Method for labeling antibody by fluorophore generated by combination of iridium coordination compound and histidine

A biomarker and coordination compound technology, applied in instruments, analytical materials, peptides, etc., can solve the problems of fluorescent labeling with huge structures and inability to carry out polypeptides and proteins, and achieve stable labeling products, good photostability, and stable properties Effect

Inactive Publication Date: 2010-06-23
苏州纳凯科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On this basis, by cross-linking fluorescent groups on Ni-NTA, His 6 Sequences with high affinity to Ni-NTA were also developed for His 6 Fluorescent labeling of tagged proteins, but the reported labeling groups have no change in fluorescence intensity before and after binding to the protein. Not only does the sequence need to contain six consecutive histidine residues, but the fluorescent labeling structure is too large, so the application is limited 【4】【5】
The

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  • Method for labeling antibody by fluorophore generated by combination of iridium coordination compound and histidine
  • Method for labeling antibody by fluorophore generated by combination of iridium coordination compound and histidine
  • Method for labeling antibody by fluorophore generated by combination of iridium coordination compound and histidine

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Embodiment 1

[0030] Implementation Example 1: Complexation reaction of iridium coordination compound with four compounds such as histidine

[0031] [(mppy) 2 Ir(CH 3 EN) 2When ](OTf) reacts with four compounds containing imidazole groups such as histidine (L-histidine, histamine, urocanic acid and imidazole), [(mppy) 2 Ir(CH 3 EN) 2 ](OTf) and the concentration of the four substances are all 50 μM, and the reaction is carried out in PBS (pH 7.4). After mixing in the test tube, the reaction is allowed to stand at room temperature for about 10 minutes. figure 2 The chemical structures of the four substances are shown respectively, as well as the experimental results after the respective reactions are completed. Under the irradiation of ultraviolet light (365nm), L-histidine and histamine are the same as [(mppy) 2 Ir(CH 3 EN) 2 ](OTf) can produce fluorescence, while the other two compounds can not produce fluorescence. The result speculated from this is that -NH in histidine 2 Part...

Embodiment 2

[0032] Implementation Example 2: Complexation reaction of iridium coordination compound with histidine-containing polypeptide

[0033] [(mppy) 2 Ir(CH 3 EN) 2 ](OTf) and two polypeptide chains (one of which contains histidine residues: KQEA H GSTA, another as a control, does not contain histidine residues: KQEASGSTA) when reacting, [(mppy) 2 Ir(CH 3 EN) 2 ](OTf) and the concentration of the polypeptide are both 50 μM, and the reaction is carried out in PBS (pH 7.4). After the reactants were mixed in the test tube, they were allowed to stand at room temperature for about 10 minutes. After the two polypeptides reacted with the iridium coordination compound, the fluorescence ( Figure 6 ). Figure 7 The fluorescence emission spectrum under 328nm excitation of the histidine-containing polypeptide combined with the iridium coordination compound is shown. Since the peptide [1] does not contain a histidine residue and the sequence of the polypeptide [2] contains a histidine ...

Embodiment 3

[0034] Implementation Example 3: Complexation reaction of iridium coordination compound with rabbit anti-sheep immunoglobulin IgG

[0035] [(mppy) 2 Ir(CH 3 EN) 2 ] (OTf) concentration was 50 μM, the concentration of rabbit anti-goat immunoglobulin IgG was 1 mg / mL, and the reaction was carried out in PBS (pH 7.4). After the above substances or the control group were mixed in the test tube, they were allowed to stand at room temperature for about 10 minutes to react. Figure 9 After showing that the reaction is over, under the irradiation of ultraviolet light (365nm) [(mppy) 2 Ir(CH 3 EN) 2 ](OTf) Fluorescence generated after binding rabbit anti-goat IgG. Figure 10 Fluorescence emission spectra of IgG-bound iridium-emitting compounds under excitation at 328 nm are shown.

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Abstract

The invention discloses a novel biomolecule labeling method, i.e. taking cyclic iridium coordination compound containing solvent molecules to combine with histidine residue in protein or polypeptide to generate a fluorophore. The method can label polypeptide and bioactive molecules of protein including antibody and the like, and can be further applied to the application field of bioinstrumentation, cell and tissue labeling, biomolecule tracing and the like. Since the fluorescence color of the compound is rich, and the labeling reaction can be completed in normal-temperature and normal butter solution in one step, thus having advantage compared with the commonly-used labeled compound and the labeling method in market, and having wide application prospect.

Description

technical field [0001] The invention relates to a novel biomarking method, that is, through the combination of a kind of self-nonfluorescent metal iridium coordination compound with the histidine residue on the protein to form an excitable fluorescent group, thereby marking the antibody molecule, It is also used in biotechnology fields such as immunoassay detection and immunofluorescence imaging. Background technique [0002] Fluorescent labeling of biomolecules is an important part of applied biotechnology. Biomolecules labeled with fluorescent groups, such as antibodies, polypeptides, and nucleic acids, recognize target molecules through intermolecular interactions, and then use highly sensitive optical methods or electrochemical methods to identify target molecules. Methods (photoluminescence, fluorescence resonance energy transfer, time-resolved, electrochemiluminescence) to obtain signals generated by labeled molecules, in order to form a series of detection methods bas...

Claims

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Application Information

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IPC IPC(8): G01N33/533C07K2/00C07K16/00
Inventor 费浩周明贾俊丽王小波黄泽柱
Owner 苏州纳凯科技有限公司
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