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

Method for the diagnosis of c3nef-mediated membranoproliferative glomerulonephritis

A glomerulonephritis, proliferative technology, applied in the direction of disease diagnosis, biological testing, biomaterial analysis, etc., can solve the problem of not describing the evaluation of C3 convertase decay

Pending Publication Date: 2020-12-22
红公里科学技术园股份公司
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the reported method does not describe the assessment of C3 convertase decay

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
  • Method for the diagnosis of c3nef-mediated membranoproliferative glomerulonephritis
  • Method for the diagnosis of c3nef-mediated membranoproliferative glomerulonephritis
  • Method for the diagnosis of c3nef-mediated membranoproliferative glomerulonephritis

Examples

Experimental program
Comparison scheme
Effect test

no. 1 approach

[0024] In a first embodiment, the method according to the invention comprises:

[0025] a) immobilizing C3b in plate wells, preferably 96 microwell plates;

[0026] b) adding IgG purified from biological samples isolated from patients and / or healthy subjects to the wells to form C3bBb complexes in the presence of FB, FD, BSA and magnesium ions;

[0027] c) detachment of the complex formed;

[0028] d) the complex is subjected to electrophoresis, and due to denaturing conditions (presence of SDS), the two components of C3 convertase, C3b and Bb, dissociate and are transferred to the membrane;

[0029] e) Detection of the protein of interest Bb to obtain a defined baseline value

[0030] It is characterized in that, after said step b) of adding purified IgG, in one or more wells, after a washing step, further incubation in the presence or absence of FH, wherein in the sample subjected to said further incubation , the ratio of the measured intensity of the Bb band to the basel...

no. 2 approach

[0039] The second embodiment includes the following steps:

[0040] a) immobilization of C3b in the wells of the plate;

[0041] b) adding FB, FD, BSA and magnesium ions in the pores;

[0042] c) in at least a first of said wells, a complex formation and decay step comprising washing and incubating in the absence and / or presence of FH with IgG purified from a biological sample of a subject, and in said In at least a second of the wells, the complex formation and decay step comprises washing and incubation with IgG purified from a control sample in the absence and / or presence of FH;

[0043] d) detachment of the complex formed;

[0044] e) electrophoresis of the complex under denaturing conditions to dissociate the two components C3b and Bb and transfer to the membrane;

[0045] f) detection of the protein of interest Bb to obtain values ​​for samples obtained in wells in which the decay step was performed in the presence of IgG isolated from the patient, and for said decay ...

Embodiment 1

[0051] Example 1: Analysis of C3NeF activity in samples from healthy or pathological subjects

[0052] In the presence of C3NeF-IgG purified from MPGN patients (IgG patients) or from healthy controls (IgG controls), in FB, FD and MgCl 2 In this case, the C3 convertase complex C3bBb (Mg 2+ ). Spontaneous or FH-mediated decay was monitored after further incubation at 25°C for 32 min in the absence or presence of FH, respectively. The results of WB analysis are shown in figure 1 middle. The amount of C3 convertase formed was calculated as the ratio of the density measurement of the Bb band after decay to the density measurement of the band corresponding to the baseline (*100).

[0053] Analysis Mean ± 2Dev Std of the results obtained by testing 35 samples from healthy donors in which a residual Bb higher than 36% relative to baseline was observed after decay in the absence of FH in which said decay occurred Samples were considered positive for bands, or in the case of said...

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

No PUM Login to View More

Abstract

A method for the diagnosis of C3NeF-mediated membranoproliferative glomerulonephritis (MPGN) in a sample comprising: a) immobilization of C3b in at least two wells of a plate; b) formation in said wells of the C3bBb complex, and, optionally, denaturation of the same complex, optionally with the addition of Factor H (FH), wherein said formation of the complex is mediated by the addition of IgGs purified from said sample and / or control in the presence of Factor B (FB), Factor D (FD), bovine serum albumin (BSA), magnesium ions; d) detachment of the formed complexes; e) electrophoretic run under denaturing conditions of said complexes and transfer on a membrane; detection of the protein of interest, Bb, which is one of the two components of C3 convertase; wherein the ratio of the intensities measured for the Bb band in the samples collected by each of said wells is indicative of C3NeF activity in said sample.

Description

[0001] The present invention relates to methods of diagnosing C3NeF-mediated membranoproliferative glomerulonephritis in a subject. The method includes: [0002] a) Immobilizing C3b in at least two wells of the plate; [0003] b) Formation of the C3bBb complex representing the C3 convertase of the alternative pathway in the pore, wherein said formation of said complex is achieved by the presence of factor B (FB), factor D (FD), bovine serum albumin (BSA ), in the presence of magnesium ions, mediated by the addition of IgG purified from biological samples isolated from patients and / or healthy subjects; [0004] c) detachment of the complex formed; [0005] d) electrophoresis of the complex under denaturing conditions and transfer to a membrane; [0006] e) detecting the protein of interest Bb, which is one of the two components C3b and Bb of the C3 convertase; [0007] wherein the measured ratio of the intensity of the Bb band in each collected sample in said well represents ...

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
Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/564G01N33/68
CPCG01N33/564G01N33/6854G01N33/6893G01N2800/347G01N2333/4716
Inventor 罗伯塔·多纳代利玛丽娜·诺里斯艾瑞拉·贝尼尼朱塞佩·雷穆齐
Owner 红公里科学技术园股份公司
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