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

Saturation binding ratiometric assay

Inactive Publication Date: 2020-09-10
LUMIRADX UK LTD
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present patent introduces a new method for measuring the ratio of beta-chain amino-terminal peptides in hemoglobin A1c (HbA1c) in a more accurate and reliable way. This is accomplished by using consistently prepared surfaces, such as beads, that bind to Hb with saturation. By measuring the signal level from the HbA1c subpopulation, the method provides an effective normalization of the amount of HbA1c in a sample. The method is simpler to run and can be used in simplified assay devices. The patent also describes the use of two specific binding agents that help in the detection of HbA1c. The first specific binding agent is chosen to expose the amino-terminal peptides of beta-chain in a way that facilitates binding of anti-A1c. The method involves contacting magnetic beads bearing the first specific binding agent with sample containing Hb and an excess of second labeled binding agent specific for A1c, thereby forming a sandwich comprising magnetic beinsolated total Hb and the second labeled binding agent. The intensity of the signals from the magnetic beads is used to determine the total Hb and the fractions of HbA1c and other hemoglobin isoforms. The ratio of A1c subpopulation to total Hb can then be calculated from the respective measures of total particles and the A1c subpopulation signal. The technical effects of this patent include improved accuracy and reliability in measuring the ratio of beta-chain amino-terminal peptides in HbA1c, as well as simplification of the assay methodology and devices.

Problems solved by technology

The non-specific adsorption used in Maruo has the drawback that such adsorption is subject to many interferences and requires a substantial wash step to remove unbound A1c before adding the anti-A1c.
Such wash step commonly results in greater imprecision.

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
  • Saturation binding ratiometric assay
  • Saturation binding ratiometric assay
  • Saturation binding ratiometric assay

Examples

Experimental program
Comparison scheme
Effect test

example 1

h Magnetic Anti-Hemoglobin Particles

[0135]Assays were carried out using magnetic beads bearing surface anti-total hemoglobin antibodies as schematically shown in FIG. 2. The antibodies were obtained from mice using an immunogen containing hemoglobin beta chain amino terminal peptide and selected as described above, such that the antibodies bind with an epitope within amino acids 7-25 of the hemoglobin beta chain and cause exposure of the beta chain amino terminus. The magnetic beads used were 200 nm. The assays were carried out as dry A1c assays, meaning reagents were provided in dry form (as fast reconstituting reagent pellets) and were reconstituted in the assay device during the performance of the assay. To carry out the assays, sample was introduced into a first assay device chamber, thereby reconstituting dry anti-Hb magnetic beads within the chamber, and incubated for 60 s, followed by mixing in the same chamber for 90 s. The magnetic beads were then moved to a second chamber ...

example 2

h Magnetic Anti-Hemoglobin Particles and Undiluted Blood Sample

[0141]A magnetic bead assay has been carried out using undiluted blood. For these assays, we used six different undiluted frozen blood samples, which contained 0, 3.9, 6.6, 9.7, 12.7, and 14.4% A1c respectively. The assays were carried out per FIG. 2, wherein magnetic beads modified with anti-total hemoglobin antibody were mixed with hemolyzed whole blood at 14 g / dL hemoglobin. After 5 min incubation, the excess blood was removed from magnetic beads by magnetic separation and the beads were washed 2 more times with a buffer. Time resolved fluorescent (TRF) beads modified with anti-A1c antibody were added to the washed magnetic beads. After 5 min incubation the unbound TRF beads were washed thrice with buffer with an aid of a magnet. The TRF signal from magnetic-TRF sandwich immunocomplexes was recorded in a TRF reader. The results shown in FIG. 7 demonstrate that the present assays can be effectively carried out using un...

example 3

low Hb A1c Assay

[0142]Another illustrative assay has been carried out using TRF label in lateral flow configuration.

[0143]In lateral flow assay format, 5 uL of hemolyzed A1c calibrators were mixed with 3 uL of 100 ug / mL time resolved fluorescent (TRF) beads modified with anti-total hemoglobin antibody. After 5 min incubation at room temperature (rt), 2 uL of 35 ug / mL biotinylated A1c antibody was added followed by additional 5 min incubation at room temperature (rt). The reaction mixture was then allowed to run by capillary forces up a nitrocellulose membrane strip containing test streptavidin zone and a reference anti-mouse antibody striped zone. The reference zone is designed to capture any excess of unbound beads modified with mouse anti-total hemoglobin antibody. The reaction mixture was finally chased with an elution buffer to clear unbound particles. The finished and dried strips were read in a TRF reader to provide results shown in FIG. 8. Dose responses from both test and co...

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

Methods, devices, and reagents are described for performing ratiometric assays for hemoglobin A1c. The methods involve a direct ratio determination between Hb A1c and normalized total hemoglobin utilizing a saturating amount of hemoglobin so that Hb A1c binds proportionately to a substrate. In some applications, the assay utilizes a proximity label system for signal generation and / or labeled magnetic beads. The methods can be configured as homogeneous or heterogeneous assays.

Description

FIELD OF THE INVENTION[0001]The present invention relates to an assay for glycated hemoglobin.SEQUENCE LISTING[0002]The sequence listing submitted herewith as ASCII text file entitled XENB2204_SeqListing_ST25.txt, created on Mar. 3, 2019, with a size of 1690 byte, is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[0003]The following discussion is provided solely to assist the understanding of the reader, and does not constitute an admission any of the information discussed or references cited constitute prior art to the present invention.[0004]Control of blood glucose concentrations in diabetic patients has been shown to decrease the frequency and severity of long-term microvascular and neurologic complications of the disease. It has been found that the rate of formation of glycated hemoglobin is directly related to the glucose concentration in blood. As a result, in the management of glucose levels, measurement of glycated hemoglobin is used to determin...

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): G01N33/72G01N33/543
CPCG01N33/54326G01N2333/805G01N33/723G01N33/543
Inventor MANNEH, VICTORSVAROVSKY, SERGEI
Owner LUMIRADX UK LTD
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