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B cell epitope peptide of human neutrophil gelatinase associated lipocalin and application thereof

A lipocalin and neutrophil technology, applied in the medical field, can solve problems such as affecting the application of AKI diagnosis and failing to develop antibodies, and achieve the effects of high titer, good specificity and high purity

Active Publication Date: 2012-11-14
重庆业为基生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In fact, there is too much variation in the test results of these kits in the population. For example, Bioporto Diagnostic reported that the concentration of NGAL in the urine of ICU patients in a hospital is between 110 and 40,000 ng / ml, and the concentration of NGAL in blood is between 25 and 3491 ng / ml. The huge difference among individuals may be due to the fact that when AKI is injured, the main form of NGAL in the urine is the monomeric NGAL re-synthesized and secreted by the injured epithelium, and the specific antibody against the characteristic NGAL of the renal epithelium has not been developed, which affects the Its application to the diagnosis of AKI

Method used

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  • B cell epitope peptide of human neutrophil gelatinase associated lipocalin and application thereof
  • B cell epitope peptide of human neutrophil gelatinase associated lipocalin and application thereof
  • B cell epitope peptide of human neutrophil gelatinase associated lipocalin and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0032] The preparation of embodiment 1NGAL recombinant protein

[0033] On the basis of obtaining the NGAL coding gene from GENBANK, carry out codon preference modification, chemically synthesize the coding gene fragment, clone it into a prokaryotic expression vector and induce protein expression after DNA sequencing and identification, and then purify and prepare NGAL recombinant protein in large quantities. The protein can be used as an immunogen and screening agent for antibody preparation, and can also be used as a calibrator for the establishment of NGAL quantitative detection in subsequent experiments, specifically:

[0034] One NGAL recombinant protein gene clone:

[0035] Obtain the gene sequence of human NGAL protein from GENBANK (accession number is NM 005564.3), and submit it to Graphical codon usage analyzer (http: / / guca.schoedl.del) to analyze its codon bias; specifically, human The synonymous replacement of NGAL gene codons with a usage rate of <10% in E. coli i...

Embodiment 2

[0058] Example 2 NGAL molecular B cell epitope prediction and polypeptide synthesis

[0059] The sequence of the chemically synthesized B cell epitope peptide is obtained from the results of bioinformatics analysis, and the NGAL protein sequence is obtained from the NCBI Protein database, and the sequence is shown in SEQ ID NO:2. Comprehensively analyze the secondary structure, antigenicity, hydrophilicity and hydrophobicity, accessibility, flexibility and possible glycosylation sites of NGAL protein, analyze and score each segment, and select the region with a high score as the B cell epitope region. Specifically, use Chou & Fasman to predict β-turn angle, Emini method to predict antigen surface accessibility, Karplus & Schulz method to predict protein flexibility, Kolaskar & Tongaonkar protein antigenicity analysis, Parker method protein hydrophobicity analysis, Bepipred linear epitope prediction and The glycosylation information of NGAL was obtained from the UniProt databas...

Embodiment 3

[0062] Example 3 Preparation and Identification of Monoclonal Antibody

[0063] Immunization of Balb / c mice with a peptide derivative of NGAL B cell epitope

[0064] The NGAL B cell epitope peptide antigen obtained in Example 2 was taken out from the -80°C refrigerator as an antigen, dissolved and then filtered. Female Balb / c mice aged 6 weeks and weighing about 20 g were selected for immunization. Antigen emulsification adopts double syringe mutual pushing method. For the first immunization, the NGAL B cell epitope peptide antigen shown in SEQ ID NO: 3 was emulsified and mixed with an equal volume of Freund's complete adjuvant to obtain the antigen mixture, and each mouse was intradermally multi-pointed in an amount of 100 μg Plus intraperitoneal injection. The second and third immunizations were carried out on the 14th and 28th days respectively. The adjuvant was changed to incomplete Freund's adjuvant. The amount of antigen, injection volume and route remained unchanged....

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Abstract

The invention belongs to the medical field and particularly relates to an acute kidney injury diagnosis technology, a B cell epitope peptide of human NGAL (Neutrophil Gelatinase Associated Lipocalin) and a hybridoma cell prepared by same, and an application of the B cell epitope peptide and a specific antibody thereof and an immune complex of the specific antibody in preparing diagnostic reagent for acute kidney injury. The B cell epitope peptide prepared by the invention immunizes a monoclonal antibody prepared by adopting a rat and has the advantages of high purity (The SDS (Sodium Dodecyl Sulfate)-PAGE (PolyAcrylamide Gel Electrophoresi) detection purity is more than 96 percent), high valence (the ELISA (Enzyme-Linked Immuno Sorbent Assay) valence reaches 1:256000), good specificity, mass preparation and the like; and through the monoclonal antibody and a polyclonal antibody prepared by the B cell epitope peptide, the B cell epitope peptide can be used for detecting the content of the NGAL in the urine of a patient, for example, by adopting a double antibody sandwich ELISA reaction mode, a double antibody sandwich structure formed by an enzyme labeled human anti-NGAL monoclonal antibody, an enzyme labeled plate package anti-NGAL polyclonal antibody and a measured sample NGAL antigen is detected.

Description

technical field [0001] The invention belongs to the field of medicine, in particular to a diagnosis technique for acute kidney injury. Background technique [0002] Acute kidney injury (acute kidney injury, AKI), sometimes confused with acute renal failure (acute renal failure, ARF), there is no uniform definition. The currently generally accepted concept of AKI refers to the whole process of renal structural changes, renal function decline, and eventually renal failure caused by various primary or secondary injury factors. Studies have shown that the incidence of AKI in patients after coronary artery bypass grafting reaches 26.6%, the incidence of AKI after severe trauma reaches 50%, and the mortality rate of AKI in the intensive care unit (ICU) is as high as 37% to 76.19%. A clinical syndrome with high morbidity and mortality. Early treatment is very important for the reversal of AKI, and early diagnosis is the premise and key of early treatment. [0003] The current di...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K16/18C12N5/20G01N33/577G01N33/543C12R1/91
Inventor 黄洪涛石延宾姚静张宪胡伟秦川魏勇
Owner 重庆业为基生物科技有限公司
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