Ultra-sensitive superparamagnetic nano immunization microsphere and GP73 antigen detection method

The technology of immune microspheres and nano-microspheres is applied in the field of magnetic immunological in vitro diagnostic reagents to detect GP73 antigen in human serum or plasma, and in the field of superparamagnetic nano-immune microspheres. Time, poor stability, etc.

Active Publication Date: 2016-06-22
BEIJING INST OF HEPATOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Ultra-sensitive superparamagnetic nano immunization microsphere and GP73 antigen detection method

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 (0.02MPBS, containing 0.1% Tween20, 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 0.1MPBS) 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 en...

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 4153 Cases of Liver Cancer Serum Specimens Test Results Statistical Table

[0183]

[0184]

[0185] The results in Table 4 above show that compared with the AFP method using the GP73 antibody and 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 present invention relates to the technical field of diagnostic reagents, and particularly relates to a magnetic immunization in-vitro diagnostic reagent. In order to solve the technical problems of smaller capacity of an immobilized antibody, long reaction time, relatively low sensitivity and low stability of the immobilized antibody of a conventional enzyme immunoassay, the present invention provides a GP73 monoclonal antibody and a superparamagnetic nano immunization microsphere with the GP73 monoclonal antibody coupled with the surface, and a human serum or plasma GP73 antigen detection method using the superparamagnetic nano immunization microsphere by double-antibody sandwich enzyme immunoassay or chemiluminescence method. An antigen for producing the GP73 monoclonal antibody has the following amino acid sequence AAAERGAVELKK. The superparamagnetic nano immunization microsphere has the characteristics of being capable of coupling more antibodies, fast in immunoreaction speed, high in specificity, good in repeatability, low in cost, and simple in experimental condition requirements 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 Applications(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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