Kit for detecting specificity platelet antoantibody by combination of monoclonal antibody and nano-microspheres

A technology of nano-microspheres and autoantibodies, which can be used in measurement devices, instruments, fluorescence/phosphorescence, etc., can solve the problems that cannot be used as a routine inspection for diagnosing ITP, the steps are cumbersome, and the standardization is difficult.

Inactive Publication Date: 2010-05-26
苏州苏大赛尔免疫生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 1995, Koksch et al. used flow cytometry fluorescence resonance energy transfer to measure autoantibodies against specific sites on platelet membrane glycoproteins. However, this method is cumbersome and difficult to standardize, and cannot be used as a routine test for the diagnosis of ITP.

Method used

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  • Kit for detecting specificity platelet antoantibody by combination of monoclonal antibody and nano-microspheres
  • Kit for detecting specificity platelet antoantibody by combination of monoclonal antibody and nano-microspheres
  • Kit for detecting specificity platelet antoantibody by combination of monoclonal antibody and nano-microspheres

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The preparation of embodiment 1 kit

[0034] 1. Preparation and purification of monoclonal antibodies

[0035] The monoclonal antibodies SZ-2, SZ-21, SZ-22 and 7E3 in this example were self-made by the inventors. Monoclonal antibodies SZ-2, SZ-21, SZ-22 and 7E3 ascites were respectively at pH8.0, 0.1M PB (0.1mol / L Na 2 HPO 4 , 0.1mol / LNaH 2 PO 4, pH8.0)), centrifuge (8000rpm), take about 1ml of the supernatant and pass it through the column (affinity chromatography column ProteinG-Sepharose 4B) to fully bind each monoclonal antibody to protein G, buffer with 0.1M glycine-hydrochloric acid solution (pH2.8) to collect each monoclonal antibody peak. The collected monoclonal antibodies were concentrated with polyethylene glycol 20000, and then dialyzed in 0.01M PBS for 4 hours to obtain pure monoclonal antibodies SZ-2, SZ-21, SZ-22 and 7E3, which were stored in a low-temperature refrigerator for later use .

[0036] 2. Connection of monoclonal antibody to nanospheres...

Embodiment 2

[0044] Example 2 Detection of platelet-specific autoantibodies

[0045] 1. Preparation of platelet lysate

[0046] According to the platelet counts of patients and healthy people, 2ml of peripheral venous blood was collected with EDTA anticoagulated siliconized tubes, mixed gently and then centrifuged at 800rpm for 10min, and the upper layer of platelet-rich plasma was taken, centrifuged at 3000rpm for 10min, and the separated plasma was stored at -20°C (used as MAIPA) ), take the platelet pellet, wash the platelet 3 times with 0.05% EDTA-PBS, count the platelet, adjust the platelet concentration to 1×10 7 per ml, absorb 400 μl, centrifuge at 3000rpm for 5min, discard the supernatant, add 240μl Triton X-100 with a concentration of 0.5% (20min at room temperature), centrifuge at 3000rpm for 5min, draw the supernatant into 4 Eppendorf tubes, 50μl per tube, namely Obtain platelet lysate.

[0047] 2. Detection by flow cytometry

[0048] Pipette 50 μl of monoclonal antibody nano...

Embodiment 3

[0049] Example 3 Detection of the Stability of the Monoclonal Antibody Connected on the Microsphere

[0050] Place the four monoclonal antibody-nanospheres (SZ-2-nanospheres, SZ-22-nanospheres, SZ-21-nanospheres and 7E3-nanospheres) at 4°C for 45 days, every 9 Take 50 μl of monoclonal antibody-nanospheres to react with 10 μl of FITC-GAH-IgG (0.75 mg / ml) every day, and use flow cytometry to detect the average fluorescence intensity of the microspheres, which is used to reflect the stability of the connection between the antibody and the microspheres . The results showed that the amount of antibody on the surface of the detected microspheres changed very little, and the coefficients of variation were 2.15%, 2.65%, 4.22%, and 3.05%, respectively, and they could be stored at 4°C for at least 45 days.

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Abstract

The invention relates to a set of a kit for detecting specificity platelet antoantibody by the combination of monoclonal antibody and nano-microspheres. The kit comprises each 3 ml of four types of monoclonal antibody-polystyrene nano-microspheres PBS solution, 6 ml goat-anti-human polyclonal antibody and PBS solution, wherein the four types of the monoclonal antibody-polystyrene nano-microspheres PBS solution are sealed by bovine serum albumin and are respectively connected with a monoclonal antibody SZ-2, a monoclonal antibody SZ-22, a monoclonal antibody SZ-21, a monoclonal antibody 7E3, and the concentration of polystyrene nano-microspheres is 2 mg / ml; the goat-anti-human polyclonal antibody is connected with fluorescein isothiocyanate and with the concentration of the goat-anti-human polyclonal antibody is 15 mug / ml. A detection process of the specificity platelet antoantibody comprises the following steps of: oscillating monoclonal antibody--microspheres and platelet lysis buffer sample for incubation; washing by PBS; adding the goat-anti-human polyclonal antibody connected with the fluorescein isothiocyanate; washing, re-suspending by the PBS; detecting on a flow cytometry to obtain average fluorescence intensity values of the four types of the nano-microspheres respectively; and confirming whether the specificity platelet antoantibody is positive or not according to a ratio of the obtained values to a basic contrast value of that of a healthy person, in order to provide foundation for early diagnosis of ITP. The detection has the advantages of simple process, high sensitivity and good specificity.

Description

technical field [0001] The invention relates to a kit for combined detection of platelet-specific autoantibodies with monoclonal antibody nano-microspheres, which is used for detecting platelet-specific autoantibodies in blood and providing diagnostic basis for immune-mediated thrombocytopenia syndrome. The invention also relates to the preparation of the kit and the method for detecting platelet-specific autoantibodies. Background technique [0002] Idiopathic thrombocytopenic purpura (Idiopathic thrombocytopenic purpura, ITP) is an immune-mediated thrombocytopenia syndrome, which is the most common clinical hemorrhagic disease, accounting for about 30% of the total number of hemorrhagic diseases. It is generally believed that the disease is caused by excessive destruction of autoantibody-sensitized platelets by the mononuclear macrophage system, with extensive skin, mucous membrane or visceral bleeding, thrombocytopenia, development and maturation of bone marrow megakaryoc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/546G01N33/531G01N21/64G01N33/564
Inventor 何杨赵益明费敏阮长耿
Owner 苏州苏大赛尔免疫生物技术有限公司
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