Anti-human neutrophil gelatinase-associated lipocalin antibodies and use thereof

A technology of lipocalin and neutrophils, applied in the fields of biotechnology and immunology, can solve the problems of inability to timely and accurately reflect glomerular filtration rate, low specificity and sensitivity, and reduced clinical effect

Active Publication Date: 2014-12-03
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, creatinine is not specific and sensitive as a diagnostic criterion for acute kidney injury
There are two main reasons: First, serum creatinine concentration is easily affected by many factors, such as age, gender, muscle metabolic rate and hydration status, etc., and cannot reflect changes in glomerular filtration rate in a timely and accurate manner
Second, due to the strong compensatory ability of the kidneys, the serum creatinine concentration only rises significantly when the glomerular filtration rate drops to 1 / 3 of normal people, and at this time the kidney function has lost 50% [P.Devarajan, Update on mechanisms of ischemic acute kidney injury.Journal of the American Society of Nephrology:JASN17(2006)1503-1520], thus missing the best opportunity for intervention
However, due to the lack of early diagnostic markers for AKI, the clinical effects of some promising treatment options have been greatly reduced [A. Urbschat, N. Obermuller, A. Haferkamp, ​​Biomarkers of kidney injury. Biomarkers: biochemical indicators of exposure, response , and susceptibility to chemicals16Suppl1(2011)S22-30]

Method used

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  • Anti-human neutrophil gelatinase-associated lipocalin antibodies and use thereof
  • Anti-human neutrophil gelatinase-associated lipocalin antibodies and use thereof
  • Anti-human neutrophil gelatinase-associated lipocalin antibodies and use thereof

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Experimental program
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preparation example Construction

[0055] In an example of the present invention, the monoclonal antibody can be prepared by the following preparation method, which includes the steps of: (1) providing mice pretreated with adjuvant; (2) intraperitoneally inoculating the mouse with the said hybridoma cells and secrete monoclonal antibody; (3) extract ascites and separate and obtain said monoclonal antibody. As a preferred method, the method for isolating monoclonal antibody from ascites is as follows: collect ascites, precipitate with ammonium sulfate and octanoic acid, and then purify with Protein G prepacked chromatography column to obtain high-purity PSA monoclonal antibody.

[0056] In addition, the hybridoma cells can also be cultured and expanded in vitro according to conventional animal cell culture methods, so as to secrete the monoclonal antibody.

[0057] Reagent test kit

[0058] The present invention utilizes monoclonal antibody and polyclonal antibody specifically recognizing NGAL protein, and acco...

Embodiment 1

[0075] Embodiment 1, the preparation of hybridoma cell line

[0076] 1. Construction of DNA antigen for immunization

[0077] pBudCE4.1 was used to construct the pBud-Dse dual-promoter secretory expression vector as follows: the coding sequence of human IgE heavy chain signal peptide (Ig E-leader peptide) was codon-optimized to improve expression. The eukaryotic cell ribosome recognition site Kozak sequence GCCGCCACC was introduced before the start codon ATG; the signal peptide sequence was followed by a multiple cloning site, and two optimized signal peptides and multiple cloning site sequences were synthesized in the whole gene, and one segment was used in a restriction internal Dicer NotI and BglII were digested, and then connected to the multiple cloning site behind the hEF1α promoter of the pBudCE4.1 vector, and the other nucleotide sequence was digested with restriction endonucleases BamHI and HindIII, and connected to the previously constructed In the vector, the isola...

Embodiment 2

[0116] Embodiment 2, mammalian cell recombinant NGAL protein

[0117] In order to evaluate the affinity of the monoclonal antibody and provide a standard for the assembly of the detection kit, the present invention uses the FreeStyle MAX CHO expression system provided by Invitrogen to express and purify the secretable NGAL protein. The protein is a fusion protein with a 6×His tag at the C-terminus and a size of 28kD. The pBud-Dse-NGAL plasmid was transiently transfected into CHO-S cells (Invitrogen) according to the instructions provided by Invitrogen. After 72 hours of suspension culture, the cell culture supernatant was collected, centrifuged at 4°C and 6000rpm for half an hour, and the cell debris was removed. Purifier UPC100 fast protein purification system (FPLC), through Ni Sepharose High Performance chromatography column (GE), the C-terminal NGAL protein with 6×His tag secreted by cells in the cell culture supernatant is efficiently bound, and finally 300mM imidazole is...

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Abstract

The invention relates to anti-human neutrophil gelatinase-associated lipocalin (NGAL) antibodies and a use thereof, and discloses the monoclonal antibodies for specific detection of NGAL and a detection kit containing the monoclonal antibodies. Compared with traditional diagnostic methods and nucleic acid diagnostic methods, the kit not only has the characteristics of fast diagnostic speed, high accuracy degree and high throughput, but also has the characteristics of low cost, simple operation and the like.

Description

technical field [0001] The invention belongs to the fields of biotechnology and immunology; more particularly, the invention relates to an anti-human neutrophil gelatinase-associated lipocalin antibody and its application. Background technique [0002] Acute kidney injury (AKI) is a clinical syndrome caused by a sudden decline in renal function in a short period of time (within hours to days). So far, AKI is still a very common and serious clinical problem. The incidence of AKI in hospitalized patients accounts for 5%, and the incidence of AKI in intensive care units accounts for 30-50%. Rate and morbidity remain high [P. Devarajan, Biomarkers for the early detection of acute kidney injury. Current opinion in pediatrics23(2011) 194-200]. [0003] The lack of specific early clinical diagnostic indicators is the main obstacle to the treatment of this disease. In current clinical practice, the standard for diagnosing AKI is serum creatinine level. Unfortunately, creatinine i...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577C07K16/18C12N5/20
CPCG01N33/566G01N33/92
Inventor 孙兵伊春艳凌志洋边超
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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