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Difunctional tumor diagnosis reagent and method thereof

A tumor diagnosis, dual-function technology, applied in the intersection of immunology and biomedicine, nanotechnology, and biomimetic fields, it can solve the problems of complex steps and long operation time, and achieve the effect of simple operation.

Active Publication Date: 2012-11-14
重庆康巨全弘生物科技有限公司
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  • Abstract
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  • Claims
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Problems solved by technology

Immunohistochemistry or immunofluorescence can provide detailed information on the distribution, content and cell morphology of a specific antigen, which is very meaningful for in-depth research on pathology. However, immunostaining requires multi-step incubation of primary antibody, secondary antibody or even third antibody. Repeated washing with PBS, and labeling of enzymes or fluorescent molecules, the steps are complicated and the operation time is long

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  • Difunctional tumor diagnosis reagent and method thereof
  • Difunctional tumor diagnosis reagent and method thereof
  • Difunctional tumor diagnosis reagent and method thereof

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Embodiment 1

[0029] The preparation of embodiment 1 bionic ferritin

[0030] By genetically recombining the full-heavy chain human ferritin shell, using the principle of human biomineralization, iron ions are transferred into the protein shell, and then oxidized into iron oxide nanoparticles. Ferritin, named biomimetic ferritin, for tumor detection. The specific operation is as follows: First, construct the recombinant plasmid pET12b-HFn (Santambrogio P. et al., (2000), Protein Expr. Purif., 19: 212-218) containing the gene sequence of human full heavy chain subunit ferritin. The human skeletal muscle total cDNA library was purchased from Invitrogen (D8090-01, Carlsbad, CA, USA). The full heavy chain human ferritin gene was isolated and amplified from the cDNA using PCR technology. Two primers were designed as follows: forward PCR primer: 5'-A GTC GCC CAT ATG ACG ACCGCG TCC-3', the restriction site is NdeI (underlined), with 7 protection bases; reverse PCR primer: 5'-GCC GGA TCC TTA...

Embodiment 2

[0036] The peroxidase activity of embodiment 2 bionic ferritin

[0037] Based on the report of our research group, magnetic iron oxide nanoparticles have catalytic activity similar to that of peroxidase (Gao L, et al. (2007) Nature Nanotech., 2: 577-583.), because biomimetic ferritin is wrapped with The inner core of iron oxide should therefore have oxidase-like activity. We use the substrate of horseradish peroxidase to detect the enzymatic activity of biomimetic ferritin, the specific method is as follows: add 30% H to biomimetic ferritin 2 o 2 And TMB or DAB, observe the color change. Such as figure 1 As shown in B, when biomimetic ferritin and H 2 o 2 After that, it turns dark blue, and DAB is added with biomimetic ferritin and H 2o 2 After that, it turns dark brown, and the same color reaction as horseradish peroxidase appears, indicating that biomimetic ferritin has peroxidase-like activity.

Embodiment 3

[0038] Example 3 Biomimetic Ferritin Binding to Tumor Cells

[0039] In order to study the binding of biomimetic ferritin to human tumor cells, common human tumor cells were selected to incubate with fluorescent molecularly labeled biomimetic ferritin, and cell flow cytometry was used to detect the binding of biomimetic ferritin to each tumor cell.

[0040] The experimental method is as follows: according to the labeling method provided in the manual, NHS-activated Cy5.5 (Cy5.5-NHS, purchased from GE Healthcare) was labeled on biomimetic ferritin. Culture each cell line to 1×10 5 Left and right, trypsinize, wash the cells three times with 0.3% BSA / PBS, add 50 μg / ml Cy5.5-labeled biomimetic ferritin, and incubate at 4°C for 45 minutes. Then the cells were washed three times with 0.3% BSA / PBS, and finally resuspended in PBS, and the fluorescence of the samples was detected by flow cytometry.

[0041] The results are shown in Table 1. Among the 12 tumor cell lines tested, biomi...

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Abstract

The invention relates to a difunctional tumor diagnosis reagent and a method thereof, and belongs to the nano-technological field, the biological bionic field, and the immunology and biomedicine crossing field. The invention especially provides the difunctional tumor diagnosis reagent which is composed of a protein shell for specifically identifying cancer tissues and / or cancer cells and an inorganic nanometer core having a peroxidase catalytic activity. The invention also provides a kit comprising the reagent, and a method for detecting tumors in an individual through using the reagent. The difunctional tumor diagnosis reagent and the method can be used for cancer screening, early-stage diagnosis, treatment monitoring, cancer cell metastasis analysis, postoperative recurrence evaluation or basic cancer researches. The difunctional tumor diagnosis reagent integrates two functions comprising tumor specific identification and coloration into one, and enables the tumor specific identification and the coloration to be completely in a one step manner without the marking of a secondary antibody, a tertiary antibody or HRP or a signal molecule, and the operation is simple and convenient.

Description

technical field [0001] The invention belongs to the intersecting fields of nanotechnology, bionics, immunology and biomedicine. In particular, the present invention provides a tumor diagnostic reagent that integrates dual functions of cancer focus specific recognition and color development, and a method for using the tumor diagnostic reagent in the diagnosis of cancer tissue and cancer cells. Background technique [0002] Malignant tumors have become one of the major diseases that are increasingly common and seriously threaten human life and quality of life. Pathological section examination is the most accurate and reliable, and is recognized as the "gold standard" for tumor diagnosis at home and abroad (Shi, et al., (2008) Am.J.Clin.Pathol., 129:358-366; Taylor et al.( 2006) Biotech Histochem., 81:3-12.; Larsson, et al., (1988) Immunocytochemistry: Theory and Practice. Boca Raton, FL: CRC Press 41-73). Currently, the main staining methods for pathological sections include...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/531C12Q1/28A61K51/065G01N33/54326G01N33/54346G01N33/574G01N2333/005G01N2800/7028
Inventor 阎锡蕴梁敏敏范克龙潘永信曹长乾杨东玲卢迪冯静
Owner 重庆康巨全弘生物科技有限公司
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