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Combination measuring

A technology of binding activity and LAG-3, applied in the field of probes and kits, can solve the problems that cannot be used to determine the specific binding of IMP321 to immobilized cells

Inactive Publication Date: 2018-06-26
伊缪泰普有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, well-to-well signal variation was found to be unacceptable
Given this, it was concluded that, among the quality control assays used to test GMP-grade products, neither the MSD ECL assay nor the ELISA assay could be used to determine the specific binding of IMP321 to immobilized cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Evaluation of the use of a fluorescence-activated cell sorting (FACS) assay for determining the binding of IMP321 to Raji cells

[0095] FACS assays were performed to determine the binding of IMP321 to Raji cells. IMP321 samples with 100%, 75% and 50% MHC class II binding activity were tested. A sample with 100% activity is a reference sample with known MHC class II binding activity at a predetermined concentration. Samples with 75% and 50% activity were prepared by diluting the reference sample.

[0096] The obtained binding curves are shown in figure 2 middle. They show that no upper plateau is reached, so there is no parallelism between the binding curves of the reference sample with 100% activity and the other samples. This prevents calculation of relative potencies of different samples.

Embodiment 2

[0098] Evaluation of the use of the Meso Scale Discovery (MSD) assay for determining the binding of IMP321 to Raji cells

[0099] This example describes the evaluation of the Meso Scale Discovery (MSD) assay used to determine the binding of IMP321 to Raji cells.

[0100] The Meso Scale Discovery platform (MSD-ECL) uses electrochemiluminescent labels conjugated to detection antibodies. When electrically stimulated in the appropriate chemical environment, these tags generate light, which can then be used to measure key proteins and molecules.

[0101] Power is applied to the plate electrodes via the Meso Scale Discovery platform (MSD-ECL), causing the markers to emit light. The light intensity is then measured to quantify the analyte in the sample.

[0102] The detection process starts at electrodes located in the bottom of a Meso Scale Discovery (MSD-ECL) microplate, and only labels near the electrodes are excited and detected. The system employs a buffer solution with a h...

Embodiment 3

[0109] Evaluation of non-specific binding of IMP321 to ELISA plates

[0110] This example describes the evaluation of non-specific binding of IMP321 and Rituxan to plates for enzyme-linked immunosorbent assay (ELISA) using different blocking reagents.

[0111] Briefly, microplates were blocked with blocking reagent for 2 hours at 25°C. Samples and rituxan controls were diluted to 2 μg / ml with dilution buffer and then further diluted by two-fold serial dilution. Before and after addition of diluted samples and incubation, the microplates were washed and drained well. After incubation with secondary antibodies, the signal was measured by spectrometry using a SpectraMax M2 (450-650 nm).

[0112]

[0113] The test results are in Figure 5 shown in . Figure 5 (a) shows the results of ELISA using increasing concentrations of IMP321 and ELISA plates blocked with 5% BSA or 10% FBS. Figure 5 (b) shows the results of ELISA using increasing concentrations of IMP321 or Rituxan...

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PUM

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Abstract

The invention discloses combination measuring, and discloses a method used for measuring the MHC II combination activity of Lymphocyte activationgene-3(LAG-3) protein, or fragments, derivatives, or analogues of Lymphocyte activationgene-3(LAG-3) protein. The method comprises measuring of the LAG-3 protein, the fragments, the derivatives, or the analogues using biolayer interferometry (BLI). The method can be used for quality control measuring of the compounds in good manufacturing practice (GMP) grade production. The invention also discloses a probe and a kit of the method.

Description

technical field [0001] The present invention relates to a method for determining the MHC class II binding activity of a lymphocyte activation gene-3 (LAG-3) protein or a fragment, derivative or analog preparation thereof, as well as probes and kits used in said method . Background technique [0002] The LAG-3 protein is a CD4I homologous membrane protein with four extracellular immunoglobulin superfamily domains. Like CD4, LAG-3 oligomerizes at the surface of T cells and binds MHC class II molecules on antigen presenting cells (APCs), but with significantly higher affinity than CD4. LAG-3 in activated CD4 + and CD8 + Expressed on T lymphocytes, where it associates with the CD3 / T cell receptor complex at the cell surface and negatively regulates signal transduction. Thus, it negatively regulates T cell proliferation, function and homeostasis. LAG-3 is upregulated on exhausted T cells compared with effector or memory T cells. LAG-3 is also upregulated on tumor infiltrati...

Claims

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Application Information

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IPC IPC(8): G01N21/45C07K14/705G01N33/68
CPCC07K14/70514G01N21/45G01N33/6854G01N33/6857G01N2021/458G01N2333/70514C07K14/70503G01N33/6872G01N2333/70503G01N33/68G01N2333/70539G01N2333/70596G01N2021/772G01N33/54393G01N33/52
Inventor 贾晓青陈敏
Owner 伊缪泰普有限公司
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