Detection kit for neutrophil gelatinase-associated lipocalin

A lipocalin, neutrophil technology, applied in the biological field, can solve the problems of long time, low detection sensitivity, low sensitivity and accuracy, etc.

Active Publication Date: 2017-05-10
FAPON BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The detection of NGAL initially used the western blotting method, which played an important role in the early quantitative research of NGAL, but due to the disadvantages of low sensitivity and accuracy, complicated operation, and poor repeatability, it is rarely used now. Quantitative research of NGAL; radioimmunoassay (Radioimmunoassay, RIA) is established and perfected in laboratory research. May cause potential pollution and injury to the environment and experiment personnel
In 2005, Danish BioPorto company first launched ELISA commercial kit, which can be used for NGAL detection of serum (serum) and urine. Its latest ELISA kit can be used for rapid laboratory diagnosis. Clinical diagnosis and monitoring of functional impairment, but compared with the latex-enhanced immune turbidimetric assay, it takes a long time and cannot be automated. Later, the company developed a latex-enhanced immune turbidimetric kit, but it is expensive
[0007] Based on this, the detection sensitivity of the current traditional NGAL detection kit is low

Method used

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  • Detection kit for neutrophil gelatinase-associated lipocalin
  • Detection kit for neutrophil gelatinase-associated lipocalin
  • Detection kit for neutrophil gelatinase-associated lipocalin

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, the preparation of immune source and standard product.

[0058] Using genetic engineering technology, the dominant epitope gene segment of NGAL protein was analyzed and screened through a large number of molecular biology analysis software, and the dominant codon of E. coli was optimized. The sequence is SEQ ID No.1, and the overlap PCR primers were designed , by means of PCR polymerase amplification to artificially synthesize the NGAL activity dominant epitope DNA fragment. The upstream primer has a BamHI site, the downstream primer has an EcoRI site and an EcoRI site, and the fragment of the PCR is digested with BamHI and EcoRI after recovery, and connected to the expression vector PK2 (Fei Peng Bio) after digestion with BamHI and EcoRI. Co., Ltd.), the recombinant plasmid PK2-NGAL was obtained, and cultured in 500 mL LB containing 100 μg / mL kanamycin sulfate (Shanghai Sangon Bioengineering Technology Service Co., Ltd., hereinafter referred to as Sangon,...

Embodiment 2

[0060] Example 2, the establishment of anti-human NGAL hybridoma cell line and the preparation of its monoclonal antibody

[0061] 1. Immunization of mice with recombinant NGAL

[0062] The recombinant NGAL was diluted to 1.0 mg / mL, mixed with an equal volume of Freund's complete adjuvant (Sigma-Aldrich, product number: F5881), and fully emulsified to obtain an oily emulsion. The emulsion was subcutaneously administered to the dorsal site of BALB / c mice (Guangdong Provincial Medical Experimental Animal Center: No. 119, Poyang Road, Huangqi, Nanhai, Foshan City, Guangdong Province, 6-week-old female, 5 mice) at a dose of 0.2 mL. Fourteen days after the first immunization, the intraperitoneal booster immunization was performed, that is, the same amount of antigen was mixed with an equal volume of Freund’s incomplete adjuvant (Sigma-Aldrich, F5506). The titer was determined by the method, if the titer is higher than 1:10000, it can be used for fusion.

[0063] Three days before...

Embodiment 3

[0079] Embodiment 3, preparation and debugging of NGAL detection kit

[0080] 1. Preparation of buffer

[0081] The buffer solution is 25mM Tris+25mM glycine+0.1%BSA+50mMNaCl+0.5%Tween 20+0.1%NaN3+25mMMgCl2+0.1%EDTA, pH8.2;

[0082] 2. Preparation of detection solution

[0083] The chemical cross-linking method was used to connect the antibody and latex, and one monoclonal antibody was taken from different epitopes and mixed equally. After debugging, the monoclonal antibodies secreted by the hybridoma cell line NGAL-3B5, the hybridoma cell line NGAL-2D8 and the hybridoma cell line NGAL-4F6 are evenly mixed according to the mass ratio of 1:1:1, and the product performance is better ,Proceed as follows:

[0084] 1) Activation: 20mg microspheres + 1.4mL buffer + 2mgEDC + 6mgNHS shaking reaction at room temperature for 15min, centrifugation at 20000rpm for 30min;

[0085] 2) Coupling: Discard the supernatant and add 1mL 5mM CB for ultrasound, then add the mixed antibody (40μgA...

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Abstract

The invention discloses a detection kit for neutrophil gelatinase-associated lipocalin. The detection kit comprises a detection liquid and a standard substance, wherein the detection liquid comprises latex particles coupled with an anti-human neutrophil gelatinase-associated lipocalin antibody; the standard substance is naturally configured recombinant neutrophil gelatinase-associated lipocalin. The detection kit measures the content of the neutrophil gelatinase-associated lipocalin through a latex particle intensified immunity turbidity, and compared with the traditional NGAL detection kit, the detection kit has the advantages of high detection sensitivity, high simpleness in operation, good repeatability and short time consumption, does not need sample pretreatment, and can be used for a fully automatic biochemical analyzer.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a detection kit for neutrophil gelatinase-associated lipocalin. Background technique [0002] In the past few decades, people's understanding of the pathogenesis of acute kidney injury (AKI) and the level of medical technology have greatly improved, but the morbidity and mortality of AKI are still high. One of the main reasons leading to this situation is the lack of effective early diagnostic markers. The RIFLE (Rise, Injury, Failure, Loss, End-stage renal disease) diagnostic system, which uses elevated serum creatinine (sCr) or urine output as an index, is the standard method for judging AKI in current clinical practice. However, sCr levels vary greatly due to factors such as age, sex, muscle mass, muscle metabolism, drug use, and hydration. Another fact is that sCr levels often do not rise significantly until several days after AKI. Therefore, although sCr is a reliable marker ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/92
CPCG01N33/92
Inventor 李瑞净刘俊鹏刘春燕钟冬梅孟媛魏鈡杰
Owner FAPON BIOTECH INC
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