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Preparation method and applications of sterol-based hapten-alkaline phosphatase cross-linked product

A technology of sterols and haptens, which is applied in the field of steroidal hapten-alkaline phosphatase cross-linked products and its preparation, chemiluminescent detection kits, and can solve the problem that alkaline phosphatase cannot label sterols Problems with haptens, inability to bind, formation of Schiff's bases, etc.

Inactive Publication Date: 2018-01-19
SHANGHAI KEHUA BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since sterol haptens do not contain primary amino groups, they cannot form Schiff's bases with alkaline phosphatase, and the traditional sodium periodate method cannot be used to label alkaline phosphatase on sterol haptens; similarly, Sterol haptens also fail to bind alkaline phosphatase via glutaraldehyde

Method used

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  • Preparation method and applications of sterol-based hapten-alkaline phosphatase cross-linked product
  • Preparation method and applications of sterol-based hapten-alkaline phosphatase cross-linked product
  • Preparation method and applications of sterol-based hapten-alkaline phosphatase cross-linked product

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preparation example Construction

[0116] 1. The method for preparing alkaline phosphatase-labeled sterol hapten provided by the present invention solves the existing technical difficulties, and efficiently labels alkaline phosphatase on the sterol hapten, and the activity of each component of the cross-linked product is good , the sensitivity of the marker is excellent, which is higher than that of the alkaline phosphatase sterol hapten marker purchased abroad;

[0117] 2. The cross-linking technology provided by the present invention does not require additional chemical separation operations, and only needs to be operated according to the process parameters provided by the present invention to efficiently label sterol haptens to alkaline phosphatase molecules. Moreover, the cross-linking technology provided by the present invention has a very short preparation period, and the preparation process can be completed in only one day.

[0118] 3. The sensitivity of the sterol hapten-alkaline phosphatase cross-linke...

Embodiment 1

[0122] Dissolve 10mg of alkaline phosphatase in 4ml of 10mM PBS buffer (pH7.5), so that the final concentration of alkaline phosphatase solution is 2.5mg / ml; Sterols were dissolved in 0.5ml DMF solvent to make the final concentration 10mg / ml; 15mg EDAC was dissolved in 0.5ml DMF solvent to make the final concentration 30mg / ml; 15mg NHS was dissolved in 0.5ml DMF solvent, Make its final concentration reach 30mg / ml.

[0123] Mix the sterol compound with the O-carboxymethyl oxime structure modified at the 6-position above and the EDAC solution thoroughly, and react at room temperature for 15 minutes; then add the above NHS solution, and continue the reaction at room temperature for 15 minutes; 2.5mg / ml alkaline phosphatase solution, mix thoroughly and react at room temperature for 2 hours.

[0124] The reacted alkaline phosphatase solution was desalted and purified. The desalted column was 3 HiTrap 5ml Desalting in series (source: GE Healthcare), and the desalted equilibrium buf...

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Abstract

The invention discloses a sterol-based hapten-alkaline phosphatase cross-linked product and a preparation method thereof. According to the present invention, labeling is performed with the carboxyl contained in modified sterol or a sterol-based molecule derivative, and the sterol or the sterol-based molecule derivative is labeled on alkaline phosphatase molecules by using carbodiimide and N-hydroxysuccinimide; the efficiency of the method for preparing the alkaline phosphatase-labeled sterol-based hapten is high; the activities of various components of the cross-linked product are good; the cross-linked product has high sensitivity; the method has characteristics of simple operation and short preparation period; and the sterol-based hapten-alkaline phosphatase cross-linked product is provided.

Description

technical field [0001] The present invention relates to the field of in vitro diagnostic immunoassays. Specifically, the present invention relates to a steroidal hapten-alkaline phosphatase crosslinked product, a preparation method thereof, and a chemiluminescent detection kit comprising the crosslinked product. Background technique [0002] Immunoassay is an analytical method for detecting various substances (such as drugs, hormones, proteins, microorganisms, etc.) by using the specific binding reaction of antigen-antibody. [0003] At present, clinical immunoassay methods mainly include colloidal gold method, immunoturbidimetric method, immunofluorescence method, chemiluminescence immunoassay method and so on. Chemiluminescence immunoassay is a relatively advanced immunological method, which has the advantages of short reaction time, high sensitivity, good specificity, wide linear range and good repeatability, so it is the most promising and most reliable method for diagn...

Claims

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Application Information

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IPC IPC(8): G01N33/535G01N33/74G01N33/64G01N21/76C09K11/07
Inventor 杨晨辰彭珊何建文姚姣妮
Owner SHANGHAI KEHUA BIO ENG
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