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 3von 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 lipo￾somes 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 compo￾nent 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, how￾ever, 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 compo￾nent 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