New diagnostical methods in immuology Serologic Diagnosis Immunologic techniques - to detect antigen 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 TechniquePurposeClinical Examples Ouchterlony immuno-double-diffusionDetect and compare antigen and antibody Fungal antigen and antibody ImmunofluorescenceDetection and localization of antigenViral antigen in biopsy (e.g., rabies, herpes simplex virus) Enzyme immunoassay (EIA)Same as immunofluorescence Immunofluorescence flow cytometryPopulation analysis of antigen-positive cells Immunophenotyping ELISAQuantitation of antigen or antibodyViral antigen (rotavirus); viral antibody (anti-HIV) Western blotDetection of antigen-specific antibodyConfirmation of anti-HIV seropositivity Radioimmunoassay (RIA)Same as ELISASame as for ELISA Complement fixationQuantitate specific antibody titerFungal, viral antibody Hemagglutination inhibitionAntiviral antibody titer; serotype of virus strain Seroconversion to current influenza strain; identification of influenza Latex agglutinationQuantitation and detection of antigen and antibody Rheumatoid factor; fungal antigens; streptococcal antigens ELISA Enzyme-Linked Immuno Sorbent Assay Detection of antibodies: specific antigen is bound - surface of the wall in well The serum of patient is added to the well Incubation - detection of specific Ab. The conjugate (antibodies to the Ag-Ab complex) - labeled by ensyme Chromogen - color product positive reaction ELISAVQ100 ELISA (detection of Ag)_8-M ELISA 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 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). IFA - immunofluorescence analysis conjugate is labelled by fluorochrom positivity is identified by fluorescence of the complex direct immunofluorescence indirect immunofluorescence Direct immunofluorescenceoutube.com/ watch?v=O H2GFeaGV 6w Indirect immunofluorescence. Western blot Detection of specific antibodies against individual antigens Agens is divided by electorphoresis to separate antigens The serum of patient - to react with antigens the complex ag-ab is formed. Next steps are identical as in ELISA: conjugate labelled with ensyme substrate. Color reaction Western Blot Procedure electrophoresis Western Blot Procedure Electroblotting transfers the separated proteins from the gel to nitrocellulose membrane. Western Blot Procedure The blot is incubated with a serum (Ab1) the Ab1-antigen complex - incubated with a second antibody (Ab2, radioactively labeled or enzyme labeled)solutions.blogspot.sk/2008/07/western- blot.html ELISPOT enzyme linked immnosorbent spot assay Detection of low quantities of cytokines produced by stimulized T cells Typical Areas of ELISpot assays Characterization of the TH1/TH2 response Vaccine Development Infectious diseases, i.e. HCV Therapy monitoring Epitope mapping Autoimmunity Transplantation 5C84A Patient A is positive for TB infection whilst patient B is negative 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. Principle of the PCR Wne6E PCR Real time PCR 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; lymphocyte transformation test a patients own lymphocytes are brought into contact with various pathogens or materials, to assess the specific reaction of these important cells. Thereby, this elaborate test enables the diagnosis of existing inflammatory processes in our body as well as, the potential response to various medications or, amongst others, dental materials. lymphocyte transformation test T cells cultured in vitro by non-specific mitogen (eg PHA Phytohemagglutimin, PHA) or specific antigen stimulation, there may be increased cell volume, metabolism, protein and nucleic acid synthesis and can be split into lymphoblastoid cells, namely the phenomenon of lymphocyte transformation. Lymphocyte transformation rate level can reflect the level of cellular immunity. Therefore, determination of lymphocyte transformation test can be used as an indicator of immune function. Lymphocyte transformation test (LTT) normal: Morphological lymphocyte transformation rate (LTT) to 60.1% 7.6%. Lymphocyte transformation test (LTT) Clinical significance: Abnormal results: reduced lymphocyte transformation test, seen in hepatitis, hemorrhagic fever with renal syndrome (epidemic hemorrhagic fever), measles, cytomegalovirus infections, severe fungal disease, severe tuberculosis, lepromatous leprosy, systemic lupus erythematosus, the liver parenchymal disease, immune deficiency diseases. Type I. - ALLERGY Allergic reactions occur when a person's immune system reacts to normally harmless substances in the environment.environment A substance that causes a reaction is called an allergen.allergen Type/alternative name Often mentioned disorders Mediators Allergy Allergy (immediate) Atopy Anaphylaxis Asthma IgEIgE and IgG4IgG4 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. Type/alternat ive name Often mentioned disorders Mediators Cytotoxic, antibody- dependent Autoimmune hemolytic anemia Thrombocytopenia Erythroblastosis fetalis Goodpasture's syndrome Graves' disease Myasthenia Gravis IgMIgM or IgGIgG (Complement)Complement 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.glomeruli Type/alternative name Often mentioned disorders Mediators Immune complex disease Serum sickness Arthus reaction Rheumatoid arthritis Systemic lupus erythematosusSystemic lupus erythematosus (SLE)SLE Extrinsic allergic alveolitis Extrinsic allergic alveolitis (Hypersensitivity pneumonitis)Hypersensitivity pneumonitis IgG (Complement)Complement 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.helper T cells major histocompatibility complexIL-12IL-2interferon Activated CD8+ T cells destroy target cells on contact, whereas activated macrophages produce hydrolytic enzymes and, on presentation with certain intracellular pathogens, transform into multinucleated giant cells.hydrolyticenzymesgiant cells Type/alternative name Often mentioned disorders Mediators Delayed-type hypersensitivity (DTH), cell-mediated immune memory response, antibody-independent cell-mediated immune memory response Contact dermatitis Mantoux test Chronic transplant rejection Multiple sclerosis T-cells 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. Skin Tests skin test positive: - antibody (IgE) on specialized cells in the skin - release histamine = redness and itching. mast cells + IgE antibody Skin Patch Tests the allergen is placed on the skin. Positive - skin reddens, swells Negative (-), Irritant reaction (IR), Equivocal / uncertain (+/-) Skin Patch Tests Interpretation of the results Weak positive (+) Skin Patch Tests Strong positive (++) 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. Skin Prick Testing The skin prick test introduces such a tiny amount of allergen into the skin that testing is quite safe. These tests can be carried out on all age groups, including babies, although the response will be considerably smaller than in an adult. 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