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Questions and Answers
What component is placed in the gel during Single Radial Immunodiffusion?
Which test is primarily used for the detection of specific HIV antibodies?
What indicates a positive result in the Western Blot Test?
Which test involves the use of toxin-antitoxin precipitation on the surface of the agar?
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What can be measured using Single Radial Immunodiffusion?
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What is the reaction called when an antibody reacts with a particulate antigen?
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Which method is most commonly used for rapid blood grouping?
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What is used to make invisible antigen-antibody reactions visible?
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In the Tube Method, what determines the endpoint of the agglutination test?
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Which test is NOT listed under the types of antigen-antibody binding reactions?
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What is the primary use of the Tube Method (Semi-Quantitative agglutination test)?
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Which condition can be diagnosed using the Widal test?
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What characterizes passive agglutination?
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What is the purpose of mixing the mother's serum with prepared RH positive RBCs in the Indirect Coombs Test?
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Why is anti-human gamma globulin added during the Indirect Coombs Test?
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In the Coagglutination (COA) test, what is the main function of protein A on the surface of Staph aureus?
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How does the Coagglutination (COA) test detect bacterial antigens directly in body fluids?
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What is the primary use of the antiglobulin agglutination test?
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At what temperature are the mixtures incubated in both the Indirect Coombs Test and the Coagglutination (COA) test?
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Which phenomenon describes the incomplete antibodies that cannot bridge between RBCs to cause visible agglutination?
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What is detected by the Direct Coombs Test?
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What is the incubation condition for the Indirect Coombs Test to detect anti-Rh antibodies in the mother's serum?
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What type of antibodies are detected by the coagglutination (CoA) test?
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Which specimens can be tested using the coagglutination process?
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What is essential for an antigen-antibody reaction in a complement fixation assay?
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In an indirect ELISA, what is typically done after the test serum is incubated and washed?
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Which step is unique to the double antibody technique in ELISA?
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Which method relies on colorimetric measurement to detect enzyme activity?
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Where is the antigen fixed in a direct immunofluorescence assay?
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Which method is now considered historical for antigen-antibody reactions?
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What type of reaction occurs when an antigen in solution reacts with an antibody?
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Which method is commonly used in blood grouping?
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The tube method is a semi-quantitative agglutination test used to determine what?
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What is the purpose of the antiglobulin test (e.g., Coombs test)?
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What does a rising antibody titer by 4 folds or more indicate?
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Which of the following is an example of an antibody labeled assay?
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In the tube method, what determines the endpoint of the test?
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Which assay involves using fluorescent dyes to make the reactions visible?
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What type of agglutination test uses latex particles to measure antibodies?
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The Widal test is used to detect antibodies against which organism?
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What is the purpose of the Western Blot Test?
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What is the principle behind the appearance of bands on the nitrocellulose paper strip in the Western Blot Test?
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Which method is used to confirm the toxigenicity of diphtheria bacilli?
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What indicates a positive result in Elek's Test?
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Which characteristic is essential for the medium used in Elek's Test?
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How are HIV proteins separated in the Western Blot Test?
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What is one major requirement for conducting a Radioimmunoassay (RIA)?
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Which one of these is a primary characteristic of Radioimmunoassay (RIA)?
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Why does Radioimmunoassay (RIA) require special licensing?
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What is the main use of Radioimmunoassay (RIA)?
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Which of the following is NOT a challenge associated with Radioimmunoassay (RIA)?
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What indicates a successful antigen-antibody reaction in direct immunofluorescence?
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In indirect immunofluorescence, what is the role of the second labeled antibody?
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Which step is common to both direct and indirect immunofluorescence?
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In indirect immunofluorescence, what is added to the known antigen on the slide first?
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What type of microscope is typically used to examine the slides in both direct and indirect immunofluorescence?
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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.
