Diagnostic of infectious diseases

1. Diagnostic of infectious diseases Direct diagnostic . Visualisation -microscopy -antigen detection -cultivation -genetic matierial detection Indirect diagnostic: - detection of the reaction of macroorganism to the presence of microorganism Specific reaction of immune system *cell immunity – skin tests *humoral immunity – specific antibodies detection - serological reactions

2. Serological reactions • Reaction of antigen and antibody in laboratory conditions in different (stated) environment • Antigen – substance with ability to activise immune system to specific antibodies production. Non self substance. Immunogenicity. Protective antibodies • Antigenic determinants – areas of microorganism with a structure eliciting the antibodies production • Specificity of antigen – determined by production of antibodies, that reacts only with it. Non specific antigens – heterofil – if antibodies agains them react with other antigens

3. Antibodies • In blood serum of immunised annimals there are specific proteins – immuneglobulins, that bind antigens causing their production (behring, Kitaso, 19 th century) • Serum – liquid that will remain at the top of the tube after centrifugation of coagulated blood • Plasma – liquid that remains at the toop of tube after centrifugation of not coagulated blood • Antiserum – serum with specific antibodies • Function of antibodies – to bind specifically antigen, neutralise its function via other reactions that are activised by the antigen antibody binding (complement activation....)

4. Reaction of antigen with specific antibody depends on the type of antigen – immunecomplexes of different quality *corpuscular antigens – microorganism, erytrocytes – agglutination *soluble antigens – small imunecomplexes – in solution, - big complexes - precipitation

5. Electrophoresis of proteins • Alfa, beta, gama fraction • Immuneglobulines – 5 classes IgG, IgA, IgM, IgDm IgE – subclasses IgG1, IgG2, IgG3, IgG4,, types – kappa, lambda, subtypes : lambda 1,2,3,4,

6. Antibodies production - dynamics • Acute antibodies IgM • Persistent antibodies igG • First response IgM • Booster effect second response • Total antibodies

7. Serological reactions • Agglutination – antigen + several dilution of serum = visible agglutination • Latex agglutination – antibody bound on latex particles + Ag = big agglutination • Precipitation – Ag+Ab = ring in the touch zone • Immunediffusion – diffusion of Ag and Ab in agar. In the meeting point – precipitation ring • Neutralisation – serum with antibodies can neutralise the activity of microorganisme in living model • Hemagglutination – passive agglutination – ag is bound on the surface of RBC • CF – complement fixation- Ag +Ab +C´+ Ery + antieryab –lysis • ELISA, RIA, IFT

8. ELISA, RIA, IFT • Ag + séru (Ab) + antibody against the complex Ag+Ab*(labelled with enzyme, radioactive labell or fluerescent labell) • Detection of enzyme activity, radioactivity or fluorescence

9. Reading of serological reactions • Qualitative reaction – positive or negative (comparison with bordeline value) • Quantitative reaction – titer of antibodies, or concentration Determination of total antibodies (CF,) – dynamics of antibody production – 2 samples in the interval of 14-21 days. Results – titer of antibodies = turned out dilution of serum Determination of immuneglobulin classes – IgA, igM – acute IgG – long lasting, protective, later Results – in concentration of antibodies – g/l Confirmation of acute infection: Seroconversion – from negativity to positivity, fourfold increase of total antibody titer, or presence of IgM