n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/58G01N33/577G01N33/543G01N21/76
CPCG01N33/6893G01N33/581G01N33/577G01N33/54393G01N21/76G01N2333/47G01N2800/26
Inventor 王睿瑞张磊
Owner SHANGHAI UNIV OF T C M
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