Serological diagnosis of infectious diseases

Serological diagnosis of infectious diseases1Involves detection of antigens and antibodies in the patient's serumAntibodies indirect evidence of infectionAntigen detection detection of a part of the infectious organism that evokes an immune responseFirst antibody to be produced in any infection is IgM, this is followed by IgG a week later, which replaces itTitre = the reciprocal of the highest dilution of patient's serum to give a positive test2AffinityThe strength of attraction between and antigen and it's antibodyAvidityThe strength of binding between an antigen and it's antibodySpecificityThe ability of an individual antibody combining site to react with only one antigenic determinantThe ability of a population of antibody molecules to react with only one antigen3SensitivityAbility of a test to detect very minute quantities of antigens or antibodiesCross reactivityThe ability of an individual antibody combining site to react with more than one antigenic determinant.The ability of a population of antibody molecules to react with more than one antigen4UsesDetection of IgM to detect recent infectionIgM detection is also very useful to diagnose neonatal infectionsRising titres (four fold rise) in paired sera indicate current infectionUseful to diagnose infections by hard-to-cultivate or uncultivable pathogensAs an epidemiologic tool Detection of antibody levels (IgG) to determine prevalence of a disease5Some serological testsPrecipitationAgglutinationComplement fixationImmunofluorescenceELISAWestern blot6Precipitationa soluble antigen combines with its antibody at optimal proportionsin the presence of electrolytesat optimum temperature and pHthe antigen-antibody molecules clump together to form an insoluble precipitateSlide precipitation VDRLPrecipitation in gelElek's gel precipitation test for Diphtheria toxin7Agglutinationa particulate antigen combines with their specific antibodiesat optimal proportions in the presence of electrolytesat a suitable temperature and pHthe antigen-antibody complex gets clumped together (agglutinated)8Slide agglutination testsA drop of the antigen and antibody suspension is mixed on a slide and observed for agglutinationExamplesbacterial slide agglutination tests for various pathogens like Salmonella, Shigella, Vibrioslide haemagglutination in blood grouping where RBCs are the antigens9Serial dilutions of the antibody suspension (here patients serum) is made in test tubes and to each of these dilutions an equal volume of antigen suspensionThis mixture is then incubated at a suitable temperature for a required period of time after which agglutination is observedExamplesWIDAL test, to detect antibodies to Salmonella typhi and S. paratyphi in the patients serum.Standard agglutination test for brucellosis10Cold agglutination testhuman O blood group RBCs are used to detect cold antibodies (cold agglutinins) in the patients serumCold agglutinins are IgM antibodies present in several disease conditions and have the ability to agglutinate human O group RBCs at 4CSome of these conditions are malaria, trypanosomiasis, acquired haemolytic disease, primary atypical pneumonia11Coombs testDirect Coombs test is used to detect anti Rh antibodies in the babyIndirect Coombs test is used to detect anti Rh antibodies in the maternal blood12

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16Indirect (passive) agglutination reactionsSoluble antigens are coated on to carrier particles thus converting them to particulate antigensThese then react with antibodies to give agglutinationThus a precipitation test can be converted into an agglutination test Example: RPR test for syphilis17

18Reverse passive agglutination testAntibodies are coated on to carrier particles and this complex is used to detect various antigens in biological fluidse.g. Using latex particles detection of HBsAg in the serumusing RBCs detection of HBsAg in the serum19

20Co-agglutination test:This test uses a killed strain of Staphylococcus aureus that is very rich in protein A (on its cell surface)The strain used is cowan strain IThe protein A of Staphylococcus aureus has a high affinity for the Fc fragment of immunoglobulinsTherefore it can bind large number of immunoglobulin molecules by their Fc portion leaving the Fab portion free to bind with antigens21

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23Complement fixation test24

Positive test25

Negative test26ImmunofluorescenceFluorochromes bound to antigens or antibodies, illuminated by UV light, are used to demonstrate the specific reaction occurring between these labelled antigens/antibodies and their corresponding antibodies/antigens present in the clinical sample27

Directimmunofluorescence28

IndirectImmunofluorescence29Enzyme ImmunoassayThis test makes use of enzyme systems to demonstrate specific reaction between antigens and antibodies, thereby helping in detection of antigens and antibodiesAn enzyme system contains:Enzyme tagged to antigen/antibodySubstrate for enzyme A colour change indicates the presence of antigens/antibodies, and the intensity of colour change gives a measure of the antigens or antibodies present.30

Direct ELISA31

Indirect ELISA32Western blottingThis test detects the presence of antibodies in the specimen. It is highly specific because it detects multiple antibodies against different antigens of the same micro organism.33

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