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Compositions and methods for detecting antibodies against natural human leukocyte antigen

A human leukocyte antigen, leukocyte antigen technology, applied in the detection of programmed cell death, receptor/cell surface antigen/cell surface determinant, compound screening, etc., can solve the problem of distinguishing between natural HLA antibodies and denatured HLA antibodies, etc.

Active Publication Date: 2016-11-16
美国莱姆德有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, these substrates are limited in their ability to distinguish native HLA antibodies from denatured HLA antibodies

Method used

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  • Compositions and methods for detecting antibodies against natural human leukocyte antigen
  • Compositions and methods for detecting antibodies against natural human leukocyte antigen
  • Compositions and methods for detecting antibodies against natural human leukocyte antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] 7.1 Example 1: Preparation of Microbeads Linked to Native Class I HLA

[0109] This example provides an exemplary preparation of microbeads linked to native HLA class I ("native HLA class I microbeads"). LABScreen Single Antigen Beads (LSAB), specifically Single Antigen HLA Class I – Combi (Catalogue ID: LS1A04) was purchased from One Lambda, Inc. Untreated, LSABs contain native and denatured class I HLA bound to the bead surface.

[0110] Figures 1A-1C The reactivity of W6 / 32 and HC10 with LSAB is shown. Monoclonal antibody W6 / 32 specifically binds native class I HLA. Monoclonal antibody HC10 specifically binds to denatured class I HLA. Figures 1A-1C described for the A-locus ( Figure 1A ), B-locus ( Figure 1B ) and the C-locus ( Figure 1C) mean fluorescence intensity (MFI) of class I HLA alleles. The MFI level of the detected antibody corresponds to the level of binding of the antibody to native or denatured class I HLA. These figures show that LSAB is r...

Embodiment 2

[0115] 7.2 Example 2: Detection of natural class I HLA microbeads

[0116] This example demonstrates the high purity of exemplary microbeads prepared according to the methods described in this application. To confirm that proteolytic digestion removed denatured class I HLA, bead panels prepared according to Example 1 were incubated with W6 / 32 or HC10 to detect native or denatured class I HLA, respectively.

[0117] About 1 μl of the diluted antibody dissolved in about 100 μl of about 1X PBS was incubated with native HLA class I beads on a shaker at room temperature for about 30 minutes. Then use about 1mL Washing Solution The beads were washed three times and then incubated with about 100 μl of phycoerythrin-conjugated (PE-conjugated) goat anti-mouse secondary antibody for about 30 minutes at room temperature on a shaker. The beads were then washed three times and analyzed with a Luminex flow cytometer.

[0118] Figures 2A-2C The reactivity of W6 / 32 (specific for native ...

Embodiment 3

[0119] 7.3 Example 3: Purity of Natural Class I HLA Microbeads

[0120] This example provides the purity of several exemplary samples of native HLA class I microbeads prepared according to the methods described in this disclosure. Tables 1-3 below show the percentages of remaining denatured class I HLA in microbeads prepared according to the preparation method provided in Example 1 above and detected using the method provided in Example 2 above. Calculate the percentage of denatured class I HLA according to the formula: [HC10 MFI / (HC10 MFI+W6 / 32 MFI)]*100. Data are from 98 bead samples (5 batches).

[0121] The data in Table 1 below are the specificity of native HLA class I microsphere samples for the A-locus class I HLA alleles. The data showed that the mean percentage of denatured class I HLA was 0.81% with a standard deviation of 0.97%.

[0122] Table 1: Percentage of denatured HLA on native HLA class I beads (A-locus)

[0123] Sample ID

[0124] The data in T...

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Abstract

The present disclosure provides compositions comprising native and denatured human leukocyte antigens (HLA) and methods of making the compositions. The present disclosure also provides methods and kits for detecting anti-native HLA antibodies. So far, native HLA is the main component of the composition, which is prepared by enriching native HLA in a mixed composition by means of a selective enzymatic reaction.

Description

[0001] 1. Cross-references to related applications [0002] This application claims priority to US Provisional Application No. 61 / 338,258, the entire contents of which are hereby incorporated by reference. 2. Field of invention [0003] The compositions and methods provided in the present disclosure generally relate to compositions, such as compositions for detecting antibodies against native human leukocyte antigen (HLA), and methods of making the same. 3. Background technology [0004] Human leukocyte antigens (Human leukocyte antigens, HLA) can bind and display antigens on the surface of human cells to effector T cells. The two main classes of HLA, class I and class II HLA, present both alloantigens and native antigens. Class I HLAs can bind and present intracellularly produced peptide antigens, including viral and tumor-specific proteins, to CD8+ effector T cells (eg, Cytotoxic T cells (CTLs)). In response to alloantigens presented by cells containing HLA class I, CD8+...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/705G01N33/68C12P21/00
CPCG01N2333/70539G01N2800/24G01N2800/245G01N2500/04Y10T436/25375Y10T436/25125G01N33/566G01N33/564G01N33/532G01N33/6854
Inventor P·I·泰萨奇邓纯灿A·伊迪卡
Owner 美国莱姆德有限公司
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