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Complex of labeled probe and water-soluble carrier

一种水溶性载体、复合物的技术，应用在标记化探针-水溶性载体复合物领域，能够解决难以得到高灵敏度且稳定的复合物等问题，达到再现性良好、灵敏度高、保持经时稳定性的效果

Inactive Publication Date: 2013-01-23
EIKEN KAGAKU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, in the above-mentioned prior art, there is a technical problem that the response at the blank value also increases as the reactivity increases, and it is difficult to obtain a highly sensitive and stable Complex

Method used

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  • Complex of labeled probe and water-soluble carrier
  • Complex of labeled probe and water-soluble carrier
  • Complex of labeled probe and water-soluble carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Preparation of IgG with Thiols by Reductive Treatment

[0061] Anti-HCV core antigen mouse monoclonal IgG antibodies c11-9 and c11-14 were dissolved in 0.1 M phosphate buffer (pH 7.2) to prepare 5 mg / mL solutions, respectively. 30 µL of 0.2M 2-mercaptoethanol (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 300 µL of these IgG solutions, and the reaction was carried out at 37° C. for 1.5 hours. After the reaction, gel filtration purification was performed using PD-10 (manufactured by GE Healthcare) to obtain IgG having a thiol group obtained by reducing a disulfide bond present in the antibody. As a result of quantifying the number of each thiol group, it was confirmed that 8.0 thiol groups existed per IgG molecule.

Embodiment 2

[0063] Preparation of Fab' with thiol groups by reduction treatment

[0064] Anti-HCV core antigen mouse monoclonal IgG antibodies c11-9 and c11-14 were dissolved in 0.1 M sodium acetate buffer (pH 4.5) to prepare 10 mg / mL. Next, 0.2 mg of pepsin was added to 1 mL of these IgG solutions, and stirred at 37° C. for 6 hours to digest the IgG with pepsin. After digestion with pepsin, it was neutralized with 2NNaOH, and further purified with a Superdex 200 column (manufactured by GE Healthcare) to obtain F(ab') 2 .

[0065] To each 3.0mg / mL F(ab') 2 130 μL of 0.2 M 2-mercaptoethanol (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 1300 μL, and the reaction was carried out at 37° C. for 1.5 hours. After the reaction, gel filtration purification was performed using PD-10 (manufactured by GE Healthcare) to obtain Fab' having a thiol group obtained by reducing a disulfide bond present in the body. The number of each thiol group was quantified, and as a result, 3...

Embodiment 3

[0067] Introduction of maleimide groups into streptavidin

[0068] 20 mg of streptavidin (manufactured by MP Bio) was dissolved in 2 mL of 0.1 M phosphate buffer (pH 7.2), and a maleimide reagent dissolved at 6 mg / mL was added to dimethylformamide Sulfo-KMUS (manufactured by Dojin Chemical Co., Ltd.) 133 μL. After reacting at 30° C. for 1 hour, it was purified by gel filtration using PD-10 (manufactured by GE Healthcare) to obtain streptavidin having a maleimide group introduced therein. As a result of quantifying the number of maleimide groups, it was confirmed that 3.7 maleimide groups were present per one molecule of streptavidin.

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Abstract

The object is to construct, at a high reproducibility, a complex composed of a labeled probe and a carrier, said complex being to be used for detecting and measuring a target substance to be measured at a high sensitivity and high stability. In constructing a complex composed of an avidinized probe and a water-soluble carrier, a label is bound to a probe-water-soluble carrier using specific binding of avidin or an analog thereof such as streptoavidin to biotin and the binding of the avidin or an analog thereof to the probe is performed before the binding to the carrier. Namely, a complex of an avidinized probe and a water-soluble carrier is constructed by binding avidin or an analog thereof to a substance, which is capable of binding to the target substance, and then binding the bound product thus obtained to a high-molecule water-soluble carrier. Next, the complex of the avidinized probe and the water-soluble carrier is mixed with a biotinylated label. Thus, a stable complex of the labeled probe and the water-soluble carrier, which enables the highly sensitive detection and measurement of the target substance, can be obtained, via the specific binding of avidin or an analog thereof to biotin, at a high reproducibility.

Description

technical field [0001] The invention relates to a method for making a labeled probe-water-soluble carrier complex formed by combining two or more probes on a water-soluble carrier and further combining a marker; the labeled probe-water-soluble carrier produced by the method compounds; and methods of use thereof. By using this labeled probe-water-soluble carrier complex, stable detection and measurement can be performed with high sensitivity. Background technique [0002] In the method of detecting and measuring the analyte by using the combination of the probe and the marker that specifically binds to the analyte and taking the binding amount of the analyte and the marker through the probe as an index to detect and measure the analyte, its sensitivity Usually determined by the number of molecules of probes and labels. That is, there is a limit to the number of molecules that a label can bind to one molecule of probe, and this ratio determines the sensitivity. [0003] The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/548
CPCG01N33/54353G01N33/532G01N33/581G01N33/582
Inventor 大广义幸高安进
Owner EIKEN KAGAKU
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