A method for detecting the activity of the Fc segment of intravenously injected human immunoglobulin

Abstract

The invention discloses a human intravenous immunoglobulin Fc fragment activity detection method. The method adopts a more easily available stable antigen, adds system applicability verification, employs cytometry for visual detection of red blood cell hemolysis, reduces the result fluctuation caused by sample clarity, impurity protein, reagents, equipment consumables and the like in traditional OD value indirect detection, improves the detection result precision, at the same time reduces the detection system, improves the flux, and is more convenient to operate.

Description

technical field [0001] The invention relates to the technical field of drug analysis, in particular to a method for detecting the Fc segment activity of intravenously injected human immunoglobulin. Background technique [0002] Intravenous injection of human immunoglobulin (Human intravenous immunoglobulin, IVIG) is purified from human fresh frozen plasma and used clinically for treatment. The main ingredient is immunoglobulin G (IgG). The third volume of the Chinese Pharmacopoeia 2015 Edition (hereinafter referred to as the "Pharmacopoeia") stipulates that the purity of IgG should be >95%. A complete IgG molecule includes an Fc segment and two Fab segments bound together by disulfide bonds. The Fab segment is the main antigen-binding site, and the Fc segment is involved in opsonization (promoting the contact and phagocytosis of microorganisms by phagocytes) , Immune regulation of autoimmune diseases (competitive inhibition of effector cell Fc receptors, induction of inh...

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Problems solved by technology

[0005] 1) The antigen used to sensitize red blood cells is bacterial toxoid or virus (Chinese Pharmacopoeia recommends diphtheria toxoid or mumps virus, and European Pharmacopoeia recommends rubella virus), which has certain environmental hazards, is difficult to obtain, and has poor activity uniformity of antigenic components. It is difficult to control the quality between batches, and the difference is large, resulting in unstable sensitization effect

[0006] 2) Only single-point standard data is used for comparison, and there is no standard calibration or system suitability experiment, so the accuracy and validity of each data cannot be guaranteed

[0007] 3) The hemolysis of red blood cells is reflected by the side of the light absorbed at OD540. Samples with poor clarity will easily cause the initial absorbance (used to correct the slope of the hemolysis kinetic curve) to be higher than that of the standard product, resulting in lower results

[0008] 4) Hemoglobin produced after red blood cell hemolysis has light absorption at OD540, which may lead to low test results

[0009] 5) Complement, as a reaction system reagent that must be added, has light absorption at OD540, and it is easy to cause fluctuations in experimental data when replacing batches of complement or changing manufacturers, affecting retrospective analysis between multiple detection samples

[0011] 7) The experiment uses a quartz cuvette, which is not disposable. It is difficult to clean the cuvette after containing red blood cells. The red blood cells gather on the surface of the dirty inner wall, resulting in a high initial absorbance and a low result, or the initial absorbance fluctuates up and down. , seriously affecting the repeatability of the same sample

The inner wall of the cuvette that has not been dried in time after cleaning has water to dilute the sample, resulting in a low initial absorbance and a high result

In addition, the disposable cuvette has been verified to have poor light transmittance and sensitivity, and cannot be used for drawing the kinetic curve of Fc activity

[0012] 8) Pharmacopoeia conventional method, the detection system is 1ml, a single sample detection requires 900μl of samples and standards, 100μl of sensitized red blood cells, 200μl of complement, and 1ml of domestic standard lyophilized preparations can only be used for one batch of sample detection, reagents especially standard The product cost is huge

[0013] 9) The conventional method of the Pharmacopoeia requires a single sample to be measured, and the average time-consuming is 30min / sample, and the throughput is low

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