Biological activity detection method for VEGF targeted therapy drugs

Abstract

The present invention provides a biological activity detection method for VEGF targeted therapy drugs. The biological activity detection method comprises: taking HUVEC cells being in a logarithmic growth phase, adjusting to achieve an appropriate cell density, plating, and culturing to make cells be adhered on the plate; carrying out series gradient dilution on a reference substance and a sample, sequentially adding to a culture plate with paved cells, and continuously incubating for a certain time; and carrying out a coloration reaction on the cell culture plate by using a detection system for detecting the amount of living cells or dead cells, and obtaining the biological activity of the sample according to the coloration reaction results. According to the present invention, the requirements on the specificity, the accuracy, the precision (including repeatability, day difference, personnel operating error, and the like) and other verifications can be met, and the method can be effectively applied for screening and quality control of VEGF targeted therapy drugs.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a biological activity detection method for VEGF-targeted therapeutic drugs. Background technique [0002] Vascular endothelial growth factor (VEGF) is a kind of growth factor that can promote the division and proliferation of endothelial cells, promote the formation of new blood vessels, and improve the permeability of blood vessels. It can promote the proliferation of endothelial cells by binding to the corresponding receptors on the vascular endothelium, increase the permeability of blood vessels, make endothelial cells migrate, induce tumor angiogenesis, and maintain the continued growth of tumors. It is the strongest angiogenesis found so far. Factors are related to many physiological and pathological processes. Studies have shown that tumor growth has two distinct phases, namely the slow growth phase without blood vessels and the rapid proliferation phase wi...

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Problems solved by technology

[0004] At present, the commonly used method for measuring the biological activity of VEGF-targeted therapeutic drugs is designed based on the principle that VEGF can promote cell proliferation and differentiation, and drugs can neutralize VEGF activity, thereby inhibiting cell proliferation. The cells used The main line is HUVEC cells. According to literature reports, the biological activity method of cell proliferation inhibition has not been recognized or standardized. Since HUVEC cells are primary cells and the cell adhesion is not good, ordinary cell culture plates are used. Unreliable experimental results can not be obtained, so a layer of gelatin needs to be pre-laid on the 96-well plate in advance during the experiment to help HUVEC adhere to the wall, and the gelatin laying situation directly affects the cell state and experimental results, making the experiment complicated and difficult standardization

In addition, the color development methods and color development conditions are also different. From the literature reports, most of them use radioactive isotopes ( 3 H-TdR) labeling method, AlamarBlue or CelltiterGlo chromogenic method, the former is an isotope labeling method, which has certain radiation and damage to the environment and personnel, which is not conducive to method promotion and quality control; AlamarBlue is a fluorescent chromogenic method, color development The operation is convenient, but the experiment must be carried out on an opaque 96-well cell plate, and it is inconvenient to observe the cell state during the experiment, and the coefficient of variation of the RLU value between multiple wells is relatively large; the CelltiterGlo chromogenic method is a chemiluminescence detection method based on ATP , it is also necessary to use an opaque 96-well cell plate for the experiment, which is not convenient for observing the cell state during the experiment; in addition, since the storage condition of the substrate and buffer is -20°C, it must be thawed before color development, and the The substrate and the buffer solution are mixed and returned to room temperature before they can be used for color development. The prepared color development solution can only be stable for 48 hours at 4°C.

In addition, more importantly, among the reported biological activity detection methods for VEGF-targeted therapeutic drugs, there is a lack of method validation and evaluation of the specificity, precision, accuracy, linearity and range of the detection system. , it is difficult to ensure the accuracy and reliability of the test results

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