Immunodiffusion and HIV Testing
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Questions and Answers

What component is placed in the gel during Single Radial Immunodiffusion?

  • Serum
  • Antibody (correct)
  • Enzyme
  • Antigen

    • Which test is primarily used for the detection of specific HIV antibodies?

  • Double Diffusion
  • ELISA
  • Single Radial Immunodiffusion
  • Western Blot Test (correct)

    • What indicates a positive result in the Western Blot Test?

  • Positive colorimetric reaction (correct)
  • Formation of cloudiness
  • Formation of sediment
  • Line of precipitation

    • Which test involves the use of toxin-antitoxin precipitation on the surface of the agar?

    <p>Double Diffusion (Elek's Test)</p> Signup and view all the answers

    What can be measured using Single Radial Immunodiffusion?

    <p>Immunoglobulin levels</p> Signup and view all the answers

    What is the reaction called when an antibody reacts with a particulate antigen?

    <p>Agglutination</p> Signup and view all the answers

    Which method is most commonly used for rapid blood grouping?

    <p>Slide Method</p> Signup and view all the answers

    What is used to make invisible antigen-antibody reactions visible?

    <p>Fluorescent dyes or enzymes</p> Signup and view all the answers

    In the Tube Method, what determines the endpoint of the agglutination test?

    <p>The highest dilution (lowest concentration) showing visible clumps</p> Signup and view all the answers

    Which test is NOT listed under the types of antigen-antibody binding reactions?

    <p>Western Blot</p> Signup and view all the answers

    What is the primary use of the Tube Method (Semi-Quantitative agglutination test)?

    <p>Measuring the amount of antibodies in patient serum</p> Signup and view all the answers

    Which condition can be diagnosed using the Widal test?

    <p>Enteric fever</p> Signup and view all the answers

    What characterizes passive agglutination?

    <p>Soluble antigen coated onto an inert particle</p> Signup and view all the answers

    What is the purpose of mixing the mother's serum with prepared RH positive RBCs in the Indirect Coombs Test?

    <p>To detect anti-RH antibodies in the mother's serum</p> Signup and view all the answers

    Why is anti-human gamma globulin added during the Indirect Coombs Test?

    <p>To link the anti-RH antibodies together causing agglutination</p> Signup and view all the answers

    In the Coagglutination (COA) test, what is the main function of protein A on the surface of Staph aureus?

    <p>To bind IgG nonspecifically through the Fc region</p> Signup and view all the answers

    How does the Coagglutination (COA) test detect bacterial antigens directly in body fluids?

    <p>By using staphylococci coated with known antibodies to agglutinate unknown antigens</p> Signup and view all the answers

    What is the primary use of the antiglobulin agglutination test?

    <p>Detection of anti-Rh antibodies</p> Signup and view all the answers

    At what temperature are the mixtures incubated in both the Indirect Coombs Test and the Coagglutination (COA) test?

    <p>37°C</p> Signup and view all the answers

    Which phenomenon describes the incomplete antibodies that cannot bridge between RBCs to cause visible agglutination?

    <p>Prozone phenomenon</p> Signup and view all the answers

    What is detected by the Direct Coombs Test?

    <p>Antibodies on newborn erythrocytes</p> Signup and view all the answers

    What is the incubation condition for the Indirect Coombs Test to detect anti-Rh antibodies in the mother's serum?

    <p>Rh positive RBCs for 60 minutes</p> Signup and view all the answers

    What type of antibodies are detected by the coagglutination (CoA) test?

    <p>By using killed <em>Staphylococcus aureus</em> to nonspecifically detect antibodies</p> Signup and view all the answers

    Which specimens can be tested using the coagglutination process?

    <p>Serum, urine, and cerebrospinal fluid</p> Signup and view all the answers

    What is essential for an antigen-antibody reaction in a complement fixation assay?

    <p>A complement component</p> Signup and view all the answers

    In an indirect ELISA, what is typically done after the test serum is incubated and washed?

    <p>Reincubation with enzyme-labeled antiglobulin</p> Signup and view all the answers

    Which step is unique to the double antibody technique in ELISA?

    <p>Fixation of known antibody to the solid phase</p> Signup and view all the answers

    Which method relies on colorimetric measurement to detect enzyme activity?

    <p>Indirect ELISA</p> Signup and view all the answers

    Where is the antigen fixed in a direct immunofluorescence assay?

    <p>On a slide</p> Signup and view all the answers

    Which method is now considered historical for antigen-antibody reactions?

    <p>Complement fixation</p> Signup and view all the answers

    What type of reaction occurs when an antigen in solution reacts with an antibody?

    <p>Precipitation</p> Signup and view all the answers

    Which method is commonly used in blood grouping?

    <p>Slide agglutination</p> Signup and view all the answers

    The tube method is a semi-quantitative agglutination test used to determine what?

    <p>The amount of antibodies</p> Signup and view all the answers

    What is the purpose of the antiglobulin test (e.g., Coombs test)?

    <p>To detect antigen-antibody complexes</p> Signup and view all the answers

    What does a rising antibody titer by 4 folds or more indicate?

    <p>Acute infection</p> Signup and view all the answers

    Which of the following is an example of an antibody labeled assay?

    <p>ELISA</p> Signup and view all the answers

    In the tube method, what determines the endpoint of the test?

    <p>Lowest antibody concentration showing clumping</p> Signup and view all the answers

    Which assay involves using fluorescent dyes to make the reactions visible?

    <p>Immunofluorescence</p> Signup and view all the answers

    What type of agglutination test uses latex particles to measure antibodies?

    <p>Passive agglutination</p> Signup and view all the answers

    The Widal test is used to detect antibodies against which organism?

    <p>Salmonella</p> Signup and view all the answers

    What is the purpose of the Western Blot Test?

    <p>To detect specific HIV antibodies and confirm a positive ELISA test</p> Signup and view all the answers

    What is the principle behind the appearance of bands on the nitrocellulose paper strip in the Western Blot Test?

    <p>Binding of HIV antibodies with corresponding antigens followed by detection with an enzyme-linked anti-human IgG</p> Signup and view all the answers

    Which method is used to confirm the toxigenicity of diphtheria bacilli?

    <p>Double diffusion (Elek's Test)</p> Signup and view all the answers

    What indicates a positive result in Elek's Test?

    <p>Appearance of a white line of toxin-antitoxin precipitate at a 45° angle within 24-48 hours</p> Signup and view all the answers

    Which characteristic is essential for the medium used in Elek's Test?

    <p>20% serum agar</p> Signup and view all the answers

    How are HIV proteins separated in the Western Blot Test?

    <p>Electrophoretic separation on a nitrocellulose strip</p> Signup and view all the answers

    What is one major requirement for conducting a Radioimmunoassay (RIA)?

    <p>Specialized equipment</p> Signup and view all the answers

    Which one of these is a primary characteristic of Radioimmunoassay (RIA)?

    <p>High sensitivity</p> Signup and view all the answers

    Why does Radioimmunoassay (RIA) require special licensing?

    <p>Due to radiation hazards</p> Signup and view all the answers

    What is the main use of Radioimmunoassay (RIA)?

    <p>Detection of unknown antigens or antibodies</p> Signup and view all the answers

    Which of the following is NOT a challenge associated with Radioimmunoassay (RIA)?

    <p>High sensitivity</p> Signup and view all the answers

    What indicates a successful antigen-antibody reaction in direct immunofluorescence?

    <p>The formation of fluorescent sites</p> Signup and view all the answers

    In indirect immunofluorescence, what is the role of the second labeled antibody?

    <p>To react with the first antibody</p> Signup and view all the answers

    Which step is common to both direct and indirect immunofluorescence?

    <p>Washing away excess antibodies</p> Signup and view all the answers

    In indirect immunofluorescence, what is added to the known antigen on the slide first?

    <p>The serum of the patient</p> Signup and view all the answers

    What type of microscope is typically used to examine the slides in both direct and indirect immunofluorescence?

    <p>Fluorescent microscope</p> Signup and view all the answers

    Study Notes

    Antigen-Antibody Reactions

    • Antigen-antibody reaction occurs between an antigen and the antibody elicited by that antigen or by a closely related antigen (Lock and Key Concept).
    • If the antigen is particulate, the reaction is called agglutination.
    • If the antigen is in solution, the reaction is precipitation.
    • If the reactants are invisible, substances like fluorescent dyes or enzyme are added to make the reaction visible.

    Types of Antigen-Antibody Binding Reactions

    • Agglutination
    • Precipitation
    • Antibody-labeled assays
      • Enzyme-Linked Immunosorbent Assays (ELISA)
      • Immunofluorescence
      • Radioimmunoassay (RIA)

    Agglutination Reactions

    • Definition: An antigen-antibody reaction in which the antigen is in the form of particles, such as microorganisms or cells, or latex particles coated with the antigen.
    • These particles become clumped, i.e., agglutinated, when mixed with its specific antibody.

    Types of Agglutination

    • Direct Agglutination
      • Slide method (Qualitative agglutination tests)
      • Tube method (Semi-Quantitative agglutination test)
    • Passive Agglutination
      • e.g., Latex agglutination
    • Antiglobulin test
      • e.g., Coomb's test
    • Co-Agglutination

    Slide Method (Qualitative Agglutination Tests)

    • A rapid test commonly used in blood grouping and useful in identification of organisms isolated from clinical specimens.

    Tube Method (Semi-Quantitative Agglutination Test)

    • Determines the amount of antibodies in the serum of patients.
    • Example: Widal test used to detect antibodies against Salmonella in the serum of patients suspected to have enteric fever.

    Passive Agglutination

    • An agglutination test done with a soluble antigen coated onto an inert particle, such as latex or RBCs, for measurement of antibodies to this antigen.
    • Examples: Rheumatoid factor latex test, C-reactive protein (CRP), and anti-streptolysin "O" (ASO) test.

    Antiglobulin Tests

    • Antiglobulin agglutination test, also known as Coomb's test, is used for:
      • Detection of anti-Rh Ab
      • Autoimmune hemolytic anemia

    Coomb's Test

    • Principle: Incomplete Antibodies
    • Rh IgG antibodies are incomplete (unagglutinable) and can only coat Rh positive RBCs.
    • However, they cannot bridge between RBCs to cause visible agglutination, known as the prozone phenomenon.
    • Coomb's test can be performed in two ways:
      • Direct Coombs Test
      • Indirect Coombs Test

    Coagglutination (CoA)

    • Killed Staphylococcus aureus can be used to detect antibodies nonspecifically through the process of coagglutination.
    • If Staphylococci are coated with the antigen, they will agglutinate.
    • This test can be used in various specimens like serum, urine, and cerebrospinal fluid (CSF).

    Precipitation

    • In these reactions, the antigen is soluble, and the reaction can be done in agar gel.
    • Single Radial Immunodiffusion:
      • Ab is put in gel, and the Ag is in wells punched in the agar.
      • The diameter of the ring is proportional to the concentration.
      • It can measure the immunoglobulin level, i.e., Quantitative.

    Western Blot Test

    • Used for detection of specific HIV antibodies and to confirm a positive ELISA test.
    • HIV proteins (antigens) are separated electrophoretically on a nitrocellulose strip.
    • The strip is incubated with the patient's serum.
    • If serum contains HIV antibodies, they will bind with the corresponding viral antigens.
    • These are then detected by using an enzyme-linked antihuman IgG, as in ELISA.
    • A positive colorimetric reaction forms bands on the nitrocellulose paper strip corresponding to specific HIV antigens.

    Double Diffusion (Elek's Test)

    • Used to prove toxigenicity of diphtheria bacilli.
    • It is a gel diffusion, toxin-antitoxin precipitation test.
    • Antitoxin-soaked strip of filter paper is placed on the surface of a 20% serum agar medium.
    • The plate is heavily inoculated by streaking a line of inoculums perpendicular to the strip.
    • The appearance of a white line of toxin-antitoxin precipitate at a 45° angle, within 24-48 hours, is diagnostic of a toxigenic organism.

    Assays Based on Complement

    • Complement Fixation
      • This is an antigen-antibody reaction that occurs in the presence of a third component known as complement.
      • Considered now a historical test.

    Antibody-Labeled Assays

    • Enzyme-Linked Immunosorbent Assays (ELISA)
      • This method depends on the conjugation of an enzyme with either antigen or antibody, then the enzyme activity on a substrate is used as a quantitative measure.
      • Indirect Method:
        • A known antigen is fixed to a solid phase (plastic cup or microplate) and incubated with the test serum and washed to remove excess unattached antibody.
        • Then reincubated with enzyme-labeled antiglobulin.
        • The latter will attach to the antibody bound to the fixed antigen.
        • After washing, a specific substrate is added, and the enzyme activity is measured (the degree of color change).

    Immunofluorescence

    • Direct Immunofluorescence
      • Labeled antibodies are added to the unknown antigen fixed on a slide, left to react, and the excess antibody is washed.
      • Then the slide is examined under the fluorescent microscope.
      • The appearance of a fluorescent site indicates the antigen-antibody reaction.
    • Indirect Immunofluorescence
      • To detect antibodies in the serum of the patient, the serum is added to known antigen fixed on a slide, left to react, and the excess antibody is washed.
      • A second labeled anti-immunoglobulin antibody directed toward the first antibody is added.
      • Then the slide is examined under the fluorescent microscope.

    Radioimmunoassay (RIA)

    • Although it is an extremely sensitive and specific technique, it requires specialized equipment, special precautions, and licensing due to the hazards of radiation.
    • Used for the detection of unknown Ag or Ab.

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    Description

    This quiz covers various immunodiffusion techniques, including Single Radial Immunodiffusion, and their application in HIV testing, including the Western Blot Test.

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