Manual of basic techniques for a health laboratory - libdoc.who.int

11. Immunological and serological techniques 335A quantitative precipitin curve can be drawn in which theproportion of antigen and antibody determines the extent ofcross-linking and precipitation. The curve shows the followingfeatures (Fig. 11.10):● The equivalence zone in which the proportions of antigenand antibody are equivalent.● The excess antibody zone in which all available antigenicdeterminants are bound by individual antibody molecules,leaving some antibody molecules unbound.● The excess antigen zone in which all the antigen bindingsites of an antibody are bound by individual antigen molecules,leaving some molecules of antigen unbound.ExcessantibodyEquivalenceConcentration of antigenSeveral other immunological techniques employ the precipitationreaction in one form or another. These include neph-Fig. 11.10 A quantitative precipitin curveelometry, turbidimetry, radial immunodiffusion (Mancini technique), double diffusion(Ouchterlony) and some immunoelectrophoresis techniques.Mass of precipitated antibodyExcessantigenNephelometry and turbidimetryNephelometry and turbidimetry involve the measurement of the light scatteringand light absorption properties, respectively, of antigen–antibody complexes. Thesetechniques are used to measure concentrations of proteins and drugs in serum orCSF. The assays are rapid and sensitive. A constant, excessive amount of antibodyis incubated with an antigen in a cuvette.In nephelometry light is passed through the cuvette and the scatter produced bythe antigen–antibody complexes that have been formed is measured.The antigen concentration is determined from a standard curve made by measuringthe light scatter produced by a series of antigen solutions of known concentrations.In some assays, polymers are added to accelerate the formation ofantigen–antibody complexes.In turbidimetry light is passed through the cuvette andthe absorption produced by the antigen–antibody complexesthat have been formed is measured. A conventionalphotometer can be used for thisStandard 1 Standard 2purpose.Standard 3UnknownRadial immunodiffusion (Mancini technique)Radial immunodiffusion is based on the principle that aquantitative relationship exists between the amount ofantigen placed in a well cut in an agarose gel containingantibody and the diameter of the resulting ring of precipitate.The antigen concentration is proportional tothe square of the diameter of the ring of precipitate. Theconcentration of unknown samples is calculated with theaid of a standard curve which is prepared by plottingthe diameter 2 of the resulting ring of precipitate producedby a series of antigen solutions of known concentration(Fig. 11.11). This technique can be used for the quantitativemeasurement of complement factors andimmunoglobulins.Diameter 2 of ring of precipitateAntigen (or antibody) concentrationFig. 11.11 Principle of radial immunodiffusion