Complement:Measurement Other techniques tested-for nd fu 6aoatom rotein-detection method)can the amount of test component.Sera that are defi ent in omponents obtained commerially,bu those using preformed defined haemolytic intermediates and are not suitable for regulatory components(C1-inh, Detection of complement activation products factors H and I,properdin) Haemolytic assays for individual components native molecule can assess increased complement turnover. using haemolytic intermediates Before a good range of discriminatory antibodies nts can be added toe or alternative pathway are added up to the level of the products are easily appropriate will bind if &nihodiesareuseg As and A or on o ning compo is pre functional.As for the ab not been fully evaluated.Precautions to prevent generation ssays,by ensuring that all other components are in excess the degree of lysis is proportional to the amount of test ni-Ccophore or preinc o(E pure component ved.Of ah if the EACI.EAC4 to clinicians are ELISA assays for stable,macromolecular and EAC14 are IgM-sensitized sheep erythrocytes coated uanes o pathway)C3bBbe with Cl,C 4and C 1 with C4.respectively.Usinga low ion quantitation o cont 0 complexes indicates the de ree of adding CI to EA.(Note that barbitone buffer isextre pathway.The latter assays use an antibody against one poisonous.)After washing in buffer that does not contain component (e.g. CIs)to bind the complex to an ELISA plate, (gc body again her compc nt of the nd C2 fr cells.leaving EAC4.Adding back Cl in the presence of Detection of complement receptors calcium ions generates EAC14 cells.EACI and EAC14 Monoclonal antibodies to complement ors are ept a up to 2 available commercially and can be used in flow cytometry uti of the tes to detect the presence ofreceptors on cell suspensions.Care sample to EAC4 cells,adding guinea-pig C2 and,finally must be taken in obtaining the specimens usually in the form o -EDTA).Tocalculate the chemistry of tissue sections or cytospin preparation if cell gainst the dilution of the comr onent under test (Fic re 2) morphology is required. As a single complement molecule(C1-C9)can effectively yse an individual cell, Haemolytic assays for individual components using complement-deficient sera plot is not obtained then the concentration of one or more The simplest haemolytic assays for assessing both the components not under investigation is limiting. Similarly,C4 is S osen componen mpk,guinea-pigd 4 ENCYCLOPEDIA OF LIFE SCIENCES/2001 Nature Publishing Group/www.els.net Other techniques Gel rocket techniques, although time consuming and not especially sensitive, are relatively simple. Abnormal forms of complement components can be detected by western blots but Ouchterlony (a protein-detection method) can occasionally be as useful. For example, NHS and C8- deficient serum against anti-C8 may reveal lines of partial identity, suggesting C8 b chain deficiency. Detection of complement activation products Measuring products of complement activation by using antibodies that distinguish activation products from the native molecule can assess increased complement turnover. Before a good range of discriminatory antibodies was available, cleavage products and native molecules could be distinguished by different electrophoretic mobility. Com￾mercial kits are available, but inhouse assays for activation products are easily established, provided appropriate antibodies are used. ELISAs and RIAs for C3a, C4a and C5a can be established, provided standards can be obtained, but the clinical usefulness of these assays has not been fully evaluated. Precautions to prevent generation of anaphylatoxins in vitro are essential. Crossed immu￾noelectrophoresis or preincubation of serum samples with anti-C3c/polyethylene glycol (PEG) allows the quantita￾tion of C3d after the C3c has been removed. Of more help to clinicians are ELISA assays for stable, macromolecular complexes formed during complement activation. The quantitation of C1s–C1-inh (classical pathway), C3bBbP (alternative pathway), or C5b–C9 (terminal pathway) complexes indicates the degree of activation of each pathway. The latter assays use an antibody against one component (e.g. C1s) to bind the complex to an ELISA plate, and an antibody against another component of the complex (e.g. C1-inh) to detect the intact complex. Detection of complement receptors Monoclonal antibodies to complement receptors are available commercially and can be used in flow cytometry to detect the presence of receptors on cell suspensions. Care must be taken in obtaining the specimens that activation of the cells and consequent upregulation of the receptor does not occur. Antibodies are also suitable for immunohisto￾chemistry of tissue sections or cytospin preparation if cell morphology is required. Haemolytic assays for individual components using complement-deficient sera The simplest haemolytic assays for assessing both the presence and functional activity of individual components use EA and a serum deficient in the chosen component. Adding all other components in excess together with serial dilutions of a test sample will give lysis of EA only if the tested-for component is present and functional. As other components are present in excess, lysis is proportional to the amount of test component. Sera that are deficient in individual components can be obtained commercially, but can also be prepared in the laboratory, if such assays are performed regularly. These assays are not as sensitive as those using preformed defined haemolytic intermediates and are not suitable for regulatory components (C1-inh, factors H and I, properdin). Haemolytic assays for individual components using haemolytic intermediates Complement components can be added to erythrocytes in a step-wise fashion. If the components in either the classical or alternative pathway are added up to the level of the component under test, the test component will bind if subsequently added. Addition of the remaining compo￾nents in the pathway will result in lysis if the test component is present and functional. As for the above assays, by ensuring that all other components are in excess, the degree of lysis is proportional to the amount of test component. Most are functionally pure components commercially available, but all can be prepared in the laboratory if there is sufficient requirement. EAC1, EAC4 and EAC14 are IgM-sensitized sheep erythrocytes coated with C1, C4 and C1 with C4, respectively. Using a low ionic strength buffer that contains calcium and magnesium (usually veronal (barbitone) buffer), EAC1 are prepared by adding C1 to EA. (Note that barbitone buffer is extremely poisonous.) After washing in buffer that does not contain calcium, the addition of human serum in the presence of EDTA will result in the binding of C4 and C2. Subsequent incubation at 378C will cause decay of C1 and C2 from the cells, leaving EAC4. Adding back C1 in the presence of calcium ions generates EAC14 cells. EAC1 and EAC14 must be kept on ice and used within a few hours while EAC4 can be kept at 2–48C for up to 2 weeks. C1 activity is assayed by adding dilutions of the test sample to EAC4 cells, adding guinea-pig C2 and, finally, adding the remaining components, usually in the form of rat serum containing EDTA (Crat-EDTA). To calculate the concentration of effective molecules of the component under test, the Z value ( 2 ln (1 2% lysis)) is plotted against the dilution of the component under test (Figure 2). As a single complement molecule (C1–C9) can effectively lyse an individual cell, a straight line is achieved. One unit of complement is taken as that which gives 63% lysis (Z 5 1) in an assay for that component. If a straight-line plot is not obtained then the concentration of one or more components not under investigation is limiting. Similarly, C4 is assayed using EAC1, dilutions of the test sample, guinea-pig C2 and Crat-EDTA as for C1. C2 Complement: Measurement 4 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net