Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Latex enhanced immunoturbidimetric detection kit for retinol binding protein and clinical application thereof

A protein-binding and retinol-binding technology, applied in the field of medical immunodiagnosis, can solve the problems of low cost, difficulty, and difficult cloning of natural coding sequences, and achieve the effect of low cost, high accuracy, and good clinical application prospects

Active Publication Date: 2019-05-07
ZONHON BIOPHARMA INST
View PDF5 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As we all know, Escherichia coli has a clear genetic background, simple culture method, short growth cycle, and low cost. It is an ideal tool for expressing proteins, but there are also operational difficulties that the natural coding sequence is not easy to clone.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Latex enhanced immunoturbidimetric detection kit for retinol binding protein and clinical application thereof
  • Latex enhanced immunoturbidimetric detection kit for retinol binding protein and clinical application thereof
  • Latex enhanced immunoturbidimetric detection kit for retinol binding protein and clinical application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Preparation of embodiment 1 rhRBP4 protein

[0041] 1. Construction of expression vector of rhRBP4 gene

[0042] According to the cDNA sequence of hRBP4 published by NCBI (GenBank accession number: NM_006744.3), the inventor obtained the rhRBP4 gene of the present invention after codon optimization, as shown in SEQ ID No:2.

[0043] The optimized rhRBP4 whole gene was introduced into the NdeI restriction site sequence at the 5' end, and the XhoI restriction site sequence was introduced at the 3' end, and the whole gene synthesis was carried out, and the synthetic gene fragment was constructed into the pUC57 plasmid (provided by Nanjing Jin Provided by Sri Technology Co., Ltd.), a long-term storage plasmid was obtained, which was designated as pUC57-rhRBP4 plasmid. The pUC57-rhRBP4 plasmid was used as a template for PCR amplification, and the primer sequences used were as follows:

[0044] Upstream primers:

[0045] M13F: TGT AAA ACG ACG GCC AGT

[0046] Downstream...

Embodiment 2

[0076] Preparation of Example 2 Rabbit Polyclonal Antibody

[0077] 1. Three days before immunization, blood was collected from the ear vein as a negative control (500ul per mouse, let stand at 37°C for 1 hour, centrifuge at 1500rpm for 15 minutes, draw serum and store at -20°C);

[0078] 2. After 3 days, mix the recombinant human RBP4 protein (1 mg / rabbit) with complete Freund's adjuvant in an equal volume to form a "water-in-oil" form, and inject it subcutaneously at multiple points on both sides of the rabbit's spine;

[0079] 3. The second immunization after 2 weeks: mix the recombinant human RBP4 protein (1 mg / rabbit) with incomplete Freund's adjuvant in an equal volume to form a "water-in-oil" form, and subcutaneously on both sides of the spine at the injection site (injection The site should be different from the first time); one week after the immunization, blood was collected from the ear vein (the blood processing method is the same as step 1);

[0080] 4. The third...

Embodiment 3

[0087] Embodiment 3 The preparation and use method of detection kit

[0088] The detection kit of the present invention is a liquid double reagent, which includes reagent R1, reagent R2 and calibrator, see below for details:

[0089] 1. Reagent R1: Add stabilizer (for example: NaCl), coagulant (for example: PEG6000), protective agent (for example: BSA), preservative (for example: Proclin 300), chelating agent (for example: EDTA), stirred and mixed, that is the R1 reagent.

[0090] 2. R2 reagent: Dilute the rabbit anti-human retinol-binding protein polyclonal antibody to 2 mg / mL with phosphate buffer to prepare antibody dilution; use MES buffer to dilute latex microspheres (polystyrene latex, Purchased from Japan JSR Company, particle size 80-300nm) was diluted to 1% (mass concentration), then added 0.5%-2% (mass concentration) of EDC, stirred and reacted at 30°C for 30min, after the reaction was completed, centrifuged to remove untreated The reacted EDC, then add the rabbit ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Dominant wavelengthaaaaaaaaaa
Sensitivityaaaaaaaaaa
Login to View More

Abstract

The invention relates to a latex enhanced immunoturbidimetric detection kit for retinol binding protein and an application thereof in clinical detection of content of retinol binding protein (RBP4) inserum, and belongs to the field of in vitro diagnosis of medical immunology. The RBP latex enhanced immunoturbidimetric detection kit provided by the invention adopts recombinant expressed rhRBP4 asa calibration product, a quality control product and related rabbit polyantibody immune antigens, and the rabbit polyantibody, the rhRBP4 calibration product and the quality control product are used for preparing the latex enhanced immunoturbidimetric detection kit, and the content of RBP4 in blood can be accurately detected. Compared with commercial products, the kit has better specificity and higher accuracy, and shows better clinical application prospect. Besides, the recombinant expression method of the rhRBP has the characteristics of short period, large expression amount and low cost, and improves the quality uniformity of related products to a great extent.

Description

technical field [0001] The invention belongs to the field of in vitro medical immunodiagnosis, in particular to a recombinant retinol-binding protein and its application in a detection kit for detecting the content of retinol-binding protein (Retinol-Binding Protein, RBP4) in blood samples. Background technique [0002] Retinol-binding protein (RBP4), with a molecular weight of 21kDa, is a transporter of vitamin A in blood and is widely distributed in blood, cerebrospinal fluid, urine and other body fluids. In the blood, RBP4 exists in the complex form of 1:1:1 (molar ratio) with retinol and prealbumin, and transports 90% of retinol in the body to body tissues. When RBP4 binds to the RBP receptor on the cell surface, retinol enters the cell, the complex disintegrates, and free RBP4 is filtered out from the glomerulus, most of which are reabsorbed by proximal renal tubular epithelial cells and decomposed, For tissue utilization, only a small amount is excreted in urine. Dis...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K14/47C12N15/70C12N1/21C07K16/18G01N33/68C12R1/19
Inventor 马永杨芸赵利利赵百学
Owner ZONHON BIOPHARMA INST
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com