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New diagnostical methods in immuology

This article explores new techniques in immunology for serologic diagnosis, including immunologic techniques to detect antigen or antibody, the specificity of the antibody-antigen interaction, and quantitation of antibody. The article also discusses the seroconversion ELISA, RIA, IFA, Western blot, ELISPOT, PCR, and lymphocyte transformation test. These methods are used to detect various infections, measure hormone levels, and screen donated blood for viral contamination.

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New diagnostical methods in immuology

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  1. New diagnostical methods in immuology

  2. Serologic Diagnosis • Immunologic techniques - to detectantigen or antibody • specificity of the antibody-antigen interaction • In most cases, the same technique can be adapted to evaluate antigen and antibody. • quantitation of the antibody • The titer of an antibody is defined as the lowest dilution of the sample that retains a detectable activity. • Seroconversion

  3. ELISA – Enzyme-Linked Immuno Sorbent Assay • Detectionofantibodies: • specificantigenisbound - surfaceofthewall in well • Theserumofpatientisadded to thewell • Incubation - detectionofspecificAb. • Theconjugate (antibodies to theAg-Abcomplex) – - labeled by ensyme • Chromogen - colorproduct – positivereaction

  4. ELISA http://www.youtube.com/watch?v=RRbuz3VQ100

  5. ELISA (detection of Ag) http://www.youtube.com/watch?v=70TPrfL_8-M

  6. ELISA

  7. ELISA : • screening donated blood for evidence of viral contamination by • HIV-1 and HIV-2 (presence of anti-HIV antibodies) • hepatitis C (presence of antibodies) • hepatitis B (testing for both antibodies and a viral antigen) • measuring hormone levels • HCG (as a test for pregnancy) • LH (determining the time of ovulation) • TSH, T3 and T4 (for thyroid function) • hormones (e.g., anabolic steroids, HGH) that may have been used illicitly by athletes • detecting infections • sexually-transmitted agents like HIV, syphilis, and chlamydia • hepatitis B and C • Toxoplasma gondii

  8. RIA : • plasma levels of: • most of our hormones; • digitoxin or digoxin in patients receiving these drugs; • certain abused drugs • for the presence of hepatitis B surface antigen (HBsAg) in donated blood; • anti-DNA antibodies in systemic lupus erythematosus (SLE).

  9. IFA - immunofluorescence analysis • conjugate is labelled by fluorochrom • positivity is identified by fluorescence of the complex • direct immunofluorescence • indirect immunofluorescence

  10. Direct immunofluorescence http://www.youtube.com/watch?v=OH2GFeaGV6w

  11. Indirect immunofluorescence.

  12. Western blot • Detectionofspecificantibodiesagainstindividualantigens • Agens isdivided by electorphoresis to separateantigens • Theserumofpatient - to reactwithantigens thecomplexag-ab isformed. • Nextsteps are identical as in ELISA: • conjugatelabelledwithensyme • substrate. • Colorreaction

  13. Western Blot Procedure • electrophoresis

  14. Western Blot Procedure • Electroblotting – • transfers the separated proteins from the gel to nitrocellulose membrane.

  15. Western Blot Procedure • Theblotisincubatedwith a serum (Ab1) • the Ab1-antigen complex - incubatedwith a second antibody (Ab2, radioactively labeled or enzymelabeled) http://bio-solutions.blogspot.sk/2008/07/western-blot.html

  16. ELISPOT – enzyme linked immnosorbent spot assay • Detection of low quantities of cytokines produced by stimulized T cells

  17. Typical Areas of ELISpot assays • Characterization of the TH1/TH2 response Vaccine Development • Infectious diseases, i.e. HCV • Therapy monitoring • Epitope mapping • Autoimmunity • Transplantation

  18. http://www.youtube.com/watch?v=lkHmyC5C84A

  19. Patient A is positive for TB infection whilst patient B is negative

  20. PCR – polymerase chain reaction • Every organisme – microbes included contain specific sequention of aminoacids • With the help of ensymes this sequention can be „cut out“ and millions of copies can be synthetised in the infectious material if present there. • These can be then identified via „antibodies“ against this sequency – labelled by ensymes or fluorochrom ... • The purpose of a PCR is to make a huge number of copies of a gene. This is necessary to have enough starting template for sequencing.

  21. Principle of the PCR

  22. Principle of the PCR http://www.youtube.com/watch?v=DkT6XHWne6E

  23. PCR

  24. Real time PCR

  25. lymphocyte transformation test • in vitro test of lymphocyte function. • increased DNA synthesis followed by cell division and differentiation of lymphocytes in response to antigens or mitogens;

  26. Type I. - ALLERGY • Allergic reactions occur when a person's immune system reacts to normally harmless substances in the environment. • A substance that causes a reaction is called an allergen.

  27. Type II. - Cytotoxic, antibody-dependent • the antibodies produced by the immune response bind to antigens on the patient's own cell surfaces. • the reaction to penicillin wherein the drug can bind to red blood cells, causing them to be recognized as different; B cell proliferation will take place and antibodies to the drug are produced.

  28. Type III • when there is little antibody and an excess of antigen, leading to small immune complexes being formed that do not fix complement and are not cleared from the circulation. • These immune complexes insert themselves into small blood vessels, joints, and glomeruli, causing symptoms.

  29. Type IV. • the reaction takes two to three days to develop. • CD4+ helper T cells recognize antigen in a complex with either type 1 or 2 major histocompatibility complex. The antigen-presenting cells in this case are macrophages that secrete IL-12, which stimulates the proliferation of further CD4+ T cells. CD4+ T cells secrete IL-2 and interferon gamma, further inducing the release of other Type 1 cytokines, thus mediating the immune response. • Activated CD8+ T cells destroy target cells on contact, whereas activated macrophages produce hydrolyticenzymes and, on presentation with certain intracellular pathogens, transform into multinucleated giant cells.

  30. Skin Tests • What are the advantages of skin tests? • Skin tests are rapid, simple, and relatively safe. • They can be very helpful in specifically identifying causes of allergies.

  31. Skin Tests • skin test – positive: - antibody (IgE) on specialized cells in the skin - release histamine = redness and itching. • mast cells +IgE antibody

  32. Skin Patch Tests • the allergen is placed on the skin. • Positive - skin reddens, swells • Negative (-), • Irritant reaction (IR), • Equivocal / uncertain (+/-)

  33. Skin Patch Tests • Interpretation of the results • Weak positive (+)

  34. Skin Patch Tests • Strong positive (++)

  35. Skin Prick Testing • Skin prick testing measures specific IgE attached to cells in the skin. • This is probably the most commonly used allergy test and is appropriate for both inhaled and ingested (eaten) allergies.

  36. Skin Tests • A small drop of liquid extract is placed on the skin (forearm or back). • underlying skin - gently scratched through the small drop (with sterile needle). • Positive - skin reddens, swells, • Negative – no reaction • Two control samples are included

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