General Microbiology and Immunology Lecture 4B PDF

Level 2, Semester 3 General Microbiology and Immunology PMB201 Lecture 4B Prof. Mona Ibrahem Shaaban Prof. of Microbiology and Immunology, Faculty of Pharmacy, Mansoura University  Serology Se...

Level 2, Semester 3 General Microbiology and Immunology PMB201 Lecture 4B Prof. Mona Ibrahem Shaaban Prof. of Microbiology and Immunology, Faculty of Pharmacy, Mansoura University  Serology Serology is a branch of laboratory medicine that test infection in blood serum or in other body fluids by evaluating antigen-antibody reactions in vitro It can detect and identify unknown antibody using known antigen It can detect and identify unknown antigen using known antibody The titer of antigen antibody reaction ❑ Titer refers to highest dilution of antibody produce visible reaction with antigen. Types of Serological Reactions 1. Enzyme linked immuno sorbent assay (ELISA) 2. Precipitation 3. Agglutination 4. Neutralization test 5. Complement fixation test (CFT) 6. Immunofluorescence (IF) 7. Radioimmunoassay (RIA) 8. Blotting techniques 1. Enzyme-linked immunosorbent assay (ELISA) It is an immunoassay technique involving the reaction of antigen and antibody in vitro. ELISA is a sensitive and specific assay for the detection and quantitation of antigens or antibodies. Principle: The antigen is immobilized to a solid surface. (directly or by the use of a capture antibody ). The antigen is detected by antibody conjugated with an enzyme horseradish peroxidase (HRP) and alkaline phosphatase (AP). The enzyme is detected by substate such as 3,3'5,5'- Tetramethylbenzidine (TMB) and PNPP (p- Nitrophenyl Phosphate HRP TMB AP PNPP  APPLICATIONS OF ELISA Serum Antibody Concentrations Disease outbreaks- tracking the spread of disease e.g. HIV, bird flu, common colds, cholera Detection of antibodies in blood sample for previous infection e.g. Antibodies against Schistosoma Detections of antigens e.g. pregnancy hormones, drug allergen Detecting potential food allergens (milk, peanuts, walnuts, almonds and eggs) 7 TYPES OF ELISA ❑ DIRECT ELISA ❑ INDIRECT ELISA ❑ SANDWICH ELISA I- The direct ELISA Uses a labeled primary antibody that reacts directly with the antigen Disadvantages; Immunoreactivity of the primary antibody is affected by labeling with enzymes or tags. II- Indirect” ELISA Antigen bound to solid phase Add antibody tested serum (primary antibody) Add known labeled sec antibody bind with enzyme Measure enzyme label  III-A sandwich ELISA. (1)Plate is coated with a capture antibody; (2)sample is added, and any antigen present binds to capture antibody; (3)Primary antibody is added, and binds to antigen; (4)Enzyme-linked secondary antibody is added, and binds to detecting antibody; (5)Substrate is added, and is converted by enzyme to detectable form http://upload.wikimedia.org/wikipedia/commons/thumb/4/4b/ELISA-sandwich.svg/300px-ELISA-sandwich.svg.png  IV. Immunofluorescence test Fluorescent dyes (fluorescein or rhodamine) attached to known specific Abs, used to detect presence of Ag in serum or (microorganisms) in a sample It is valuable for identifying and locating antigens on the surface of cells or tissues. Types 1.Direct immunofluorescence The antibody specific to the antigen is directly tagged with the fluorochrome. It is used in the detection of rabies virus in brain smear. 2 Indirect immunofluorescence The antibody specific for the antigen is unlabeled A second anti-immunoglobulin antibody directed toward the first antibody is tagged with the fluorochrome. Indirect fluorescence is more sensitive than direct immunofluorescence since there is amplification of the signal. Detection of syphilis. V) Immunochromatography (Rapid test) It is a speed method for the etiologic diagnosis of infections Principle Dye-labelled antibody, specific for target antigen, is present on the lower end of nitrocellulose strip or in a plastic well provided with the strip. Antibody, is specific for the target antigen, is bound to the strip in a thin (test) line Either antibody specific for the labelled antibody, or antigen, is bound at the control line Test (T): the test is applied and indicates the presence or absence of Ag or AB. Control (C): test the validity of the test -If antigen is present, labelled antibody will be trapped on the test line -Excess-labelled antibody bind with the control line Applications Diagnosis of HCV (serum antibody) Diagnosis of HBC (serum antigen, surface antigen) Hormone pregnancy, (HCG hormones)  II. Precipitation reactions: Interaction between antigen and antibody molecules, result in precipitate out of solution 1. Ring test Ascoli’s thermoprecipitin test, which is used in the diagnosis of anthrax 2. Tube test:A simple quantitative tube precipitation test for syphilis Kahn's test for syphilis 3 Slide test Antigen and antibody are mixed on a slide In a positive test floccules appear. The VDRL (Venereal disease research laboratory) test for syphilis 4. Immunodiffusion Elk test for toxigenicity in diphtheria bacilli.  III. Agglutination reactions: Interaction between antibody and antigen result in visible clumping called agglutination If the antigen is an erythrocyte the interaction called hemagglutination. Types of agglutination reactions – Direct agglutination tests – Indirect agglutination tests – Hemagglutination inhibition 2.1 Direct Agglutination reaction This technique is used for the detection of antibody (soluble) in a using specific antigen (soluble). The serum to be tested is serially diluted. The standardized amount of specific antigen is added, mixed well. Incubated at a suitable temperature and examined for clamping. Results are expressed as titer. The highest dilution of test serum at which positive agglutination occurs. Application: Example: Blood Grouping Used for brucellosis test. Typhoid (enteric fever), In this test, dead or inactivated bacteria suspensions are used as antigens  S. Typhi———-O antigen S. Tyhhi———- H antigen S. Paratyphi —–AH antigen S. Paratyphi —–BH antigen  2- Indirect Agglutination Principle To test patient serum for the presence of antibodies Using Ag that bound to artificial surface, polystyrene latex, RBCs (Hemagglutination), bentonite or charcoal. Ab in patient serum react to antigens adsorbed on the surface of carriers results in the agglutination of carrier particles forming visible clumps Applications; Assay of pregnancy hormone in urine Diagnosis of rheumatoid arthritis (Rose-Waaler test). Rapid Plasma Reagin test is used to screen for syphilis. Antigenic cardiolipin released from spirochete-damaged host cells stimulates the production of antibodies. These antibodies, collected from syphilis-suspect patients, will agglutinate syphilis bacteria in vitro, yielding a positive test.  3- Agglutination Inhibition Used to test soluble antigens in patient serum. Using reagent antibody specific for antigen, and reagent antigen with (latex or RBCs). If antigen was present in the sample all reagent antibody binds to it so no antibody is present to react with antigens coating the particles Thereby, inhibiting the agglutination of the RBC Example; detection of virus infection such as influenza, measles virus Detection of illegal drugs, such as cocaine or heroin. Thank you