Complement:Measurement Secondary article Jonathan North,City Hospital NHS Trust,Birmingham,UK Artice Contents K Whaley,University of Kuwait,Safat,Kuwait The complement system comprises a group of serum proteins and cell membrane receptors that function primarily to fight infection.Clinically,measurement of Introduction complex proteolysis.Cleaved C4(C4b)will bind to the activating membr function complex. The sent an the central results of activation of these pathways are to C3 by limited pr iActivated C3(C3)will bind deposit the opsonin C3b on bacteria to promote phago cytosis,to lyse by t assembly of the terminal valently to certain surfaces (see below)and act as a high- afinity ligand for C3b receptors on phagoeytes. to the In ren :C4b2a dual com onent levels is,therefore.of value in cases of immunodeficiency inhibito and conditions that pdmheimtialswages CI-inhibitor (CI-inh).which binds to CIr and CIs and volve complemic lupus erythematosus (SIE prevents C4 activation.C4-binding protein (C4bp)dis and acts as a cofactor for the readily achieved using standard immunochemical meth ods.Nephelometry and turbidimetry can be used to assay Mannan-binding lectin activation of (CI-i h)).Enzyme-linked say (ELISA) complement or radioimmunoassay(RIA)are used to measure compo several molecules nents present in rela tively low concentrations,but s m In addition to immune c plasma centrati 0 bndheolhgemous.eobCigamdogrQhso of th jority of the uch s DNA. olecul onents To fully app eciate the condi- tions required for the functional assays,it is important to polysaccharides understand he process occurring. Accordingly In contrast,mannan-binding lectin (MBL)is a protein nditions p necess is in the dy an q when bo following section,with more detailed descriptions to be found in the individual assay sections.Full methodologies in this chapter are given in the vith two MBL-associated serine protea es(MASPs I and 2)that activate C4 and C2 to form C4b2a;the classical I here i ctly.The MR The classical pathway uire scalcium ions for its formation and binding to carbohydrate but will activate C4 in the absence of calcium once bound. ecomea that pontain imunopo on Once bound.the activated and this,in turn.activates Cls.The integrity of ENCYCLOPEDIA OF LIFE SCIENCES/2001 Nat ishing Group/www.els.net
Complement: Measurement Jonathan North, City Hospital NHS Trust, Birmingham, UK K Whaley, University of Kuwait, Safat, Kuwait The complement system comprises a group of serum proteins and cell membrane receptors that function primarily to fight infection. Clinically, measurement of complement pathway activity and individual component levels is of value in cases of immunodeficiency and inflammatory conditions that involve complement activation. Introduction The complement system comprises a group of serum proteins and cell membrane receptors that function primarily to fight infection. These components interact in three activation pathways and one final pathway. The central results of activation of these pathways are to deposit the opsonin C3b on bacteria to promote phagocytosis, to lyse bacteria by the assembly of the terminal membrane attack complex and to promote inflammation. Clinically, measurement of pathway activity and individual component levels is, therefore, of value in cases of immunodeficiency and inflammatory conditions that involve complement activation, the prime example of the latter being systemic lupus erythematosus (SLE). The measurement of complement protein levels is readily achieved using standard immunochemical methods. Nephelometry and turbidimetry can be used to assay levels of serum complement components that are present in relatively high concentrations (e.g. C3, C4, C1-inhibitor (C1-inh)). Enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA) are used to measure components present in relatively low concentrations, but serum/ plasma concentrations of all components have been successfully measured by ELISA. It is also possible to determine the functional activity of the majority of the complement components. To fully appreciate the conditions required for the functional assays, it is important to understand the process that is occurring. Accordingly a summary of the complement pathways, stressing certain conditions necessary for activation, is given in the following section, with more detailed descriptions to be found in the individual assay sections. Full methodologies for the assays described in this chapter are given in the literature listed for further reading. The classical pathway Immune complexes that contain immunoglobulin G (IgG) or IgM can activate the classical pathway by the binding of the first component of complement (C1) via the C1q subcomponent. Once bound, the subcomponent C1r is activated and this, in turn, activates C1s. The integrity of the C1q–C1r–C1s complex is calcium-dependent. Activated C1s is a serine protease that activates C4 by limited proteolysis. Cleaved C4 (C4b) will bind to the activating complex. C2 binds to C4b when magnesium ions are present and C1s will then activate C2 to form the C4b2a complex (classical pathway C3 convertase) that activates C3 by limited proteolysis. Activated C3 (C3b) will bind covalently to certain surfaces (see below) and act as a highaffinity ligand for C3b receptors on phagocytes. In addition to the inherent instability of C4b2a, spontaneous activation of the classical pathway is controlled in the initial stages by the serine protease inhibitor C1-inhibitor (C1-inh), which binds to C1r and C1s and prevents C4 activation. C4-binding protein (C4bp) dissociates the C4b2a complex and acts as a cofactor for the cleavage of C4b by factor I. Mannan-binding lectin activation of complement In addition to immune complexes, several molecules can bind the collagenous region of C1q and cause classical pathway activation. Such molecules include C-reactive protein and fibronectin, and molecules that have an anionic nature such as DNA, lipopolysaccharide and mucopolysaccharides. In contrast, mannan-binding lectin (MBL) is a protein that structurally resembles C1q itself and can activate C4 in the absence of antibody and C1q when bound to specific carbohydrates (e.g. those of many bacteria). MBL circulates as a macromolecular complex in association with two MBL-associated serine proteases (MASPs 1 and 2) that activate C4 and C2 to form C4b2a; the classical pathway C3 convertase. There is also evidence that the MBL/MASP complex can activate C3 directly. The MBL/ MASP complex requires calcium ions for its formation and binding to carbohydrate but will activate C4 in the absence of calcium once bound. Article Contents Secondary article . Introduction . Measurement of Total Complement Activity . Quantitation of Individual Components . Clinical Relevance ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 1
Complement:Measurement The alternative pathway Specimen preparation and storage The As some els Fo hydrolysis of a small proportion of circulating C3.which serum.blood samples should arrive in the laboratory as permits binding of factor B in the presence of magnesium soon as possible after venepuncture.Blood should be ons.Factor D cleaves the bound The C3b g erated in this way can bind to surfaces via a thioester and,once attached,bind more factor B.The be aliquotted and stored at -70C as quickly as thioester bond preferentially reacts with hydroxyl groups practicable and adequate aliquots stored from each sample to form ester of alternative pathway- to prevent tha ng and reir reach assay.For use s Hand I Sialic acid is one a int known surface constituent which render surfaces resistant traacetic acid)(which prevents further complement activa ay-accele ples are su of on he d ed as fo to C3b on activating surfaces is cleaved by factor D to give ind C3bBbP car inhibitors such as phenylmethvlsul- s,thus amp bind C3 the below）. Haemolytic complement assays If the MACisa ne ofred blood The membrane attack complex(MAC) lls and po ur This phenomenon is used to assess the functional integrity of the ean be actnated by either cal whole of the class cal or alterative pathways with the respectively).Activated C5 binds to C6 and the remaining components,C7,C8 and C9.bind in turn.At least six and terminal complementco onents and de ends on the molecules o components being functionally active.The assay is 0 a cell on whi sas Two membrane-poundt roteins homo to proc Th logous restriction factor(HRF)and CD59,inhibit self- thway is initiated by IgM on the surface of sheep red inflicted cell lysis. blood cells(EA.antibody sensitized sheep erythrocytes) n the presence of calium and way activa Sheep red cells are inefficient at activating the alterative Measurement of Total Complement Activity thy ates cal but not n gn The Chsoand aphsoassays are used to determine whether way while permitting activation of the altemative or not a patient is genetically deficient in a complement pathway. These assays need to be ntrolled with positive contro a serum amp nown to co ,e.g.fres blood cel(classical pathway.CHor rabbit erytro control)which controls spontaneous lysis of the erythro cytes(alternative pathway,APHso).respectively.)Both the cytes.The amount of haemolysis(measured by the optical presence of a nonlunc a one-tube meth ernatively,using the 2 ENCYCLOPEDIA OF LIFE SCIENCES/2001 Nature Publishing Group/www.els.net
The alternative pathway The alternative pathway of complement activation requires C3, factor B, factor D and properdin. Activation of this pathway involves a constant, low level of spontaneous hydrolysis of a small proportion of circulating C3, which permits binding of factor B in the presence of magnesium ions. Factor D cleaves the bound factor B to form C3Bb and the latter can activate further C3 to form C3a and C3b. The C3b generated in this way can bind to surfaces via a thioester and, once attached, bind more factor B. The thioester bond preferentially reacts with hydroxyl groups to form ester bonds. Surfaces of alternative pathwayactivating particles bind C3b in a form that is resistant to the regulatory activities of factors H and I. Sialic acid is one known surface constituent which render surfaces resistant to alternative pathway activation. The membrane-associated regulatory molecules decay-accelerating factor (DAF), CR1, and membrane cofactor protein (MCP) also mediate inactivation of C3b on host cells. Factor B bound to C3b on activating surfaces is cleaved by factor D to give C3bBb. Properdin stabilizes this complex and C3bBbP can cleave further C3 molecules, thus amplifying the process. Additional C3b can bind to the original C3b to alter the specificity of the enzyme to that of a C5 convertase (see below). The membrane attack complex (MAC) C5 can be activated by either the classical or alternative pathway by C5 convertases (C4bC3b2a and (C3b)nBbP respectively). Activated C5 binds to C6 and the remaining components, C7, C8 and C9, bind in turn. At least six molecules of C9 will bind to this complex and form a pore in the membrane of a cell on which it is present. This can lead to lysis of the cell, a property that is used in haemolytic assays. Two membrane-bound regulatory proteins, homologous restriction factor (HRF) and CD59, inhibit selfinflicted cell lysis. Measurement of Total Complement Activity The CH50 and APH50 assays are used to determine whether or not a patient is genetically deficient in a complement component. (CH50 and APH50 refer to the reciprocal dilution of a serum required to produce 50% haemolysis of a standard preparation of antibody sensitized sheep red blood cells (classical pathway, CH50) or rabbit erythrocytes (alternative pathway, APH50), respectively.) Both the deficiency of a component or the presence of a nonfunctional component will be detected. Specimen preparation and storage As some components are extremely labile, incorrect storage of samples for complement assay can result in decreased levels. For assays of individual components in serum, blood samples should arrive in the laboratory as soon as possible after venepuncture. Blood should be allowed to clot at room temperature for 30 minutes, then the sample placed on ice for 1 hour for clot retraction to occur. Separation should occur at 2–48C. Samples should be aliquotted and stored at 2 708C as quickly as practicable and adequate aliquots stored from each sample to prevent thawing and refreezing for each assay. For use, samples should be thawed at 378C then immediately placed on ice. Blood collected into EDTA (ethylenediaminetetraacetic acid) (which prevents further complement activation by chelating calcium and magnesium and, hence, such samples are suitable for activation product assays) should be kept on ice for as short a time as possible before being spun to produce platelet poor plasma which is stored as for serum. Samples from tissue culture may benefit from the addition of proteinase inhibitors such as phenylmethylsulfonyl fluoride (PMSF). Haemolytic complement assays If theMAC is assembled on the cell membrane of red blood cells and polymeric C9 inserted, lysis will occur. This phenomenon is used to assess the functional integrity of the whole of the classical or alternative pathways with the terminal pathway. The simplest of these assays is the CH50. This assay depends on a sample containing all the classical and terminal complement components and depends on the components being functionally active. The assay is quantitative, with the result expressed as the reciprocal of the serum dilution required to produce lysis of 50% of defined numbers red cells under standard conditions. The pathway is initiated by IgM on the surface of sheep red blood cells (EA, antibody sensitized sheep erythrocytes) and the assay is performed in the presence of calcium and magnesium ions (required for classical pathway activation). Sheep red cells are inefficient at activating the alternative pathway, so rabbit erythrocytes are used in APH50 assays. EGTA (ethyleneglycol bis(b-aminoethyl ether)-N,N-tetraacetic acid) chelates calcium, but not magnesium ions, and will prevent any concomitant activation of the classical pathway while permitting activation of the alternative pathway. These assays need to be controlled with positive control samples known to contain all the components, e.g. fresh normal human serum (NHS), and by buffer alone (negative control) which controls spontaneous lysis of the erythrocytes. The amount of haemolysis (measured by the optical density of the cell supernatant) can be compared with a known NHS in a one-tube method. Alternatively, using the Complement: Measurement 2 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Complement:Measurement von Krogh equation which describes the curve obtained by Quantitation of Individual Components plotting the percentage lysis against the sample dilution When a serum is shown to be deficient in classical, CH (Figure 1.Controls and standard conditions are essential. as the CHso unit obtained depends on the amount and nature of the antibody use ensit e the cells,the Immunochemical assays reaction time and temperature The assay system of choice depends on the number of level of ensitivity required tubes or agar inic,eithe nedusingsipgetest ailable Fo hel anti ctduiomate used in routine clinical immunology laboratories for somes is also available. measuring C3,C4.Cl-inh and Clq.Although radial uc.fo Immunochemical assays complement components.For cell culture studies.how ELISA plates coated with a classical pathway activator ever,levels may be as low as I ngmL and ELISA or RIA be the only practical method avail ole.Ihe bu (e.g.IgM,IgG-containing complexes) or an alternative pathway activator (lipopolysaccharide)ca be use nti nd r on p usually used.For all of these assays.the choice of antibody epitopes erdino e may recognize own pro 0 0. 100 0 100( Figure 1 Log ENCYCLOPEDIA OF LIFE SCIENCES /e 2001 Nature Publishing Group /www.elsne 3
von Krogh equation which describes the curve obtained by plotting the percentage lysis against the sample dilution, the sample dilution required to obtain 50% haemolysis is calculated and, after taking the initial sample dilution into account, this value is translated into CH50 units mL 2 1 (Figure 1). Controls and standard conditions are essential, as the CH50 unit obtained depends on the amount and nature of the antibody used to sensitize the cells, the erythrocyte concentration and fragility, the ionic strength, divalent cation concentration and pH of the buffer, reaction time and temperature. The above assays can also be performed using single test tubes or agarose gels, either method being suitable for large-scale clinical screening. An automated method for screening classical pathway activity using sensitized liposomes is also available. Immunochemical assays ELISA plates coated with a classical pathway activator (e.g. IgM, IgG-containing complexes) or an alternative pathway activator (lipopolysaccharide) can be used to assess the respective pathways by looking for the appearance of neo-epitopes of C9 or properdin upon the addition of serum. Quantitation of Individual Components When a serum is shown to be deficient in classical, alternative or terminal pathway activity, then the component that is missing or nonfunctional is defined. Immunochemical assays The assay system of choice depends on the number of assays being performed, the level of sensitivity required, the level of the analyte and the quality of the antibody available. For instance, in patient samples, nephelometry is used in routine clinical immunology laboratories for measuring C3, C4, C1-inh and C1q. Although radial immunodiffusion (RID) (available commercially) can be used, ELISA is a robust and sensitive technique, especially with regard to quantitation of serum/plasma levels of other complement components. For cell culture studies, however, levels may be as low as 1 ng mL 2 1 and ELISA or RIA may be the only practical method available. The buffers used in these types of assay are those that are optimal for antibody binding and phosphate-buffered saline (PBS) is usually used. For all of these assays, the choice of antibody is critical. Polyclonal antibodies are generally used, and these may recognize breakdown products of the component under investigation and the resulting value obtained may not reflect the level of functional protein present. 10 1 0.1 100 20 30 40 50 1000 Serum volume (µL) y/(1–y) Figure 1 Log–log plot of y/1 2 y against volume of diluted serum in a CH50 assay. At the point of 50% haemolysis, y/(1 2 y) 5 1 and the volume of diluted serum giving this is shown by the vertical line. Redrawn from Whaley K (ed.) (1985) Methods in Complement for Clinical Immunologists, p. 102. Edinburgh: Churchill Livingstone. Complement: Measurement ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 3
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. Commercial 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 immunoelectrophoresis or preincubation of serum samples with anti-C3c/polyethylene glycol (PEG) allows the quantitation 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 immunohistochemistry 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 components 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
Complement:Measurement 20 1.8 1.6 12 1.0 08 0.6 04 0.2 1:8x101:4x103 1-2x103 Serum dilution 1:5x10 Figure 2 nce forivity is measured using EACu n this case the time for max for the cells must be ineme e epared to provide In a more technically demanding assay.Cl-inh activity can be measured by testing the ability of the test sample to y.C2and C3areadded to a given level of exogenou -I adde【o sensitize 1 hu开 e Cl-inh and C2 by incubation in EDTA buffer.A stable C3 can to ind in he fuid phase before convertase is formed on the resultant EAC43b if factor B. add an EDTA.S e ad d8°4品5 is A,pnor o din by a adding dilutions of the run in allel for direet cor test sample and omitting the respective pure component FactorHlvels can be assayed by measuring the ability ecay of the C3 convertase on met are rep Th terminal components up to the one under test to EAC14xy the sensitized cells with the test sample is compared to a 2.EAC1-8.used for measuring C9 activity,will undergo us lysis so must be used ol tube.Similarly.activity can be aye le wit act prepare D dded in athu and anyr haemolytic activity indicates a deficiency of the inhibitor. Functional assays of complement control proteins The ability of plasma cl-inh to inhibit Cls or a Cls analogue is used in commercial kits or inhouse assays in 5
activity is measured using EAC14 but in this case the time for maximum C4b2a formation (Tmax) for the cells must be known. TheTmax depends upon the amount of C4b present on the cells (Figure 3). As the C4b2a complex is more unstable than most, EAC14oxy 2 are prepared to provide increased haemolytic activity of C2, required for the measurement of C3 activity. For alternative pathway activity, C2 and C3 are added to EAC14 to form EAC1423 with subsequent removal of C1 and C2 by incubation in EDTA buffer. A stable C3 convertase is formed on the resultant EAC43b if factor B, factor D and properdin are added. Haemolysis is achieved by adding Crat-EDTA. Samples can be assayed for the activity of factor B or properdin by adding dilutions of the test sample and omitting the respective pure component. Factor D activity in serum cannot be measured by this method and, therefore, only pure preparations of factor D are suitable. Terminal components are assayed by adding C3 and the terminal components up to the one under test to EAC14oxy 2. EAC1–8, used for measuring C9 activity, will undergo spontaneous lysis so must be used as soon as they are prepared. Functional assays of complement control proteins The ability of plasma C1-inh to inhibit C1s or a C1s analogue is used in commercial kits or inhouse assays in colorimetric reactions. Some of these assays are easily adapted to look for the presence of anti-C1-inh antibodies. Very rarely, dysfunctional C1-inh variants may inhibit C1s in some of these assays. In a more technically demanding assay, C1-inh activity can be measured by testing the ability of the test sample to inhibit a given level of exogenous C1 added to sensitized EA. If serum is being tested then C1 first has to be removed, e.g. by precipitating C1 out using phosphate buffer. C1-inh can be allowed to bind to C1 either in the fluid phase before adding to EAC4 with C2 or on EAC14, prior to the addition of C2. For haemolytic inhibitor component assays generally, a ‘solo’ reaction that has no inhibitor is run in parallel for direct comparison. Factor H levels can be assayed by measuring the ability of serum to accelerate the decay of the C3 convertase on erythrocytes. EAC4b3bBbP are prepared by adding properdin, factor B and factor D to EAC4b3b. The amount of haemolytic activity remaining after incubating the sensitized cells with the test sample is compared to a solo tube. Similarly, factor I activity can be assayed by incubating the test sample with factor H added to EAC43 to inactivate the C3. Factors B and D are then added in order to complete the alternative pathway and any residual haemolytic activity indicates a deficiency of the inhibitor. 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1:8x105 1:4x105 1:2x105 Serum dilution x y 1:5x104 Z Figure 2 The number of functionalmoleculesper cell(Z)is plottedagainstthe serum dilutionin a typicalhaemolytictitration of an individual complement component.The concentrationof the component in this instanceis given by y/x 50 000. RedrawnfromWhaleyK (ed.) (1985) Methodsin Complement for Clinical Immunologists, p. 107. Edinburgh: Churchill Livingstone. Complement: Measurement ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 5
Complement:Measurement 1.0 0.9 0.8 0.6 z05 04 0.1 0.0 123456Tme(mn10 15 Clinical Relevance Immunodeficiency t or which no nng autoimmune haemolysis,often as part of the haemolytic- This is is also ass with recurren hic pccially important in cases of meningococcal meningitis. ommonest presentation of terminal pathway ases The inconsiste ncy in the clinical effect on individuals seen in complement deficiency suggests other factors are LAPH mportant in defining the phenotype seen but,as yet,no CIs C4or C the latter beins the commonest complement CD18)and CR4(CDIla CD18)are associated with severe recurrent bacterial one of the cidgponer infection and poor wound healing.Expression on periph- in C3 cr a components.In practice,C9 deficiency is associated with wn nor some lysis of the test cells.Low values above zero suggest the level of one or more complement compone nts state As mapy Immune complex disease complement components are acute phase proteins,the Clinical conditions resembling SLE and often involving detection of heterozygous deficiency states is best achieved glomerulonephritis are seen in over 50%of pa tients with by genotyping.rather than by measuring levels of the ssical pathway component deficiency (Pickering and Deficiencies in the classical nath ciated with ncies of Clq and total deficiency of are often ass in these patients,but arealso seen n patients with SLE that increased infection and une complex is not associated with inherited complement deficiency iiec2arnndasaeonc ciency (whic 6 ENCYCLOPEDIA OF LIFE SCIENCES/2001 Nature Publishing Group/www.els.net
Clinical Relevance Immunodeficiency The authors recommend that any individual experiencing serious and/or recurrent sepsis, for which no other cause is identified, should be tested using a CH50/APH50. This is especially important in cases of meningococcal meningitis, which is the commonest presentation of terminal pathway component deficiencies and properdin deficiency (Ross and Denson, 1984). An absence of CH50 activity, but normal APH50 activity, indicates a deficiency of C1q, C1r, C1s, C4 or C2 (the latter being the commonest complement component deficiency). In contrast, a lack of APH50 activity but normal CH50 suggests a deficiency of properdin or, less commonly, factor D. No activity in either assay indicates a deficiency in C3 or one of the terminal components. In practice, C9 deficiency is associated with some lysis of the test cells. Low values above zero suggest that the level of one or more complement components is either decreased due to consumption in a disease state or may indicate a heterozygous deficiency state. As many complement components are acute phase proteins, the detection of heterozygous deficiency states is best achieved by genotyping, rather than by measuring levels of the protein. Deficiencies in the classical pathway are associated with sepsis and/or immune complex disease (Pickering and Walport, 2000). C2 deficiency is more often associated with increased infection and less often with immune complex disease compared to deficiencies of C1 and C4. Asymptomatic deficient individuals are often defined in family studies, the exception being C3 deficiency which is invariably associated with recurrent sepsis. Factor H deficiency results in secondary C3 deficiency and an increase in infections, but is primarily associated with autoimmune haemolysis, often as part of the haemolytic– uraemic syndrome. MBL deficiency is also associated with recurrent infections in children and some adults but only in some cases. The inconsistency in the clinical effect on individuals seen in complement deficiency suggests other factors are important in defining the phenotype seen but, as yet, no clear associations have been found. Deficiencies of CR3 (CD11b/CD18) and CR4 (CD11a/ CD18) are associated with severe recurrent bacterial infection and poor wound healing. Expression on peripheral blood neutrophils and monocytes can be routinely assessed by flow cytometry, and compared with that on known normal cells. Immune complex disease Clinical conditions resembling SLE and often involving glomerulonephritis are seen in over 50% of patients with a classical pathway component deficiency (Pickering and Walport, 2000). Deficiencies of C1q and total deficiency of C4 are often associated with discoid or systemic lupus erythematosus. Increased bacterial infections are also seen in these patients, but are also seen in patients with SLE that is not associated with inherited complement deficiency. There is also an increased incidence of SLE in patients with factor I deficiency (which results in secondary C3 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 1 2 3 4 5 6 Time (min) 10 15 T max Z Figure 3 Plot of the number of haemolytic sites (Z) against the incubation time at 308C in a Tmax assay. Tmax is indicated by the arrow. Redrawn from Whaley K (ed.) (1985) Methods in Complement for Clinical Immunologists, p. 96. Edinburgh: Churchill Livingstone. Complement: Measurement 6 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Complement:Measurement deficiency).More commonly seen is the association detected by ELISA.immobilizing Cl-inh on the plate. between C4-null alleles and connective tissue disease such incubating with patient serum and detecting bound anti- body by enzyme-labelled anti-IgG,IgA or IgM. expression on r 0C3 partic C4 are consumed during active disease and serial C3 and C3 nephritic factor C4 levels are markers of disease activity.C4 may,however, Patients with membranoproliferative glomerulonephritis, phy,often st C3bBb.Th dthngcoroductu monitoring d clinical relanse in sie References C1-inh deficiency rin MC and Walport MJ (2000)Links betwee 1998).Low C4 and C2levels are seen during acute attacks temic lapus erythematosushe abnorn protein and functional assays are required for the correc diagnosis.Acquired Cl-inh deficiency(AAE)is associated Further Reading with either B cell neoplasia,connective tissue disease or cinomas.dn Dodd AWand SimsRB(eds)(1997)Complemen.A Practical Approach ersity Pres now appears that AAE is always associated with auto- antibodies to Cl-inh.The antibodies affect Cl-inh func tem.In:Rother K.Till GO tion directly and result in an inactive form of Cl-inh ent.In A Practical ENCYCLOPEDIA OF LIFE SCIENCES/001 Nature Publishing Group/www.els.net 1
deficiency). More commonly seen is the association between C4-null alleles and connective tissue disease such as SLE and systemic sclerosis. In these conditions, CR1 expression on red blood cells is also seen, particularly during episodes of disease exacerbation. C3 and especially C4 are consumed during active disease and serial C3 and C4 levels are markers of disease activity. C4 may, however, be low due to genetic reasons as mentioned above and both C3 and C4 are acute phase proteins so that increased production may mask consumption. It has now been established that measuring activation products such as C4a or C3 degradation products is a more sensitive way of monitoring disease activity. Assays for C3dg are generally robust, providing specimens are handled correctly, and there is some evidence that increases in levels may predict clinical relapse in SLE. C1-inh deficiency Patients presenting with nonpainful, nonitchy angioedema should be investigated for C1-inh deficiency (Cicardi et al., 1998). Low C4 and C2 levels are seen during acute attacks and usually between attacks. C1-inh levels are low in 85% of cases of hereditary C1-inh deficiency (HAE) but the remaining patients have normal levels of a nonfunctional protein and functional assays are required for the correct diagnosis. Acquired C1-inh deficiency (AAE) is associated with either B cell neoplasia, connective tissue disease or various carcinomas, or may arise as an autoimmune condition per se. Previously divided into two subtypes, it now appears that AAE is always associated with autoantibodies to C1-inh. The antibodies affect C1-inh function directly and result in an inactive form of C1-inh of a lower molecular weight than normal. C1q levels are usually decreased more in AAE than in HAE. The antibody can be detected by ELISA, immobilizing C1-inh on the plate, incubating with patient serum and detecting bound antibody by enzyme-labelled anti-IgG, IgA or IgM. C3 nephritic factor Patients with membranoproliferative glomerulonephritis, especially type II and associated with partial lipodystrophy, often show a secondary C3 deficiency. This is usually because of an autoantibody against C3bBb. The antibody can be detected by its ability to activate fluid phase C3 or the stabilization of sheep erythrocyte-bound C43bBb. An ELISA using stabilized C3bBb as a substrate has also been described. References Cicardi M, Bergamaschini L, Cugno M et al. (1998) Pathogenic and clinical aspects of C1 inhibitor deficiency. Immunobiology 199: 366– 376. Pickering MC and Walport MJ (2000) Links between complement abnormalities and systemic lupus erythematosus. Rheumatology (Oxford) 39: 133–141. Ross SC and Denson P (1984) Complement deficiency states and infection: epidemiology, pathogenesis and consequences of Neisseral and other infections in an immune deficiency. Medicine 63: 243–273. Further Reading Dodd AW and Sims RB (eds) (1997) Complement.A Practical Approach. Oxford: Oxford University Press. Kirschfink M (1998) The clinical laboratory: testing the complement system. In: Rother K, Till GO and Ha¨nsch GM (eds) The Complement System, 2nd revised edn, pp. 522–547. Berlin: Springer-Verlag. Phimster GM and Whaley K (1990) Measurement of complement. In: Gooi HG and Chapel H (eds) Clinical Immunology.A Practical Approach, pp. 81–109. Oxford: Oxford University Press. Complement: Measurement ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 7