Measurement of complement activation by biomaterials by means of complement convertase cleavage

Biocompatibility is an essential aspect of the medical device industry. Biocompatibility testing ensures that devices do not contain materials or substances that could be harmful to patients during initial use or over the course of time. Biocompatibility tests can be used to detect many possible negative side effects of a product on patient. These may include effects on cells and physiological systems, tissue irritation and inflammation, immunological and allergic reactions and the possibility of cellular mutations leading to cancer. Email:marketing@medicilon.com.cn web:www.medicilon.com

A process for determining complement activation due to contact between a biomaterial and a complement system, by incubating in vitro the biomaterial with the complement system and determining the formation of a complement convertase by using a substrate of the complement convertase and detecting substrate cleavage. The biomaterial after said incubation with the complement system may be separated therefrom and formation of a complement convertase may be determined with the separated biomaterial, the separated complement system, or both. The complement convertase may be Factor B convertase, C3 convertase or C5 convertase, and the substrate may be a labeled oligopeptide comprising an amino acid sequence corresponding to the cleavage site of the complement convertase. Suitable labels are dyes, fluorochromes, radioactive atoms or groups, and enzymes. Either classical or alternative pathway complement activation, or both, are determined. The complement system may be a non-clotting derivative of blood, blood plasma or blood serum, including fibrinogen depleted forms, anticoagulated forms, and derivatives containing thrombin inhibitor. Complement convertase substrates suitable for use in the process are also disclosed.

The invention is in the field of diagnostics and relates to a new diagnostic technique in medicine. More specifically it is concerned with the measurement of activation of an im- munologic system in blood, the complement system, when blood is contacting a foreign body surface (biomaterial) .

In addition to its role as an anaphylatoxin, C5a is a potent chemotactic factor. This mediator causes the directed migration of PMN and macrophages to the site of inflammation so these leukocytes will phagocytize and clear immune complexes, bacteria and viruses from the system.

In a process known as immune adherence, C3b or C4b depo¬ sited on a soluble immune complex or surface permit binding of complement receptors on PMN, macrophages, red blood cells and platelets (28,29) . In these cases C3b and C5b are consi¬ dered opsonins as their presence results in more effective phagocytosis.

Laboratory measurement of complement proteins

The following two techniques for assessing the comple¬ ment system are known.

1) Hemolytic techniques measure the functional capacity of the entire sequence - either the classical or alternative pathway.

2) Immunological techniques measure the protein concen¬ tration of a specific complement component or split product. Hemolytic techniques

In order for lysis to occur in a hemolytic technique, all of the complement components must be present and functio¬ nal. Therefore hemolytic techniques can screen both functio- nai integrity and deficiencies of the complement system (30,31).

To measure the functional capacity of the classical pathway, sheep red blood cells coated with hemolysin (rabbit IgG to sheep red blood cells) are used as target cells (sensitized cells) . These Ag-Ab complexes activate the clas¬ sical pathway and result in lysis of the target cells when the components are functional and present in adequate concen¬ tration. To determine the functional capacity of the alterna¬ tive pathway, rabbit red blood cells are used as the target cell.

The hemolytic complement measurement is applicable to detect deficiencies and functional disorders of complement proteins, since it is based on the function of complement to induce cell lysis, which requires a complete range of func- tional complement proteins. The so-called CH50 method, which determines classical pathway activation, and the AP50 method for the alternative pathway have been extended by using specific isolated complement proteins instead of whole serum, while the highly diluted test sample contains the unknown concentration of the limiting complement component. By this method a more detailed measurement of the complement system can be performed, indicating which component is deficient.

However, in order to induce deficiencies of complement proteins in serum from healthy individuals, which must be used to determine biocompatibility, a very high extent of complement activation and consumption is required. Therefore, in general the hemolytic techniques are not sensitive enough to detect complement activation by biomaterials. Some hemo¬ lytic techniques based on isolated components with a highly diluted test sample appeared to be more sensitive, but even complement activation induced by 5 m2 surface of a heart-lung machine could marginally be detected with these methods (32). Immunologic techniques

Polyclonal antibodies were raised against different epitopes of the (human) C3, C4 and C5 complement factor. With these antibodies radioimmunoassays were developed against the minor split products of these complement factors, which are particularly performed after precipitation of the native factor (33-36). Binding of the antibody with the split product in competition with a known concentration of labeled split product could then be measured. Later on also (monoclonal) antibodies were raised to epitopes of the split products, rendering a higher specificity. Nowadays, radio¬ immunoassays, ELISA's and radial diffusion assays are availa¬ ble to detect complement split products.

In contrast to the hemolytic techniques, immunologic techniques provide a high sensitivity to detect complement activation, since they allow measurement of split-product formation, while these split products are only found at very low concentrations in blood from healthy individuals. Thus, clinically the measurement of the soluble split products C3a, C4a and C5a in blood plasma has allowed a more distinct evaluation of complement activation in patients (37). Later on the soluble form of the terminal complex (SC5b-9) was found a sensitive marker of complement activation (38). For detection of in vivo complement activation these techniques are most suitable, particularly since blood samples can be collected in medium containing inhibitors of the complement system. Thus only the complement activation formed in vivo is measured in the subsequent assay.

However, these in vivo or clinical studies cannot be used to determine the biocompatibility of biomaterials. Main problem during clinical use is that during application of biomaterials the complement system is activated by a variety of material-independent factors, such as surgical damage of tissue, ischemia, blood-air contact, endotoxin and drugs which alltogether dominate complement activation induced by the biomaterial. Thus, for pure biocompatibility testing in vitro studies are required, based on exposure of the biomate¬ rial to isolated blood or blood components (usually plasma or serum). At this end difficulties arise. Starting with the isolation of blood from a donor, during preparation of plasma or serum for the test, and during the test phase itself in the test tube the complement system is activated and high concentrations of split products are formed in plasma or serum. This high concentration of split products dominates the split products eventually formed by the test biomaterials during the test procedure. Thus, the sensitive immunologic techniques appear unsuitable for in vitro testing of biocom- patibility. Moreover, it has been shown that to some biomate¬ rials the split products adsorb to the surface. By the immunologic techniques these adsorbed split products are not detected. This leads to false negative appreciation of the test sample.