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Multiplexed detection of anti-red cell alloantibodies

A technology of alloantibodies and red blood cells, applied in measuring devices, immunoassays, biological tests, etc., can solve problems such as the difficulty of constructing a large number of unique passwords

Inactive Publication Date: 2009-02-11
BIOARRAY SOLUTIONS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, it is difficult to construct a large number of unique codes using surface encoding methods, unless, using a few dyes to produce many different colors, the encoding response can be finely tuned, or using a large library of dyes with different fingerprint spectra

Method used

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  • Multiplexed detection of anti-red cell alloantibodies
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  • Multiplexed detection of anti-red cell alloantibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 Separation of red blood cells and preparation of labeled ghost cells

[0017] Red blood cell wash-storage buffer

[0018] Sodium chloride = 4.383g

[0019] Monosodium phosphate = 0.345g

[0020] Magnesium chloride hexahydrate = 0.203g

[0021] Benzylsulfonyl chloride=0.0435g, dissolved in 500ml of distilled water.

[0022] Red blood cell lysis buffer:

[0023] Monosodium phosphate = 0.69g

[0024] Magnesium chloride hexahydrate = 0.203g

[0025] Benzylsulfonyl chloride=0.0435g, dissolved in 500ml of distilled water.

[0026] RBC blocking solution:

[0027] Sodium chloride=6g, dissolved in 50ml of distilled water.

[0028] Test plan:

[0029] i) Take 1ml of storage buffer and put it into a 2ml centrifuge tube

[0030] ii) Add 1 drop of fresh blood from a finger prick (~25ul), invert gently to mix

[0031] iii) Centrifuge at ~600-100Og for 2 minutes and remove supernatant. Add 1.5 ml storage buffer, gently resuspend the RBC pellet and repeat centrifu...

Embodiment 2

[0038] Example 2: Generation of libraries of encoded ghost cells

[0039] TAMRA-labeled dextran with a molecular weight of 3000 (labeling density of 1mol / mol) was obtained from Molecular Probes, and FITC-labeled dextran with a molecular weight of 4000 (labeling density of 0.05mol / mol-0.5 / mol / mol) was obtained from Obtained from Sigma-Aldrich Company. Five different stock solutions at concentrations of 10 mg / ml, 2 mg / ml, 0.4 mg / ml, 0.08 mg / ml and 0.016 mg / ml were prepared for each dextran using storage buffer. The recipe in Example 1 was used to prepare 10 different populations of encoded ghost cells. These ghost cells were characterized for their fluorescence using flow cytometry. A 1:1 (v / v) mixture of TAMRA and FITC-dextran was used to generate encoded ghost cells with both encoding dyes, respectively.

Embodiment 3

[0040] Example 3: Generation of Libraries of Encoded Ghost Cells for Magnetic Response

[0041] Encoded ghost cells can be made magnetic by magnetic cell surface labeling methods known in the art. Several companies sell kits for magnetically labeling and isolating target cells (www.miltenyibiotec.com, www.immunicon.com, www.dvnalbiotech.com). Magnetically labeled whole blood cells are also commercially available (www.diagast.com). A particularly contemplated method utilizes anti-human IgG-coated magnetic nanobeads for labeling, which can be added after binding of antibody from the sample, e.g. figure 2 as outlined.

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Abstract

Disclosed are methods for detecting antibody in a sample, where the antibody targets an antigen expressed by red blood cells or red blood cell ghosts. Rather than detecting the binding events between a particular antigen antibody pair (as in traditional agglutination based assays) the methods herein allow for multiplexed detection of clinically important allo-immune antibodies to blood group antigens. Specifically the method involves generating fluorescently encoded red blood cells or red blood cell ghosts with known antigen presentation and using them to detect the presence of antibody in serum / plasma with a fluorescent sandwich type immunoassay. The assay results can be read using flow cytometric or fluorescent microscope based imaging techniques.

Description

Background of the invention [0001] When minor blood group antigens (such as: Jk, Rh, Kell, Kidd, and Duffy) are introduced into sensitized receptors, they can induce autoantibodies and / or alloantibodies, causing autoimmune hemolytic anemia, hemolytic anemia, Hemolytic disease of the newborn and hemolytic transfusion reactions. Sensitization can result from previous blood transfusions or pregnancy with an antigen-positive fetus. These alloantibodies are typically detected and identified by detection of recipient sera followed by control in separate agglutination assays against a panel of red blood cells (RBCs) of known phenotype (for example, anti-Jka antibodies can be detected by determining whether the patient's serum is associated with Jk a+ RBC reacts without reacting with Jk a- RBC reaction to identify). By selecting multiple samples of RBCs of various antigenic phenotypes, it is possible to detect the presence or absence of clinically important alloantibodies. [0002...

Claims

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Application Information

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IPC IPC(8): G01N33/53C12N5/07C12N5/071C12N5/078
CPCY10T436/13G01N33/80Y10T436/2525Y10T436/106664
Inventor 苏堪塔·本纳吉
Owner BIOARRAY SOLUTIONS
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