A process for determining complement activation due to contact between a biomaterial and a complemen

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.

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.

The present invention provides a technique to determine one important aspect of the biocompatibility from biomate- rials, being the extent of complement activation. The present invention allows in vitro determination of complement activa¬ tion by incubating biological fluids, blood or blood products with biomaterials, allowing the complement system to be acti- vated and complement convertases to bind to the biomaterials surface. Subsequently, the biomaterials are washed to remove unbound blood proteins and cells and then the biomaterials are incubated in medium containing specific substrate, allo- wing cleavage of this substrate by complement convertases. This cleavage can then be measured due to release of colour, fluorochromes, radioactive label, etc.

In general, substrates in the present invention are labeled tripeptides, although larger structures are not limited hereby. The substrates have or comprise an amino acid sequence resembling the cleavage site of the natural sub¬ strate for the complement convertases or any other amino acid sequence with low Km and high specificity. The concentration of the substrate in the test procedure will normally be in the order of μM in order to have no substrate limitation during the test procedure.

Blood or blood plasma must be anticoagulated during incubation with the biomaterials. Some anticoagulants, such as the Ca4"1" depleting agents citrate and EDTA, also affect the complement system. Mg++ may be used in these situations to allow alternative pathway complement activation. More preferably, serum or fibrinogen depleted plasma or blood/ plasma with specific thrombin inhibitors is used to prevent clotting during incubation with biomaterials. A technique described in the present invention may be used in the screening of biomaterials to select the proper ones for construction of medical devices. It may also be used by test laboratories for blood biocompatibility testing. Further it may be used for research purposes during clinical use of medical devices or inversily to test the ability of an unknown blood sample to react with biomaterials.

The present invention also provides conditions to ensure specificity of the technique for complement convertases rather than other enzymes to cleave the substrates, by intro- duction of inhibitors for other enzymes during substrate conversion.

The present invention comprises measurement of comple¬ ment activation both on the material surface and in the fluid phase. Dependent on the material surface, convertases are released in the biological fluid.

The present invention provides measurement of complement activation in human or animal biological fluid. The present invention allows discrimination between activation of the alternative or classical pathway of the complement system, by employing Ca++ or Mg++ in the biologi¬ cal fluid during incubation with biomaterials, by means of heat treatment of the biological fluid or by inhibition of one of these pathways e.g. with antibodies.

The present invention enables measurement of complement convertase binding to biomaterials during clinical use of these biomaterials by incubation of these used and washed biomaterials in the substrate medium. The present invention allows measurement of convertase activity with substrates labeled with chromophores or fluoro- genes or other suitable markers which are released during peptide cleavage of substrate by the complement convertase. The present invention provides the possibility to characterize complement activation by any device, independent of three-dimensional structure or size. If needed the incuba¬ tion time with substrate can be adjusted to the number of convertases formed during incubation which might be dependent on the surface area and material characteristics of the test material.