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Quantitative detection card of human neutrophil gelatinase-associated lipocalin and clinical application thereof

A technology for lipocalin and neutrophils, which is applied in the biological field, can solve the problems of narrow detection range, long detection period, small linear range of measurement, etc., and achieves the effect of simple operation and convenient mass production.

Active Publication Date: 2019-05-03
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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  • Quantitative detection card of human neutrophil gelatinase-associated lipocalin and clinical application thereof
  • Quantitative detection card of human neutrophil gelatinase-associated lipocalin and clinical application thereof
  • Quantitative detection card of human neutrophil gelatinase-associated lipocalin and clinical application thereof

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

[0042]BALB / c female mice (purchased from Changzhou Cavens Experimental Animal Co., Ltd.) were immunized with recombinant human neutrophil gelatinase-associated lipocalin (recombinantly expressed in Escherichia coli, produced by our company) according to the general immunization procedure. 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.

[0043] 2. Cell Fusion

[0044] (1). Preparation of spleen cells

[0045] Take the immunized mice, remove their eyeballs, take blood, put them to death by breaking the cervical spine, soak them in 75% (v / v) alcohol for 10 minutes, t...

Embodiment 2

[0056] Example 2. Determination of the variable region sequence of the hybridoma cell line antibody

[0057] The variable region sequences of the above-mentioned hybridoma cell lines NG02 and NG19 were determined.

[0058] a. Extraction of RNA: Extract the total RNA of the above-mentioned hybridoma cell lines NG02 and NG19 with reference to the instructions of the Total Cell RNA Extraction Kit (purchased from Roche Company) and perform reverse transcription immediately;

[0059] b. Reverse transcription of RNA into DNA: Refer to Thermo Scientific Reverted First strand cDNASynthesis Kit (purchased from Thermo Company) to reverse-transcribe the total RNA extracted in the previous step to obtain cDNA, and freeze it at -20°C for later use;

[0060] c. PCR amplification and recovery of the variable region sequence: the cDNA obtained in the above step is used as a template, and the variable region sequence of the heavy chain and light chain is sequenced with the general primer for t...

Embodiment 3

[0064] Example 3. Recombinant expression and purification of single-chain antibody

[0065] 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:

[0066] 1. Construction of single-chain antibody gene expression vector

[0067] 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...

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Abstract

The invention relates to a quantitative detection card of human neutrophil gelatinase-associated lipocalin and clinical application thereof. Several kinds of antibodies are prepared and pairing and screening are carried out; and one group of antibodies including NG02 and NG19 that have the sensitivity and specificity meeting the requirements of the quantitative detection card is obtained. Meanwhile, the large-scale production is realized conveniently and thus the need of large-scale clinical application in future is met. After debugging optimization work of the detection system on the antibodygroup, the quantitative detection card of human neutrophil gelatinase-associated lipocalin is obtained, wherein the quantitative detection card has advantages of simple operation and high sensitivityand has the specificity and related detection performance that can meet the body blood and urine sample detection requirement.

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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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
Inventor 马永丁娜
Owner ZONHON BIOPHARMA INST
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