Anti-human neutrophil gelatinase-related lipid carrier protein antibody and application thereof in detection test paper card

A technology of lipocalin and neutrophils, applied in anti-animal/human immunoglobulin, application, biological testing, etc., can solve the problems of narrow detection range, long detection cycle, small linear range of measurement, etc., to achieve Easy operation and convenient mass production

Active Publication Date: 2019-04-02
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the sensitivity of chemiluminescence method is high, the linear range of measurement is small and the detection cost is high. It requires specific instruments and is difficult to be widely used in clinical practice. Immunofluorescence chromatography), the determination of NGAL can be completed within 15 hours, and the detection range is 50-2000ng / mL
These detection systems can only detect urine or plasma alone, and the amount of specimen is large, the detection cycle is long, and the detection range is narrow, which limits its routine clinical application.

Method used

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  • Anti-human neutrophil gelatinase-related lipid carrier protein antibody and application thereof in detection test paper card
  • Anti-human neutrophil gelatinase-related lipid carrier protein antibody and application thereof in detection test paper card
  • Anti-human neutrophil gelatinase-related lipid carrier protein antibody and application thereof in detection test paper card

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Preparation of anti-human neutrophil gelatinase-associated lipocalin hybridoma cell line

[0041]1. Animal immunization BALB / c female mice (purchased from Changzhou Cavens Experimental Animal Co., Ltd. ). For specific immunization conditions, please refer to the "Experimental Guidelines for Antibody Preparation and Use". The serum titer of immunized mice was tracked by indirect ELISA method, and the immunized mouse with the highest serum titer was selected for fusion experiment of mouse splenocytes and mouse myeloma cells.

[0042] 2. Cell Fusion

[0043] (1). Preparation of spleen cells. Immunized mice were plucked from the eyeballs to take blood, put to death by breaking the cervical spine, soaked in 75% (v / v) alcohol for 10 minutes, took out the spleen in a sterile operating table, and placed it in In the cell sieve, fully grind the cells, pass through the sieve, centrifuge and wash several times with sterile 1640 medium (purchased from Gibco), resuspen...

Embodiment 3

[0058] Example 3. Recombinant expression and purification of single-chain antibody

[0059] According to the sequencing results in Example 2, a connecting peptide (GGGGS) 3 was added between the heavy chain and light chain variable regions of hybridoma cell lines NG02 and NG19, six histidines were introduced, and the entire gene was directly fused And the expression system of Pichia pastoris was used for the recombinant expression of the single chain antibody. The expressed antibodies were named as antibodies NG02 and NG19, respectively. The recombinant expression of the above-mentioned single-chain antibody is specifically as follows:

[0060] 1. Construction of single-chain antibody gene expression vector

[0061] The gene sequence of the single-chain antibody NG02 is shown in SEQ ID NO:19, and the amino acid sequence is shown in SEQ ID NO:17; the gene sequence of the single-chain antibody NG19 is shown in SEQ ID NO:20, and the amino acid sequence is shown in SEQ ID NO: 1...

Embodiment 4

[0072] Example 4. Performance evaluation of antibodies

[0073] 1. Western blot identification of antibodies NG02 and NG19

[0074] a. Polyacrylamide gel electrophoresis: configure 12% separating gel and 5% stacking gel, load standard protein and recombinant human neutrophil gelatinase-associated lipocalin protein (expressed in Escherichia coli system, prepared by our company) respectively , after the sample enters the separation gel, electrophoresis under constant pressure for 1 hour;

[0075] b. Membrane transfer: under constant flow (35mA / membrane) conditions, the membrane was transferred for 1 hour, and the protein on the polyacrylamide gel was transferred to the nitrocellulose membrane. Coomassie Brilliant Blue G250 was used to stain the SDS-PAGE gel after the transfer to observe the residual protein;

[0076] c. Blocking: blocking with 5% skimmed milk in TBST buffer (blocking solution), overnight at 4°C; after blocking, wash once with washing solution (TBST, see TaKaRa...

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Abstract

The invention relates to an anti-human neutrophil gelatinase-related lipid carrier protein antibody and an application thereof in a detection test paper card. A variety of antibodies are prepared andsubjected to paired screening, and a group of antibody combination (NG02 and NG19) with sensitivity and specificity that can meet the needs is obtained; at the same time, the antibodies are prone to mass production and can meet the needs of large-scale clinical application in the future. The antibody combination is used for debugging and optimizing the detection system, and a colloidal gold immunochromatographic quantitative detection card for human neutrophil gelatinase-related lipid carrier proteins is obtained, wherein the detection card is simple to operate and has sensitivity, specificityand relevant detection performance meeting the detection of human blood or urine samples.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to two kinds of anti-human neutrophil gelatinase-associated lipocalin antibodies and preparation methods thereof and the application of the above-mentioned antibodies in the quantitative detection of human neutrophil gelatinase-associated lipocalin application. Background technique [0002] Neutrophil gelatinase-associated lipocalin (NGAL), also known as apolipoprotein-2 (Lipocalin-2), is a member of the lipocalin superfamily and is a secreted protein with a relative molecular mass of 25 kDa. type glycoprotein. In a series of clinical studies, it has been proved that NGAL has diagnostic and prognostic value for acute kidney injury. Currently, NGAL is an important marker for the diagnosis of acute kidney injury. The levels of blood and urine NGAL increased when acute kidney injury occurred. Therefore, the detection of NGAL in urine and blood can be used as an early, sensit...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13G01N33/68G01N33/558
CPCG01N33/558G01N33/68C07K16/18C07K2317/565C07K2317/56C07K2317/622
Inventor 马永赵利利王安良
Owner ZONHON BIOPHARMA INST
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