Preparation method of O-substrate phenylene diamine of horseradish peroxidase

A technology of horseradish peroxidase and o-phenylenediamine, applied in the field of immunoassay, can solve problems such as waste, adverse effects of environmental protection, and cumbersome accurate weighing work

Inactive Publication Date: 2006-01-25
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although the price of o-phenylenediamine in the above-mentioned substrate formula is relatively low, the dosage of o-phenylenediamine is 20mg and it is difficult to accurately weigh the dosage in many units. The substance must be ready-to-use and ready-to-use, and it is sensitive to light. At the same time, this substrate is not easily dissolved by the substrate buffer when it is prepared, which brings inconvenience to laboratory research analysis and analysis and application of low-level analysis institu

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Weigh 100 mg of o-phenylenediamine, dissolve it in 100 microliters of anhydrous N,N-dimethylformamide, and then store it at low temperature. Before use, draw 10 microliters of the substrate and add 10 milliliters of In the citric acid-phosphate buffer solution of hydrogen peroxide, o-phenylenediamine is very easy to dissolve in N,N-dimethylformamide, so the volume of the solvent that substrate o-phenylenediamine is dissolved should not be too large, in order to Dissolving 10 mg of o-phenylenediamine in 10 µl of N,N-dimethylformamide is easily accomplished by pipetting the required amount of substrate directly into the substrate buffer.

Embodiment 2

[0020] Weigh 300 mg of o-phenylenediamine, dissolve it in 500 microliters of anhydrous N,N-dimethylformamide, and then store it at low temperature. Before use, absorb 10 microliters of the substrate and add 20 milliliters of hydrogen peroxide containing In the citric acid-phosphate buffer solution, o-phenylenediamine is very easy to dissolve in N,N-dimethylformamide, so the volume of solvent dissolved by substrate o-phenylenediamine should not be too large, Dissolving 3 mg of o-phenylenediamine in 5 microliters of N, N-dimethylformamide is also very easy to achieve, and only needs to absorb the required amount of substrate and directly add it to the substrate buffer.

Embodiment 3

[0022] Weigh 20 mg of o-phenylenediamine, dissolve it in 200 microliters of anhydrous N,N-dimethylformamide, and then distribute it into 10-20 ml small transparent glass bottles or brown bottles, each bottle according to needs Dispensed into 5 microliters, generally the amount of substrate per bottle is 2 mg. This substrate is weighed once, and is divided into a batch of equal amount of substrates, the packing volume is all consistent, and the volume of the solvent of o-phenylenediamine is only about 5 microliters, and the substrate in one sub-package bottle is usually The amount of substrate used in a fast 96-well enzyme labeling reaction plate. Just add 10ml of substrate buffer solution containing hydrogen peroxide into the substrate bottle and mix well before use. In this way, the substrate used is relatively consistent each time, and the substrate is easily dissolved in the substrate buffer.

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Abstract

The invention discloses a method for preparing ortho-phenylene diamine substrate of horseradish peroxidase: firstly dissolving 100 mg ortho- phenylene diamine in 100 ml N,N-dimethyl formylamine to form solution A, where the oxydo-containing citric acid-phosphate substrate buffering solution is solution B, and if use, mixing 5 ml solution A and 10 ml solution B to make the ortho-phenylene diamine substrate. The invention mainly improves the formula of the ortho-phenylene diamine substrate of horseradish peroxidase, solving the problems that accurately weighing ortho-phenyene is complex, and that the ortho-phenylene diamine is uneasy to dissolve in the substrate buffering solution, and so on; besides, this improvement can not destroy the sensitivity of original horseradish peroxidase substrate system, nor destroy the analysis and testing of the system, and other related contents.

Description

technical field [0001] The invention relates to the field of immunoassay, in particular to a preparation method of o-phenylenediamine substrate of horseradish peroxidase. Background technique [0002] Horseradish peroxidase is a very important tool enzyme in the field of immunoassay. Due to its convenient source, low price, and sensitive detection system, it is widely used in immunological analysis and testing, among which there are soluble systems , Precipitating system, soluble system is that the final product is soluble after the substrate is catalyzed by horseradish peroxidase, so the data can be read by the instrument, so it is mainly used for quantitative and qualitative analysis. There are many substrates for horseradish peroxidase, the most commonly used of which is o-phenylenediamine, and the commonly used formula for this substrate is: [0003] 0.1M citric acid 24.3ml [0004] 0.2M Na2HPO4 25.7ml [0005] o-Phenylenediamine 20mg [0006] 30% hydrogen peroxide 7...

Claims

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Application Information

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IPC IPC(8): G01N33/532
Inventor 陈正贤徐正吴建祥周雪平沈立荣叶兴乾
Owner ZHEJIANG UNIV
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