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Blood platelet antibody specificity identification method and kit thereof

A specific, platelet technology, applied in the field of medical testing, can solve the problems of inability to detect platelet antibodies, inaccurate test results, and complex testing process, and achieve the effects of accurate results, simple steps, and high efficiency.

Inactive Publication Date: 2017-06-20
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problems that the existing technology cannot detect free platelet antibodies in patients with alloimmune diseases, the detection results are inaccurate, the detection process is complicated, and the efficiency is low, and then provide a detection method with accurate detection results. A platelet antibody-specific identification method with simple and efficient detection process and its kit

Method used

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  • Blood platelet antibody specificity identification method and kit thereof
  • Blood platelet antibody specificity identification method and kit thereof
  • Blood platelet antibody specificity identification method and kit thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1 Extraction of HLA Class I Glycoprotein Antigens

[0046] Investigate clinically meaningful HLA class Ⅰ antibody categories in my country, combined with the cross-reactivity characteristics of HLA class Ⅰ antigens, minimize the probability of missed detection of HLA class Ⅰ antibodies, and design the HLA class Ⅰ glycoprotein antigen combinations to be extracted as follows: A2, A11, A31, A68, B7, B 18, B35 and B40 antigens, HLA class Ⅰ glycoproteins cover the antigen map see figure 1 . Therefore, this embodiment provides a method for extracting the HLA class I glycoprotein antigen of the above antigen combination, and the extraction steps are as follows:

[0047] (1) Cultivate and collect human leukocytes or platelets expressing the above-mentioned relevant HLA class I glycoprotein antigens, store them at -80°C, and extract 3×10 9 cells;

[0048] (2) Take 3×10 9 Each of the human leukocytes or platelet cells was washed 3 times with normal saline, and the pack...

Embodiment 2

[0053] Example 2 Extraction of Platelet Glycoproteins GPⅡb / Ⅲa, GPⅠa / Ⅱa, GPⅠb / Ⅸ and GPⅣ

[0054] This embodiment provides a method for extracting platelet glycoproteins GPⅡb / Ⅲa, GPⅠa / Ⅱa, GPⅠb / IX and GPⅣ, comprising the following steps:

[0055] (1) Take fresh platelet-rich plasma (PRP) at 1300g, centrifuge for 10 minutes, discard the supernatant, and obtain the platelet-packed cells (5×10 10 ), washed 3 times with 0.1M pH 7.4 PBS (containing 2.5mM EDTA);

[0056] (2) Cleavage: Add 50 mL of lysate (the lysate is 10 mM Tris-HCl buffer at pH 7.4, containing 0.15 M of NaCl, 1 mM of Benzylbenzene) to the platelet packed cells after the above washing Sulfonyl fluoride, 2.5mM EDTA and 0.5% (v / v) Lubrol PX), cleavage at 4°C for 45 minutes, shake and mix well during the period, then add 50mL of the above-mentioned lysate, and lyse at 4°C for 30 minutes to make it Fully lysed, ready for use;

[0057] (3) After centrifuging at 100,000g for 30 minutes at 4°C, keep the supernatant (conta...

Embodiment 3

[0061] Example 3 Establishment of anti-human platelet monoclonal antibody hybridoma cell line and preparation and purification of monoclonal antibody

[0062] This embodiment provides the establishment of an anti-human platelet monoclonal antibody hybridoma cell line and the preparation and purification of the monoclonal antibody, including the following steps:

[0063] (1) Collect fresh type O whole blood, centrifuge at 200g for 10 minutes, extract the upper platelet-rich plasma, then centrifuge at 1000g for 10 minutes, discard the supernatant, keep the packed platelets, and wash with 0.01M pH 7.4 PBS buffer (containing 0.5% (w / v) EDTA) was washed twice, and the final platelet concentration was adjusted to 10 8 platelet suspension per mL; take 0.5 mL of the above platelet suspension to immunize mice through the abdominal cavity and subcutaneously on the back, for a total of 4 times with an interval of 10 days. Two days before the fusion, 0.2 mL of the above platelet suspensi...

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Abstract

The invention provides a blood platelet antibody specificity identification method and a kit thereof. The method comprises the following steps: (1) connecting different blood platelet specificity glycoprotein to different fluorescent microsphere surfaces to obtain antigen specificity fluorescent microspheres for standby use; (2) adding the antigen specificity fluorescent microspheres into a sample to be detected to perform an incubation reaction, and washing for standby use; and (3) adding a fluorescent marked anti-human IgG into step (2) to perform the incubation reaction, washing, and performing flow cytometry detection. The blood platelet antibody specificity identification method solves the problems that the blood platelet antibody detection in the prior art mainly targets at an antigen-self blood platelet antibody complex on the surface of the blood platelet and does not detect free blood platelet antibodies in plasma or serum, and the detection result of the blood platelet antibody is imprecise; and moreover, the method is simple in steps, easy in operation, short in time comsuming and higher in efficiency.

Description

technical field [0001] The invention relates to the field of medical testing, in particular to a platelet antibody specific identification method and a kit thereof. Background technique [0002] Platelet antibodies are pathogenic antibodies produced by patients exposed to foreign platelet antigens during blood transfusion, pregnancy, or transplantation. PTR), fetal-maternal alloimmune thrombocytopenia (FMAIT), posttransfusion purpura (PTP), and transplantation-associated alloimmune thrombocytopenia (transplantation-associated alloimmune thrombocytopenia, TAAT) and other diseases, patients are prone to symptoms such as petechiae, ecchymosis, and bleeding, and severe cases may even cause death. Patients with immune diseases can also produce antibodies against their own platelets when the body's immune system is disordered, resulting in autoimmune thrombocytopenia (idiopathic thrombocytopenia, ITP). [0003] Platelet antibodies include anti-HLA class I antibodies and anti-pla...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/533G01N15/14
CPCG01N33/577G01N15/14G01N33/533
Inventor 段生宝李勇陈晔洲蒙青林王红梅丁少华魏双施刘欢
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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