A kind of ultrasensitive superparamagnetic nano immune microsphere and its method for detecting gp73 antigen

A technology of nano-microspheres and immune microspheres, which is applied in the field of superparamagnetic nano-immune microspheres, magnetic immunological in vitro diagnostic reagents, and the detection of GP73 antigen in human serum or plasma. It can solve the problem of low antibody stability and the detection of liver cancer diagnostic reagents. Long time, poor stability and other issues

Active Publication Date: 2017-08-25
BEIJING INST OF HEPATOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In order to solve the technical problems of long detection time of liver cancer diagnostic reagents in the prior art, small antibody immobilization capacity, low sensitivity and poor stability, in order to solve the small capacity of immobilized antibodies in the traditional enzyme-linked immunosorbent method, long reaction time, For the technical problems of relatively low sensitivity and low stability of fixed antibodies, the present invention provides a GP73 monoclonal antibody and a superparamagnetic nano-immune microsphere coupled with a GP73 monoclonal antibody on the surface, and using the microsphere Method for detecting GP73 antigen in human serum or plasma by double-antibody sandwich enzyme immunoassay or chemiluminescent method

Method used

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  • A kind of ultrasensitive superparamagnetic nano immune microsphere and its method for detecting gp73 antigen
  • A kind of ultrasensitive superparamagnetic nano immune microsphere and its method for detecting gp73 antigen
  • A kind of ultrasensitive superparamagnetic nano immune microsphere and its method for detecting gp73 antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] 1. Preparation of GP73 monoclonal antibody.

[0122] (1) Synthetic polypeptide AAAERGAVELKK (sequence 1), coupled with BSA (bovine serum albumin).

[0123] (2) Animal immunization: the synthetic polypeptide (GP73 antigen) obtained in step (1) was mixed with complete Freund's adjuvant in equal amounts to make an emulsifier, and a total of 5 Balb / c mice were immunized. The first immunization was 100 μg / monkey, and the second immunization with incomplete Freund's adjuvant 4 weeks later was 50 μg / bird. Two weeks after the second immunization (a total of 6 weeks), the tail vein blood was taken to measure the titer, and the antibody titer of the five mice all reached 1:32000. Four weeks after the second immunization (8 weeks in total), the third immunization was performed with incomplete Freund's adjuvant, and the dose and route were the same as the second one. Two weeks after the third immunization (10 weeks in total), blood was taken from the tail vein to measure the tite...

Embodiment 2

[0162] The ultrasensitive superparamagnetic nano-immune microspheres provided in Example 1 and the method for detecting GP73 antigen of the present invention were used to detect GP73 antigen in 402 samples of healthy blood donors.

[0163] Detection of GP73 in serum or plasma:

[0164] (1) Add 50 μl of sample diluent (0.02M PBS, containing 0.1% Tween 20, pH=7.4) to each test tube,

[0165] (2) Add 50 μl of the sample to be tested,

[0166] (3) Add 50 μl of genetic engineering GP73 solution (buffer solution is 0.1M PBS) to the positive control well,

[0167] (4) Add 50 μl of normal human serum to the negative control well,

[0168] (5) No sample diluent was added to the blank well.

[0169] (6) Mix the different substances in the test tube or well, react at 37°C for 15 minutes and take it out.

[0170] (7) Add 50 μl magnetic immune microsphere solution to each test tube (not add to the blank well), mix well, react at 37°C for 15 minutes, take it out,

[0171] (8) Add 50 μl...

Embodiment 3

[0179] The ultrasensitive superparamagnetic nano-immune microspheres provided in Example 1 and the method for detecting GP73 antigen described in the present invention were used to detect GP73 antigen in 153 liver cancer patient samples.

[0180] 1. The specific detection method is the same as in Example 2

[0181] 2. The raw data (OD value) of the test results of the serum samples of 153 liver cancer cases were analyzed, and the conclusions obtained are shown in Table 4.

[0182] Table 4 Statistical table of detection results of serum samples of 153 cases of liver cancer

[0183]

[0184]

[0185] The results in Table 4 above show that compared with the AFP method using the GP73 antibody and its detection method provided by the present invention, the method provided by the present invention has a higher detection rate and better sensitivity for liver cancer patients.

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Abstract

The invention relates to the technical field of diagnostic reagents, in particular to a magnetic immune in vitro diagnostic reagent. In order to solve the technical problems of small fixed antibody capacity, long reaction time, relatively low sensitivity and low stability of fixed antibody in the traditional ELISA method, the present invention provides a GP73 monoclonal antibody and a surface-coupled antibody A superparamagnetic nano-immune microsphere linked with GP73 monoclonal antibody, and a method for detecting GP73 antigen in human serum or plasma by using the microsphere by double-antibody sandwich enzyme immunoassay or chemiluminescent method. The antigen used to generate the GP73 monoclonal antibody has the following amino acid sequence: AAAERGAVELKK. The superparamagnetic nano-immune microspheres provided by the invention have the characteristics of coupling more antibodies, faster immune response, high specificity, good repeatability, low cost, simple requirements for experimental conditions, and the like.

Description

technical field [0001] The invention belongs to the technical field of diagnostic reagents, and relates to magnetic immunological in vitro diagnostic reagents, in particular to a superparamagnetic nano-immune microsphere coupled with GP73 monoclonal antibody on the surface, and using the microspheres to adopt double-antibody sandwich enzyme immunoassay or chemical Luminescence method for detecting GP73 antigen in human serum or plasma. Background technique [0002] Liver cancer is one of the common malignant tumors, "early diagnosis and treatment" is the key to prevent and treat liver cancer. At present, the clinical diagnosis of liver cancer is mainly based on imaging and the detection of serum marker alpha-fetoprotein (AFP). Early liver cancer is small in diameter and can hardly be detected by imaging. Examination of some patients with early liver cancer. [0003] Golgi protein 73 (GP73) is considered to be one of the best serological markers for early diagnosis of liver...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/531G01N33/543
Inventor 石英陈德喜李宁谢立刘凯黄雁翔刘芳
Owner BEIJING INST OF HEPATOLOGY
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