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A kind of micro-detection method of antibody protein content

A technology of protein content and detection method, applied in the field of micro-detection of antibody protein content

Active Publication Date: 2020-10-20
上海泰因生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The present invention provides a trace detection method of antibody protein content, a method for more trace and accurate determination of antibody protein content, so as to solve the shortcomings and deficiencies of the existing A280 ultraviolet absorption method for measuring protein concentration

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  • A kind of micro-detection method of antibody protein content
  • A kind of micro-detection method of antibody protein content
  • A kind of micro-detection method of antibody protein content

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Determination of antibody protein content of antibody 1

[0039](1) Preparation of denaturation linear curve: using denaturing solution as diluent, antibody 1 was diluted 10 times, 20 times, 40 times, 80 times, 160 times, and 320 times. Absorbance value; take the dilution factor (the reciprocal of the dilution factor) as the abscissa, and the measured absorbance value is the ordinate to draw a denaturation linear curve, such as figure 1 shown.

[0040] The denaturing liquid is a mixture of 6.3mM disodium hydrogen phosphate dodecahydrate, 13.7mM sodium dihydrogen phosphate dihydrate, 6M guanidine hydrochloride, pH=6.5; the antibody 1 is fermented by Shanghai Taiyin Biotechnology Co., Ltd. , Purified; the absorbance value of the diluted solution is between 0.056 and 0.42.

[0041] (2) According to the protein structure of antibody 1, namely the amino acid sequence, the theoretical extinction coefficient was calculated online by using the ExPASyProtParam tool. ...

Embodiment 2

[0045] Example 2 Determination of antibody protein content of antibody 2

[0046] (1) Preparation of denaturing linear curve: using denaturing solution as the diluent, dilute antibody 2 by 3.33 times, 6.66 times, 8.88 times, 13.32 times and 17.76 times, and measure the absorbance values ​​of solutions with different dilution ratios after standing at room temperature for 1 hour; Take the dilution factor (i.e. the reciprocal of the dilution factor) as the abscissa, and measure the absorbance value as the ordinate to draw a denaturation linear curve, such as image 3 shown.

[0047] The denaturing solution is a mixture of 6.3mM disodium hydrogen phosphate dodecahydrate, 13.7mM sodium dihydrogen phosphate dihydrate, 6M guanidine hydrochloride, pH=6.5; the antibody 2 is fermented by Shanghai Taiyin Biotechnology Co., Ltd. 1. Purify the obtained solution; dilute the obtained solution to measure the absorbance value between 0.056 and 0.42.

[0048] (2) According to the protein stru...

Embodiment 3

[0053] The verification of embodiment 3 method precision

[0054] 3.1 Instrument repeatability: on the same date, the same analyst made a set of standard solution for the test sample, and repeated injection and measurement of this set of standard solution 6 times to obtain a standard curve for 6 times, and calculate the protein concentration of the test sample respectively. The results are shown in Table 1, and the RSD of the protein concentration of the test sample obtained by 6 calculations is 1.30%.

[0055] Table 1 Instrument repeatability test results

[0056] Injection number Concentration (mg / mL) 1 28.36 2 27.95 3 28.29 4 28.92 5 28.32 6 28.85 average value 28.45 standard deviation 0.37 relative standard deviation% 1.30％

[0057] 3.2 Sample repeatability: on the same date, the same analyst made two sets of solutions for the same test sample, and repeated injection of these two sets of solutions 3 times, an...

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Abstract

The invention discloses a trace detection method of an antibody protein level. The trace detection method comprises the following steps: (1) performing gradient dilution on a test sample by taking denaturing solution as a diluent, putting at room temperature, and then measuring absorbance values of solutions with different dilution ratios; drawing a standard curve by taking reciprocals of the dilution ratios as horizontal ordinates and taking the measured absorbance values as vertical coordinates; (2) calculating an extinction coefficient of the test sample under a non-denaturing condition, namely an absorbance value in a normal state with protein of 1mg / mL and an optical distance of 1cm; (3) calculating antibody protein concentration when the optical distance is 1cm. According to the method, the needed sample is extremely few; in a conventional method, milligram-level antibody protein is needed to determine the extinction coefficient by amino acid composition analysis, and then concentration is measured by an ultraviolet spectrometry absorption method; in the method disclosed by the invention, microgram-level antibody protein can be used, the extinction coefficient and the corresponding concentration of the antibody protein can be obtained by only testing once, besides a standard substance is not needed, and the operation is simple and quick; the method is suitable for being used in case of no protein standard substance.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and relates to an antibody protein content detection technology, in particular to a trace detection method for antibody protein content. Background technique [0002] At present, the commonly used methods for determining protein content are: Kjeldahl method, biuret method, Lowry method, Bradford method, ultraviolet absorption method, etc. These methods have their own advantages and disadvantages, and different determination methods can be selected for different types of samples. [0003] The Kjeldahl method is commonly used in the determination of organic compounds, which has long reaction time, complicated operation and large sample volume. The biuret method, the Lowry method and the Bradford method are all colorimetric methods for the determination of protein solutions and all require standards. The biuret method is often used for quick but not very precise assays, such as those us...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/33
CPCG01N21/33
Inventor 黄峥肖楠庄超郑琛齐念民
Owner 上海泰因生物技术有限公司