Complement (C) is a group of heat-resistant proteins that exist in human and animal serum or tissue fluids, have enzymatic activity after activation, and can mediate immune and inflammatory responses. It is named “complement” because it can assist and supplement the specific antibody-mediated lytic activity that is heat-resistant. Currently, more than 50 soluble proteins and membrane-bound proteins have been identified, collectively forming the complement system and playing crucial roles in both innate immune defense and adaptive immune responses. These complement components can be further divided into intrinsic complement components (such as C1q/C1s/c1r, C2~C9, MASP-1, MASP-2, factor B (FB), and factor D, etc.), complement regulatory proteins (such as factor H, FH), factor I (FI), factor P, C1 inhibitory factor (C1-INH), decay accelerating factor (DAF), membrane accessory protein (CD46 or MCP), and membrane reactive lysis inhibitory factor (CD59)), as well as complement receptors (such as CR1~CR5, etc.) based on their roles and functions.
Under normal circumstances, most complement in serum exists in the form of inactive zymogens. In pathological conditions, the complement system can be activated through three independent and intersecting pathways: the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). Specifically, AP uses antigen-antibody complexes as the primary activators, which bind to C1q to initiate the activation process. This process sequentially involves C1r C1s, C4, C2, and C3, ultimately forming the C3 convertase (C4b2a) and the C5 convertase (C4b2a3b). Unlike CP activation, the activation of AP does not rely on antigen-antibody complexes. It crosses C1, C4, and C2 to activate C3 directly, and forms C3 convertase (C3bBb) with the participation of factors B, D, and P. Through the C3 positive feedback amplification loop, more C3 convertase and C3b are generated, and multiple C3b and C3 convertase combine to form C5 convertase (C3bB3b). In addition, unlike CP and AP, the LP pathway is mediated by mannose-binding lectin (MBL), which activates complement MASP-1 and MASP-2, as well as C4, C2, and C3, to form the C3 convertase (C4b2a or C3bRb) and the C5 convertase (C4b2a3b or C3bB3b). The above three pathways converge at the activation of C3 to form C5 convertase (C4b2a3b or C3bB3b), which then enters a common terminal pathway to cleave C5 into C5a and C5b. C5b then binds to C6-C9 to form the Terminal Complex C5b-9 (TCC). When a target membrane is present, TCC mediates irreversible damage to the target cell membrane, associated with complement activation in the form of the membrane attack complex (MAC). If there is a lack of target membrane, the formed C5b-9 complex can bind to complement regulatory proteins (such as S protein) to form stable sC5b-9. The common terminal effect of complement activation is the dissolution of target cells.
The timely and moderate activation of the complement system can mediate various biological effects, including cell lysis, bacterial and viral effects, exerting regulatory effects to enhance the phagocytic ability of phagocytic cells, clearing immune complexes, causing inflammatory reactions, and participating in adaptive immune responses. Under normal circumstances, the content of various components in the body’s complement system is relatively stable, and their activation is closely regulated to prevent self-damage. However, when there is a genetic complement deficiency or abnormal complement activation, it may cause various diseases, such as pneumonia caused by various bacterial infections, systemic lupus erythematosus, atypical hemolytic uremic syndrome, C3 glomerulopathy, paroxysmal nocturnal hemoglobinuria, and Alzheimer’s disease. Accurate detection of complement activity and component content is crucial in the diagnosis and treatment of these diseases, as well as in the development of corresponding drugs.
Complement Related Testing and Sample Processing
1. Complement activity or function testing
The determination of total complement activity mainly reflects the activity of classical pathway complement components (C1 to C9). In clinical practice, the CH50 (50% Complex Hemolytic Activity) test, which measures the amount of complement required to cause 50% lysis of red blood cells, is commonly used as an indicator to evaluate total complement activity. Because complement can activate classical pathways to cause hemolysis in sheep red blood cells sensitized with antibodies (such as hemolysin), when the concentration of sensitized sheep red blood cells is constant, the hemolysis rate is positively proportional to complement content, which can be used to detect complement activity through classical pathways. Additionally, for the detection of complement activity in the alternative pathway, EGTA chelation of calcium ions can be used to block the action of C1 and prevent classical pathway activation of complement. Add rabbit red blood cells that can activate factor B, activate the complement pathway, cause hemolysis of rabbit red blood cells, and the complement activity of the pathway (AH50) that causes 50% hemolysis of rabbit red blood cells. The aforementioned 50% red blood cell hemolysis spectrophotometric method for evaluating complement activity is relatively complex to operate, and the individual animal source of the blood sample easily influences the results. It requires a highly robust detection method and consistent detection results. Currently, commercial ELISA kits can also be used to evaluate the complement activity of the classical, alternative, and lectin pathways, which are relatively easy to operate; however, the precision of the results is relatively poor. In addition to CH50 and ELISA detection, liposome-based methods are also used to evaluate classical pathway complement activity, which can meet high-throughput requirements. In practical applications, appropriate detection methods can be selected according to clinical needs for complement activity and function testing.
2. Complement component detection
Due to C3 being a core component of the three complement activation pathways, and C4 being a shared component of the classical pathway and lectin pathway, complement levels of C3 and C4 are commonly used in clinical diagnosis of autoimmune and renal diseases. In addition, C1q is an important component that initiates the activation of the classical complement pathway, and measuring its level can further clarify the activated complement pathway during disease occurrence. On the other hand, given the critical roles played by C3 and C5 convertases in the activation of the complement system, it is of great clinical significance to detect the content of complement components such as target substrates (C3 and C5), components (such as Bb), regulatory proteins (such as FH, FI, and C1-INH), and activation products (including C3a, C3b, C3dg, C5a, and sC5b-9) around these two active enzymes. Currently, multiple detection platforms and commercial ELISA kits are available to achieve quantitative detection of various complement components, including C3, C5, Bb, FH, C3a, C5a, and sC5b-9.
3. Selection and storage of test samples
It is advisable to choose serum samples for the relative quantification of complement activity through various activation pathways; however, plasma samples with added ethylenediaminetetraacetic acid (EDTA) are often used for quantifying complement activation products in vivo. EDTA can chelate calcium and magnesium ions, block the classical and alternative pathways of complement, and effectively reduce complement reactivation in vitro after sample collection. If the test sample needs to retain complement function while being anticoagulated, specific thrombin inhibitors such as piludine can be added. In addition to anticoagulants, the temperature and time during sample collection, storage, and transportation can also affect complement activation in vitro. If samples used for complement analysis need to be stored for an extended period, they should be frozen at -70 ℃ or lower. The requirements for sample storage may vary depending on different detection indicators, but in general, samples need to be frozen within 24 hours after collection and preparation; otherwise, it will affect the results of all types of complement detection. Meanwhile, it is not recommended to freeze complement samples at -20 ℃ as it requires a longer freezing time compared to -70 ℃, during which complement activation continues. Frozen samples should be transported in dry ice and subjected to minimal freezing and thawing during testing whenever possible.
Complement Related Testing and Sample Processing
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired pluripotent hematopoietic stem cell disease characterized by chronic intravascular hemolysis due to acquired defects in red blood cells that are abnormally sensitive to complement activation. Clinically, it is characterized by intermittent episodes of sleep-related hemoglobinuria, which a decrease in whole blood cells or recurrent thrombosis may accompany. The various inhibitors developed for complement C5 can inhibit the activity of related complement, thereby efficiently treating terminal pathway-mediated diseases caused or amplified by abnormal complement activation.
To detect the complement activity of the total complement (classical pathway) and alternative pathways, we established analytical methods using CH50 and AH50 as core indicators, which can provide stable and reliable reference data for complement-related clinical disease research and treatment. And the AP activity detection was developed and validated using an ELISA kit. The validation parameters of these methods meet the predetermined acceptance criteria, which can increase the source of clinical research data for complement pathway-related diseases. For the detection of Free C5 in serum, we have established a corresponding detection method on the Gyrolab platform and successfully applied it to the content analysis of clinical research samples, which can be used for clinical drug research targeting C5. Additionally, we have a mature platform for determining C3 and C4 content in samples. We have established multiple specific methods for detecting complement activation products in blood samples, including allergen components C3a and C5a, pathway-specific component Bb, C3/C4 activation regulatory proteins FH/FI and C1-INH, as well as the terminal complement complex sC5b-9. In clinical research, the accurate and reliable quantitative detection of complement components involved in complement activation pathways at various stages can provide insight into the type and status of complement activation in diseases from multiple perspectives, and it can accelerate the development of clinical drugs targeting the complement system.
A Final Word
As our understanding of complement biology continues to expand, so too does the importance of precise, validated analytical strategies in clinical research. From detecting total complement activity to profiling key components and activation products, tailored assays play a crucial role in elucidating disease mechanisms and informing therapeutic development. With robust platforms and extensive methodological expertise, researchers can generate reliable data that supports the advancement of complement-targeted therapies and enhances patient outcomes across a broad range of immune and inflammatory conditions.
