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A kind of synthetic technique of alkaline phosphatase conjugate

A phosphatase conjugate and the technology of the conjugate are applied in the field of synthesis of alkaline phosphatase markers, and can solve the problems of complicated operation, expensive reagents and the like

Inactive Publication Date: 2011-12-07
SHENZHEN MINDRAY BIO MEDICAL ELECTRONICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reagents used in this method are expensive, and the complexes to be conjugated are often complicated to operate due to the need for thiol treatment.

Method used

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  • A kind of synthetic technique of alkaline phosphatase conjugate
  • A kind of synthetic technique of alkaline phosphatase conjugate
  • A kind of synthetic technique of alkaline phosphatase conjugate

Examples

Experimental program
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Embodiment 1

[0039] Dissolve alkaline phosphatase in 80uL 30mM MES buffer (pH 4.0), add EDC and NHS, add MES buffer to 100μl, the final concentration of alkaline phosphatase is 0.01mmol / L, the final concentration of EDC is 10mmol / L, NHS The final concentration is 50mmol / L. After reacting at 25°C for 1 hour, add 100ug mouse anti-human prolactin monoclonal antibody, add 100ul pH 8.0 phosphate buffer, and adjust the pH of the system to 8.0 with 3mol / L NaOH solution. After reacting at 37°C for 2 hours, use Protein G affinity column (GE company) purifies enzyme-labeled antibody to obtain alkaline phosphatase-prolactin antibody conjugate.

Embodiment 2

[0041] Dissolve alkaline phosphatase in 80uL 30mM MES buffer (pH 5.0), add EDC and NHS, add MES buffer to 100μl, the final concentration of alkaline phosphatase is 0.01mmol / L, the final concentration of EDC is 100mmol / L, NHS The final concentration is 50mmol / L. After reacting at 25℃ for 1 hour, add 100ug mouse anti-human prolactin monoclonal antibody, add 100ul pH8.0 phosphate buffer, adjust the system pH to 8.0 with 3mol / L NaOH solution. Reaction at 25℃ After 3 hours, the enzyme-labeled antibody was purified with a Protein G affinity column (GE Company) to obtain an alkaline phosphatase-prolactin antibody conjugate.

Embodiment 3

[0043] Dissolve alkaline phosphatase in 80uL 30mM MES buffer (pH 5.0), add EDC and NHS, add MES buffer to 100μl, the final concentration of alkaline phosphatase is 0.01mmol / L, the final concentration of EDC is 10mmol / L, The final concentration of NHS is 50mmol / L. After reacting at 37°C for 10 minutes, add 100ug mouse anti-human prolactin monoclonal antibody, add 100ul pH9.0 phosphate buffer, and adjust the system pH to 9.0 with 3mol / L NaOH solution. 37°C After 2 hours of reaction, the enzyme-labeled antibody was purified with a Protein G affinity column (GE Company) to obtain an alkaline phosphatase-prolactin antibody conjugate.

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Abstract

Methods for synthesizing an ALP conjugate are provided. The methods may include activating a carboxyl group of the ALP with a carbodiimide, to generate an active ester; and adding the substance to be conjugated such that a synthetic reaction occurs between the active ester and the substance to be conjugated, to generate an ALP conjugate.

Description

Invention field [0001] The invention relates to a method for preparing an enzyme conjugate, and more specifically, to a method for synthesizing a new alkaline phosphatase marker. Background of the invention [0002] Alkaline phosphatase is an enzyme widely found in animals, plants, microorganisms, etc. The frequently used alkaline phosphatase is extracted from calf intestinal mucosa and Escherichia coli and consists of multiple isoenzymes. In an appropriate buffer, alkaline phosphatase can catalyze the hydrolysis of nitrophenyl phosphate (PNP), sodium β-glycerophosphate, naphthyl phosphate, 3-(2-spiraladamantane)-4-methoxy-4 -(3-Phosphoroyl)-phenyl-1,2-dioxetane (AMPPD) and other chromogenic substrates and chemiluminescent substrates containing phosphate groups. In view of this, alkaline phosphatase, as a marker enzyme, is widely used in clinical immunodiagnosis. [0003] The alkaline phosphatase conjugate is a chemical synthesis method that combines alkaline phosphatase and the...

Claims

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Application Information

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IPC IPC(8): G01N33/535
CPCG01N33/535C12Q1/42
Inventor 钱纯亘李可张裕平
Owner SHENZHEN MINDRAY BIO MEDICAL ELECTRONICS CO LTD
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