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

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334 Manual of basic techniques for a health laboratoryIncubatethenwashIncubatethenwashAntigencarrierparticlesAntigen (soluble)Fig. 11.8 Principle of passive agglutinationAbsorbedantigenAntibody-containingserumAgglutinationhCG antibody(a)Adsorbed hCGantigenhCG antigen(urine specimen frompregnant woman)No precipitation = positive resulthCG antibody(b)Adsorbed hCGantigenFig. 11.9 Principle of the agglutination inhibition test for hCGPrecipitation = negative resultthe reciprocal of the final dilution of antiserum capable of producing visibleagglutination.Agglutination inhibitionAgglutination inhibition is used for determining the presence of antigen. This assayis based on competition between particulate and soluble antigen for antibody-bindingsites. The antibody and the test sample are allowed to react together. If solubleantigen is present in the sample, the antibody reacts with it and is not free to reactfurther after the subsequent addition of indicator particles or cells. Thus, the absenceof agglutination with a specimen under investigation indicates a positive testresult. An example of this kind of assay is the detection of human chorionic gonadotropin(hCG) used in the test for confirmation of pregnancy and also in otherpathological conditions where hCG levels are important (Fig. 11.9).PrecipitationUnlike agglutination reactions in which the antigen is particulate (insoluble), inprecipitation reactions the interaction is between a soluble antibody and a solubleantigen. If a soluble antibody is incubated with a soluble antigen, antibody andantigen complexes cross-link and form a precipitate. Precipitation methods can bequantitative or qualitative and the interactions are dependent on ionic strength, pHand concentration.
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
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M A N U A LO F B A S I CTECHNIQUESF
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ContentsiManualofbasic techniques f
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ContentsiiiContentsPrefacex1. Intro
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viManual of basic techniques for a
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viiiManual of basic techniques for
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xManual of basic techniques for a h
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2 Manual of basic techniques for a
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Table 3.2 Dispatch of specimens 92
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100 Manual of basic techniques for
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7. Examination of urine 231Part III
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7. Examination of urine 2337. Exami
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7. Examination of urine 2357.2.3 Me
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7. Examination of urine 237Table 7.
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7. Examination of urine 239The anal
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7. Examination of urine 241MethodCo
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7. Examination of urine 243Table 7.
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7. Examination of urine 245Fig. 7.2
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7. Examination of urine 2497.2.8 De
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7. Examination of urine 251Reuse of
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8. Examination of cerebrospinal flu
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9. Haematology 2659. HaematologyHae
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9. Haematology 2679.2 Collection of
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9. Haematology 269Fig. 9.13 Feeling
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9. Haematology 2719.3 Estimation of
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9. Haematology 273Table 9.2 Sample
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9. Haematology 275Fig. 9.25 Checkin
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9. Haematology 277Calibration of th
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9. Haematology 279Errors in haemogl
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9. Haematology 281Fig. 9.28 Micro s
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Page 297 and 298: 9. Haematology 285concentration is
Page 299 and 300: 9. Haematology 287Measurement techn
Page 301 and 302: 9. Haematology 289●●●Graduate
Page 303 and 304: 9. Haematology 2911 litre, so multi
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Page 307 and 308: 9. Haematology 295Very high ESR val
Page 309 and 310: 9. Haematology 2979.9 Observation o
Page 311 and 312: 9. Haematology 2999.10 Preparation
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Page 315 and 316: 9. Haematology 303Drying the filmAd
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Page 319 and 320: 9. Haematology 307Fig. 9.77 Normal
Page 321 and 322: 9. Haematology 309Fig. 9.85 Anisocy
Page 323 and 324: 9. Haematology 311Fig. 9.92 Polymor
Page 325 and 326: 9. Haematology 313Immature granuloc
Page 327 and 328: 9. Haematology 315Fig. 9.104 Mixing
Page 329 and 330: 9. Haematology 317Fig. 9.108 Prepar
Page 331 and 332: 9. Haematology 319Heinz bodiesHeinz
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Page 335 and 336: 10. Blood chemistry 323Fig. 10.1 De
Page 337 and 338: 10. Blood chemistry 325Table 10.1 B
Page 339 and 340: 10. Blood chemistry 32710.2.4 Resul
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Page 363 and 364: Annex. Reagents and their preparati
Page 381 and 382: IndexNote: Page numbers in bold ref
Page 383 and 384: Index 371Centrifuges 32, 69-72, 69-
Page 385 and 386: Index 373Endolimax nanus 114, 114,
Page 387 and 388: Index 375Hyaline casts, urine 243,
Page 389 and 390: Index 377Microhaematocrit equipment
Page 391 and 392: Index 379Protozoablood 173-194, 178
Page 393 and 394: Index 381Smears (continued)preparat
Page 395 and 396: Index 383Thiomersal-iodine-formalde
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