Digital PCR-based protein active concentration determination method reference method

Abstract

The invention discloses a digital PCR-based protein active concentration determination method reference method. The method comprises the following steps: (1) preparing the antibody of a target protein to be tested; (2) labeling the obtained antibody with biotin; (3) synthesizing three fragments of single-strand oligonucleotides; (4) respectively labeling the 5'-end and the 3'-end of two of the three oligonucleotide segments with avidin; (5) designing a Taqman probe and a corresponding primer; (6) labeling antibodies with the oligonucleotides by using the amplification affinity reaction between the biotin and the avidin; (7) diluting the target protein to be tested with a buffer solution until the concentration is 5000 molecules / [mu]L or less, adding the above obtained two antibodies labeled with the oligonucleotides, the third oligonucleotide fragment and a DNA ligase to form an antigen-antibody compound, carrying out a digital PCR amplification reaction, and recording the obtained copy number as c0; (8) carrying out the digital PCR amplification reaction, and marking the obtained copy number as c1; and (9) calculating the concentration of the target protein to be tested according to a formula of c0 - c1.

Description

technical field [0001] The invention relates to the field of biochemical detection, in particular to a method for measuring protein activity concentration based on digital PCR. Background technique [0002] Metrology is an activity to achieve unit unity and accurate and reliable values. Research and establishment of high-accuracy measurement methods is one of the important contents of metrology research. A reference measurement method is a measurement method of the highest metrological quality, whose operation can be fully described and understood, whose final uncertainty can be expressed in SI units, and whose results are independent of the measurement standard being measured. The benchmark method is the basis for forming the source of the quantity value. [0003] With the development of life science and biotechnology, life science has experienced the development process from "descriptive biology" to "experimental biology" and then to "creative biology". Today's biology ha...

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Problems solved by technology

[0010] The above methods also need to use a chemical standard substance as a standard when performing protein quantification, and its value is not directly traceable to the SI unit; at the same time, the determination results of the protein content of the above determination methods are based on its primary sequence The protein concentration cannot reflect the activity of the protein; in addition, in the process of isotope dilution mass spectrometry, the protein needs to be decomposed into small molecules such as amino acids or peptides for measurement, and certain uncertainties may be introduced in the process of hydrolysis or enzymatic hydrolysis

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