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Chemoluminescence immunoassay measuring kit and preparation method thereof for triiodothyronine magnetic particles

A triiodothyronine and chemiluminescence technology, applied in the field of immunoassay, can solve the problems of low sensitivity of enzyme-linked immunoassay, expensive instruments and reagents, large loss of activity, etc., and achieve good market application prospects, low price, and linear performance. Good results

Inactive Publication Date: 2009-09-30
北京科美东雅生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have shortcomings: radioimmunoassay inevitably uses radioactive elements 125 I, have a certain impact on people and the environment; enzyme-linked immunoassay has low sensitivity; fluorescent immunoassay requires expensive instruments and reagents
Simply using physical adsorption to coat the monoclonal antibody on the solid phase carrier will have the disadvantages of large amount of antibody and large loss of activity, and the use of biotin-avidin system (biotin-avidin system, BAS) can avoid such shortcomings

Method used

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  • Chemoluminescence immunoassay measuring kit and preparation method thereof for triiodothyronine magnetic particles
  • Chemoluminescence immunoassay measuring kit and preparation method thereof for triiodothyronine magnetic particles
  • Chemoluminescence immunoassay measuring kit and preparation method thereof for triiodothyronine magnetic particles

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 prepares T of the present invention 3 Magnetic particle chemiluminescence immunoassay assay kit

[0034] 1. T 3 Preparation of calibrator

[0035] T 3 The pure product was diluted into a calibrator, and divided into 6 bottles of 0, 0.5, 1.0, 2.5, 7.5, 15ng / mL.

[0036] 2. Preparation of anti-FITC monoclonal antibody coated magnetic particle solution

[0037] Activate the magnetic particles with a particle size of 2-3 μm with glutaraldehyde, stir at room temperature, mix for 2 hours, apply a magnetic field, let stand for 20-25 minutes, pour out the supernatant, and use 0.01mol / L with a pH value of 7.4 Wash with phosphate buffer three times, and suspend with this solution, the concentration is 50-100 mg / mL; add 60-100 μg of anti-FITC monoclonal antibody to each ml of suspension, stir overnight at 4°C, add a magnetic field, and let it stand for 10 ~ 15min, pour out the supernatant, and block with phosphate buffer (pH 7.2) containing 0.2% ~ 1.0% bovine ser...

Embodiment 2

[0064] Embodiment 2 The using method of kit of the present invention

[0065] 1. Sample pretreatment

[0066] The fasting morning serum samples were taken from people, centrifuged at 3000rpm for 5min, and the upper serum was taken for analysis.

[0067] 2. Detection method

[0068] Follow the steps below to use this kit:

[0069] 1) Preparation of washing buffer: dilute the washing buffer provided in the kit with distilled water 20 times;

[0070] 2) Take the calibrators, antibody-coated magnetic particles, biotin-labeled substances, FITC-labeled substances and enzyme-labeled substances out of the refrigerator at 4°C, place them for 15 minutes, and equilibrate to room temperature;

[0071] 3) Number the round-bottom polystyrene test tubes required for the experiment and fix them on the plate holder;

[0072] 4) Add 0, 0.5, 1.0, 2.5, 7.5, 15 ng / mL of 0, 0.5, 1.0, 2.5, 7.5, 15 ng / mL to the reaction wells respectively, and add 1 blank tube for each test, and then add 50 μL to...

Embodiment 3

[0078] The methodological examination of the kit of the present invention of embodiment 3

[0079] The test kit prepared in Example 1 is tested according to the routine test procedures in the art. The present invention has carried out specific experimental operations on the precision, accuracy, sensitivity, specificity and stability of the test kit, and the results are as follows :

[0080] 1. Determination of kit precision

[0081] Three batches of the kits prepared in Example 1 were used for precision experiments, and three different concentrations of serum, namely low, medium, and high, were measured respectively. 8 wells were measured in parallel, and repeated 3 times to obtain the intra-assay and multiple of each analysis. Inter-assay variability (Table 1). The intra-assay and inter-assay coefficients of variation were both less than 10%.

[0082] Table 1 Inspection of method precision

[0083]

[0084] 2. Determination of kit accuracy

[0085] Accuracy refers to ...

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Abstract

The invention provides a chemoluminescence immunoassay measuring kit and a preparation method thereof for quantificationally detect triiodothyronine (T3) magnetic particles. The kit mainly comprises a triiodothyronine serial calibration sample, magnetic particle solution coated by an anti-fluorescein isothiocyanate (FITC) monoclonal antibody, a T3 antigen marked by biotin, T3 monoclonal antibody marked by FITC, streptavidin marked by alkaline phosphatase, chemoluminescence substrate solution and 20-time concentrated washing solution. The invention adopts a competitive-method reaction mode, effectively utilizes the chemoluminescence technology combined with magnetic particles and biotin-avidin immunity magnifying technology principle to quantificationally detect the content of T3 in blood serum and blood plasma samples of human bodies and ensure the sensitivity of the detection. The kit is simple, convenient, fast, sensitive and stable to use, and provides a very valuable detection method for clinic diagnosis and scientific research works.

Description

technical field [0001] The invention relates to the field of immune analysis, in particular to a magnetic particle chemiluminescence immunoassay kit for quantitative determination of triiodothyronine. The kit of the invention combines biotin-avidin immunomagnification technology, immunomagnetic particle separation technology and chemiluminescence immunoassay technology. Background technique [0002] The thyroid gland is an important part of the endocrine system. It is the largest endocrine gland in the human body, with an average physiological weight of about 20-25g. The thyroid gland secretes two physiologically active hormones, namely thyroxine (T4) and triiodothyronine Acid (T 3 ). They play an important role in regulating human growth and development, cell metabolism and hormone balance in the body, maintaining metabolism, skeletal muscle and the development of various organ systems. However, triiodothyronine (T 3 ), does not have obvious biological activity, T below...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/78G01N33/577G01N33/543G01N21/76
Inventor 辛天兵林金明梁淑轩应希堂胡国茂郑金来
Owner 北京科美东雅生物技术有限公司
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