Immune nanometer magnetic bead enzyme-linked immunosorbent assay method for detecting blood platelet antibody

An enzyme-linked immunoassay and nano-magnetic bead technology, which is applied in the field of medical testing, can solve the problems of antigen structure destruction, cumbersome operation steps, and high technical requirements, and achieve the effects of accurate results, simple detection process, and high application value

Inactive Publication Date: 2015-04-29
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] In my country, however, no platelet donor bank has been established so far, and it is difficult to carry out genotype-compatible platelet transfusion clinically. Only a few clinical institutions use serological methods for platelet antibody detection and cross-matching, but the results are still unsatisfactory.
The main reason is that there are still many shortcomings and deficiencies in the existing detection methods. For example, the "gold standard" method for platelet antibody detection - The monoclonal antibody immobilization of platelet antigen assay (MAIPA) needs to lyse platelets, It is easy to cause the destruction of the antigen structure and the missed detection of antibodies. At the same time, the operation steps are cumbersome and the amount of sample detection is limited.
However, the common solid-phase red cell adherence (SPRCA) has high technical requirements for experimental operators, weak positive results are sometimes difficult to judge, and the indicator red blood cells used are stored for a short time, making it difficult to popularize and apply.

Method used

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  • Immune nanometer magnetic bead enzyme-linked immunosorbent assay method for detecting blood platelet antibody
  • Immune nanometer magnetic bead enzyme-linked immunosorbent assay method for detecting blood platelet antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Establishment of anti-human platelet monoclonal antibody hybridoma cell line and preparation and purification of monoclonal antibody

[0023] 1. Immunization of mice

[0024] 1. Collect fresh type O whole blood, centrifuge at 900rpm for 10min, and extract the upper platelet-rich plasma. Then centrifuge at 3800rpm for 10min, discard the supernatant, retain the packed platelets, wash twice with 0.01M PBS buffer (containing 0.5% EDTA) and finally adjust the platelet concentration to 10 8 / mL.

[0025] 2. Take 0.5 ml of the above-mentioned platelet suspension to immunize mice at multiple points through the abdominal cavity and subcutaneously on the back, etc., for a total of 4 times, with an interval of 10 days.

[0026] 3. 2 days before fusion, 0.2 ml of platelet suspension was injected once through the rat tail vein.

[0027] 2. Cell Fusion

[0028] 1. Take the spleen of the immunized mouse, prepare the spleen cell suspension, and mix the mouse spleen cell...

Embodiment 2

[0045] Example 2: Preparation of functional magnetic beads

[0046] 1. Fe 3 o 4 Synthesis of Magnetic Nanoparticles

[0047] 1. Take 1.35 g FeCl 3 ·6H 2 O, 1.50 g PEG 4000 and 1.00 g urea were added to 40 mL of ethylene glycol, stirred and dissolved completely;

[0048] 2. Add the mixed solution to the polytetrafluoroethylene lining of the reactor, then close the reactor, and place it at 200°C for 8 hours;

[0049] 3. Take out the reactant after cooling, wash repeatedly with absolute ethanol for 3 times, then wash with deionized water for 3 times, and finally resuspend the product with deionized water to obtain Fe 3 o 4 Magnetic nanoparticles, stored at room temperature for later use.

[0050] 2. Fe 3 o 4 SiO 2 Preparation and Amination Modification of Magnetic Composite Particles

[0051] 1. Take Fe 3 o 4 Magnetic nanoparticles, prepared with deionized water to a concentration of 0.1%~0.3% (w / v), ultrasonically treated for 10 min;

[0052] 2. Take 30mL of t...

Embodiment 3

[0062] Example 3: Establishment of the Immuno-Nano-Magnetic Beads ELISA Method for Platelet Antibody Detection

[0063] 1. Take 50~100 μL of magnetized platelet suspension, add an equal volume of sample to be tested (serum or plasma) and sample diluent, and incubate at 37°C for 30 minutes.

[0064] 2. Magnetic platelets were separated by magnetic adsorption to obtain platelet antigen-antibody complexes, which were washed 5 times with PBST washing buffer.

[0065] 3. Add enzyme-labeled anti-human secondary antibody 50 μL / well, and incubate at 37°C for 30 minutes.

[0066] 4. The magnetized platelet reaction complex was separated by magnetic adsorption, and washed 5 times with PBST washing buffer.

[0067] 5. Add 0.5-1.5mg / mL disodium 4-nitrophenylphosphate (4-Nitrophenyl phosphate, PNPP), 50μL / well, incubate at 37°C for 15-30min.

[0068] 6. Add stop solution 2~3mol / L NaOH solution, 50μL / well.

[0069] 7. Measurement results. According to the formula critical value (C.O.) =...

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Abstract

The invention discloses an immune nanometer magnetic bead enzyme-linked immunosorbent assay method for detecting a blood platelet antibody, and the method is characterized by comprising the following steps: (1) mixing magnetized blood platelets with a to-be-detected sample and reacting; (2) separating and washing the magnetized blood platelets which react in the step (1) by using a magnetic force; (3) adding an enzyme-labeled human secondary antibody for reaction; (4) adding a substrate, developing and ending the reaction to obtain a detection result. Through the way, the immune nanometer magnetic bead enzyme-linked immunosorbent assay method for detecting the blood platelet antibody, disclosed by the invention, is simple and convenient in detection process, high in efficiency, accurate and reliable in result and relatively high in application value when being used for clinically diagnosing blood platelet related immune diseases and preventing invalid blood platelet injection.

Description

technical field [0001] The invention relates to the field of medical testing, in particular to an immune nano magnetic bead enzyme-linked immunoassay method for platelet antibody detection. Background technique [0002] There are complex blood group antigens on the surface of platelets, including ABO antigens, HLA-I antigens and HPA antigens (present on platelet membrane glycoproteins GPⅡb / Ⅲa, GPⅠa / Ⅱa, GPⅠb / IX). These antigens can stimulate the body to produce anti-platelet antibodies, destroy platelets, and cause thrombocytopenia in patients with purpura, hemorrhage and even death. Clinically related diseases include tumors, leukemia, idiopathic thrombocytopenia, neonatal thrombocytopenia And platelet transfusion ineffective. [0003] Platelet transfusion is currently one of the most important methods for the treatment of thrombocytopenia and the above-mentioned diseases. A large number of research results have shown that the effective rate of blood transfusion is greater...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/535
CPCG01N33/577G01N33/535
Inventor 段生宝李勇田晶晶王红梅丁少华蒙青林陈晔洲魏双施刘欢史素霞
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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