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A capillary isoelectric focusing detection method for urate oxidase

A uric acid oxidase and isoelectric focusing technology, applied in the field of electric focusing detection, can solve the problems of increased eosinophils, unsafe treatment strategies for lowering uric acid, necrolysis and lysis of toxic epithelial cells, etc., and achieves good repeatability.

Active Publication Date: 2022-03-08
SHENYANG SUNSHINE PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The water solubility of xanthine is lower than that of uric acid, and its accumulation is potentially dangerous. Xanthine oxidase inhibitors have poor efficacy when the initial uric acid concentration is too high, and can also cause hypersensitivity syndrome, manifested as fever, toxic epithelial cell necrolysis , hepatitis and increased eosinophils, and the mortality rate is as high as 20%. In addition, these drugs, probenecid, benzbromarone and other uric acid excretion drugs have obvious liver and kidney toxicity
Therefore, the classic uric acid-lowering treatment strategy is not safe enough and the efficacy is not ideal.

Method used

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  • A capillary isoelectric focusing detection method for urate oxidase
  • A capillary isoelectric focusing detection method for urate oxidase
  • A capillary isoelectric focusing detection method for urate oxidase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] 1. Preparation of uric acid oxidase stock solution

[0064] According to the invention patent ZL02819387.3, the urate oxidase engineering strain was prepared.

[0065] Resuscitate the urate oxidase engineered bacteria and incubate on the slope at 37°C for 17-24 hours. Then the bacteria were inoculated into shake flasks, and cultured by shaking at 37° C. at 200-300 rpm. OD of bacterial solution 600 When it is 2.0 to 5.0, it is inoculated into a fermenter for culture. 450~700rpm, pH7.0 fermenter culture, keep dissolved oxygen ≥ 50%, OD 600 Add IPTG to induce expression for 7.5-9.5 for 2 hours. Collect the cells by centrifugation, resuspend the cells with the lysate, then process the suspension with a homogenizer, centrifuge the suspension and collect the supernatant. Pure urate oxidase stock solution was obtained by hydrophobic chromatography and ion exchange.

[0066] Second, the purity detection of the original solution

[0067] Perform CE-SDS analysis on urate o...

Embodiment 2

[0069] 1. Experimental conditions

[0070] Prepare the capillary isoelectric focusing sample loading mixture according to Table 1, place it in the autosampler of Beckman's PA800plus capillary electrophoresis instrument, set the column temperature to 30 ° C, and use N-CHO coated capillary (Beckman) for analysis, 15kV Focusing for 15 minutes, 25kV analysis for 30 minutes, and detection under a UV detector with a wavelength of 280nm.

[0071] Table 1 Proportion of loading solution for capillary isoelectric focusing

[0072] Reagent name Reagent volume (μl) Final concentration 3mol / L urea-cIEF gel solution 200 2.43mol / L 3-10 ampholyte carrier (Pharmalyte3-10) 12 500mmol / L L-arginine cathode stabilization solution 20 40.49mmol / L 200mmol / L iminodiacetic acid anode stabilization solution 2 1.62mmol / L pI 10.0 protein labeling 1 - pI 5.5 protein labeling 1 - pI 4.1 protein labeling 1 - 10mg / mL protein sample 10 ...

Embodiment 3

[0076] 1. Experimental conditions

[0077] Prepare capillary isoelectric focusing sample loading mixture according to Table 2, add different volumes of anode stabilizer according to Table 3, mix well and place in the autosampler of Beckman’s PA800plus capillary electrophoresis instrument, set the column temperature to 30 °C, N-CHO coated capillary (Beckman Company) was used for analysis, focusing at 15kV for 15 minutes, analyzing at 25kV for 30 minutes, and detecting under a UV detector with a wavelength of 280nm.

[0078] Table 2 Ratio of loading solution for capillary isoelectric focusing

[0079] Reagent name Reagent volume (μl) Final concentration 3mol / L urea-cIEF gel solution 200 1.74-2.43mol / L 3-10 ampholyte carrier (Pharmalyte3-10) 12 - 500mmol / L L-arginine cathode stabilization solution 20 28.99-40.49mmol / L pI 10.0 protein labeling 1 - pI 5.5 protein labeling 1 - pI 4.1 protein labeling 1 - 10mg / mL protein ...

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Abstract

The invention relates to a capillary isoelectric focusing method for detecting uric acid oxidase, which specifically comprises an amphoteric carrier, a capillary isoelectric focusing gel, urea, an anode stabilizer, a cathode stabilizer, an isoelectric point marker and a urate oxidase sample according to Mix at a certain concentration ratio, load the sample on the coated capillary, and detect it by capillary electrophoresis under appropriate temperature, voltage and focusing time, so that the components in the protein sample to be analyzed can be effectively separated. The method has good repeatability and can be used for quality control of urate oxidase drugs in the production process.

Description

technical field [0001] The invention relates to a capillary isoelectric focusing detection method, which is used for measuring the charge heterogeneity of urate oxidase, and the detection method can perform good quality control analysis on urate oxidase drugs. Background technique [0002] Uric acid is the end product of purine metabolism in birds, reptiles and primates including humans, because in these animals there is a lack of uric acid that uses molecular oxygen as the receptor to catalyze the further decomposition of uric acid into allantoin, carbon dioxide and hydrogen peroxide oxidase. Uric acid is excreted by the kidneys as the end product of purine metabolism in the human body. When the production of uric acid exceeds the metabolic capacity of the kidneys or when the kidneys are in a pathological state, it will cause a significant increase in plasma uric acid and form hyperuricemia. Due to the low solubility and easy deposition of urea and its salts in the blood, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447G01N1/38
CPCG01N1/38G01N27/44795
Inventor 杨丽华袁杰靳征
Owner SHENYANG SUNSHINE PHARMA CO LTD