Raman homogeneous detection method for recognizing binding antigen by utilizing antibody

A technology that combines antigen and antibody recognition, applied in the biological field, can solve the problems of insufficient detection limit, time-consuming and labor-intensive, etc., and achieve the effects of improving detection efficiency, broad application prospects, and improving solid-phase connection methods

Active Publication Date: 2020-11-17
JILIN UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0012] The purpose of the present invention is to provide a Raman homogeneous detection method that uses antibodies to recognize and bind antigens, so as to overcome the shortcomings of multiple washings and multiple incubations when the detection technology is used for antigen detection, which is time-consuming and labor-intensive, and the detection limit is insufficient.

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  • Raman homogeneous detection method for recognizing binding antigen by utilizing antibody
  • Raman homogeneous detection method for recognizing binding antigen by utilizing antibody
  • Raman homogeneous detection method for recognizing binding antigen by utilizing antibody

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Embodiment 1

[0097] In the embodiment of the present invention, the construction, expression and purification of the Domain Z expression vector are as follows:

[0098] Experimental equipment: PCR instrument (Thermo Electron, USA); high-speed centrifuge (Thermo, USA); ultra-clean workbench (Suzhou Purification Equipment Factory, China); constant temperature shaking incubator (China Zhicheng Co., Ltd.); FORMA Company); biochemical incubator (Shanghai Yiheng Technology Company); ultrapure water equipment (Porma Company of the United States); electrophoresis instrument (Bio-Rad Company of the United States); electronic balance (METTLER TOLEDO Company of Switzerland); gel imager (China Tianneng Company).

[0099] Samples and reagents: 2×Taq PCR Master Mix (Thermo Electron, USA); Axygen mini-plasmid extraction kit (Axygen, USA); DNA rapid purification / recovery kit (Axygen, USA); PCR DNA polymerase (TaKaRa Corporation, Japan ) agarose (US Sigma Company); T4 DNA ligase (US Thermo Company); prote...

Embodiment 2

[0114] Detection of PSA by Raman homogeneous detection method

[0115] Prostate-specific antigen (PSA) is a tumor marker of prostate cancer, which is closely related to the occurrence of prostate and has obvious organ specificity. In clinical diagnosis, the serum PSA level of 4.0ng / mL is generally taken as the upper limit. If the upper limit is higher than the upper limit, especially when the content is higher than 10ng / mL, it indicates the risk of cancer, and further diagnosis needs to be combined with other examination methods such as prostate biopsy.

[0116] 1. Detection of different concentrations of PSA

[0117] Take 150 μl AgNPs@Ab1 probe solution, 50 μl MNs@Ab2 / 4-ABP probe solution and 2 μl PSA with different concentrations (0.1ng / mL, 1ng / mL, 10ng / mL, 100ng / mL and 1μg / mL) and mix well After reacting for 1 h, Raman detection was performed.

[0118] 2. Detection of PSA and non-PSA

[0119] Take 150 μl AgNPs@Ab1 probe solution, 50 μl MNs@Ab2 / 4-ABP probe solution and 2 ...

Embodiment 3

[0124] Detection of TSH by Raman Homogeneous Detection Method

[0125] Thyroid-stimulating hormone (TSH) is a peptide hormone produced by the anterior pituitary gland, consisting of 2 chains: an α chain and a β chain, with a molecular weight of about 28,000 Da. Compared to other glycoprotein hormones produced by the anterior pituitary, TSH has the same alpha chain and a different beta chain. TSH is critical for regulating the release of thyroid hormones and the growth of the thyroid gland. TSH test is the clinical first-line screening test for thyroid function, and its reference value ranges from 0.4 to 4.5uIU / ml.

[0126] 1. Detection of different concentrations of TSH

[0127] Accurately pipette 150 μL of AgNPs@Ab1 / 4-ABP and 50 μL of MNs@Ab2, add different concentrations of human thyrotropin and incubate for 0.5-1 hour, and then use a Raman spectrometer to analyze the samples in capillary glass tubes and standard PCR tubes, respectively. For SERS detection, the excitation...

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Abstract

The invention is applicable to the technical field of biology, and provides a Raman homogeneous detection method for recognizing a binding antigen by using an antibody, which comprises the following steps: preparing silver nanoparticles by using a sodium citrate reduction method, and coupling the antibody and Raman reporter molecules to the surfaces of the silver nanoparticles and magnetic nanoparticles by using an EDC/NHS activator to obtain the Raman homogeneous detection method, preparing two Raman probes for targeting target antigens; and detecting a specific antigen by using a Raman probe. The detection method disclosed by the invention is based on a pair of Raman probes of an immune sandwich method, and the two probes are provided with antibodies combined with a target antigen. The two probes can be combined with a target antigen to form an immune complex, so that the surface enhanced Raman effect is excited, and the target antigen is detected. According to the method, the solid-phase connection mode of the antibody on the surfaces of the nanoparticles is improved, so that the detection efficiency of the probe is improved. The detection method has a wide application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a homogeneous detection method, in particular to a Raman homogeneous detection method using antibodies to recognize and bind antigens. Background technique [0002] Biomolecular detection technologies such as proteins, peptides, and genes are the most critical technologies in clinical detection. Fast and simple detection methods are the basis for improving hospital detection efficiency, and accurate and sensitive detection results are a necessary condition for improving physicians' diagnostic results. In clinical diagnosis, physicians often detect and analyze molecules in patient serum and other body fluids that can be used for disease diagnosis, such as tumor markers and hormones. [0003] Biomolecules closely related to diseases in serum closely reflect the occurrence, generation and changes of diseases, so they have attracted the attention of many scientific researcher...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/574G01N33/78G01N21/65
CPCG01N33/54346G01N33/54326G01N33/54353G01N33/57434G01N33/57484G01N33/78G01N33/57488G01N21/65Y02A50/30
Inventor 梁重阳徐抒平许士志方帅
Owner JILIN UNIV
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