(A) Antigen is in excess. (B) Antibody is in excess. (C) Antibody is added to the antigen. (D) Antigen and antibody are at optimal concentrations.

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1 Amount of Antibody Precipitated Immunology - Problem Drill 21: Antigen-Antibody Interactions Question No. 1 of When antigen and antibodies bind, maximal precipitation occurs when? Question #1 (A) Antigen is in excess. (B) Antibody is in excess. (C) Antibody is added to the antigen. (D) Antigen and antibody are at optimal concentrations. Antigen in excess would bind all sites. Resulting in no lattice formation. Antibody would result in lattice formation but not the maximum. The order of adding the reactant is not important in this case. When the reactants are at equivalent concentrations the maximal precipitation occurs. Amount of Antigen When a give amount of antibody is added to test tubes having increasing amounts of antigen (different dilution in each tube) the amount of precipitate increases gradually until it reaches a maximum. After the maximum the amount of precipitate declines. Test tubes have a large excess of antigen relative to the antibody will have no precipitate at all. As the lattice grows, it becomes insoluble and precipitates. In mixtures were antigen is in excess each antibody is bound to only two antigen molecules and there is no cross linkage or lattice formed. Therefore there is no precipitation.

2 Question No. 2 of An anti-antibody is used when. Question #2 (A) The antigen is an antibody. (B) An antibody is precipitating. (C) The antigen is not soluble. (D) The antibody is agglutinating. A. Correct! An antibody is the antigen when using an anti-antibody. An anti-antibody would not be used to stop precipitation. An insoluble antigen would most likely be used in an agglutination assay. D. Incorrect! An antibody is involved in an agglutination reaction would not be associated with an anti-antibody. Remember that antibodies are made in response to an antigen. If the antibody is made against HIV virus the antibody is said to be an anti-hiv antibody. Likewise an antibody made against an antibody is said to be an anti-antibody antibody. Because in this case the antibody is the antigen.

3 Question No. 3 of In the complement fixation assay which of the following is required? Question #3 (A) Hemagglutination (B) Cold treatment for the sample so as to not loose any biological activity. (C) Antibodies against complement (D) Heat inactivated serum Hemagglutination is the process of blood cells agglutinating. No in this assay a specific protein should be heat inactivated. Prosthetic groups are tightly-bound cofactors. The natural complement in the sample need to be heat inactivated. Remember that if complement is not available to lyse a cell it will remain intact. The lack of antigen-antibody complexes enables complement to bind to the anti- RBC antibodies which have attached to the sheep RBCs, triggering lysis of the RBC by MAC and turns the solution transparent red. In a sample that has antibodies against the antigen the test is positive. In a positive test the patient s serum has antibodies against the disease (antigen) of interest. The antibodies bind the antigen. This antigen-antibody complex binds complement which is therefore not available to bind the anti-rbc antibodies. As a result the sheep RBC remain intact and the solution remains cloudy.

4 Question No. 4 of In the ELISA assay which of the following is used? Question #4 (A) A dye-labeled antigen (B) A dye-labeled antibody (C) Enzyme-labeled antigen (D) Enzyme-labeled antibody Remember the antigen is what the ELISA assay is trying to detect. There are assays that use dye-labeled antibodies but it is not this one. An enzyme labeled antigen might be useful in a direct binding measurement but this is not how ELISA assay is done. Yes, the enzyme-labeled antibody binds to the antigen and an enzyme substrate added. The ELISA assay has become the test of choice for many diagnostic procedures for the determination of infection. ELISA has many advantages: ELISA can detect ether antibody or antigen. ELISA can quantify amounts of antigen or antibody. ELISAs are easy to perform, relatively inexpensive and many simultaneous assays can be done quickly Plates with wells coated with antigen can be stored for testing for a long time.

5 Question No. 5 of The direct fluorescent antibody test can be used to detect the presence of. Question #5 (A) Specific antigens (B) Agglutination (C) Radioactive antibodies (D) Complement A. Correct! Yes, the direct fluorescent antibody test can be used to detect the presence of specific antigens. Agglutination is an aggregate of molecules. Radioactive antibodies are not detected with this method. This is a fluorescence method. D. Incorrect! I could be used to detect complement if it is the antigen the antibody was raised against. However this is not the best answer. Fluorescent dyes are attached as labels for several serologic tests to antibodies. The most important of these dyes fluorescein. When exposed to a fluorescent light, as in a microscope, fluorescein glows bright green. Fluorescein labeled antibodies is used in direct and indirect fluorescent antibody tests. Direct fluorescent tests are not quantitative. The amount of fluorescence is not directly related to the amount of antigen present. The fluorescein molecule shown has been modified with a sulfur group to link it to the antibody.

6 Question No. 6 of Which of the following is correct about radial immunodiffusion? Question #6 (A) Antigen diffusing from the plate well forms a ring. (B) The ring formed around the antigen well is a function of the antigen binding the antibody. (C) The ring precipitate that forms is directly proportional to the concentration of antigen in the well. (D) All of the above are correct. This answer is partially correct and not the best answer. This answer is partially correct and not the best answer. This answer is partially correct and not the best answer. This answer is correct and is the best answer. In this technique an antigen is allowed to diffuse from a well into agar that has a known concentration of antibody in it. As the antigens diffuse out of the wells a ring will form at optimal concentration. The diameter of the ring is directly proportional to the concentration of antigen in the well. By using a series of wells each having an increasing concentration of antigen it is possible to make a standard curve by measuring the ring diameter. This method allow the determination of the amount of antigen in a solution. It is common to use radial immunodiffusion to measure the concentration of a specific antibody in a person s serum.

7 Question No. 7 of Which of the statements below is true of the difference between precipitation test and agglutination tests? Question #7 (A) There really is no difference. (B) One involves antibody excess the other antigen excess. (C) One uses anti-antibodies. (D) Agglutination involves clumping of insoluble particles; precipitation involves aggregation of soluble molecules. There is a fundamental difference between the two assays. The ratio of antibody to antigen must be optimal in precipitation. There does not always an optimal ratio in agglutination. No, this answer is not relevant to this question. Agglutination involves clumping and can be used on insoluble proteins as well as soluble one. Since not all antigens are soluble that can be precipitated by antibody an alternative test was developed. Because antibodies have multiple antigen binding sites they can also cross-link particulate antigens, like entire bacteria which results in agglutination or lumping. The difference between agglutination and precipitation is that agglutination involves clumping of insoluble particles. Precipitation is the aggregation of soluble molecules

8 Question No. 8 of Which statement is correct with respect to Neutralization tests? Question #8 (A) These tests neutralize the ph to 7. (B) Antibodies when binding an antigen may inactive the antigens function. (C) When antibody antigen binds they are neutralized. (D) None of the above The neutralization test has nothing to do with the ph. B. Correct! Neutralization tests work because antibodies can neutralize the biological activity of toxins and many pathogens. This is not correct because the binding of an antibody to an antigen does not necessarily neutralize its activity. This is only true for some binding events not all events as implied by this answer. D. Incorrect! There is a correct answer. Consider the biological activity of some antigens. Neutralization tests work because antibodies can neutralize the biological activity of many pathogens and their toxins. For instance by adding antibodies against tetanus toxin with a sample of the toxin results in the toxin being harmless. This is because the antibodies have neutralized the toxin. To determine if a patient has been exposed to a specific virus a sample of blood serum is tested. To the serum the virus of interest is added. The mixture is then added to cells growing in culture. If the cells die there were no antibodies in the serum against that virus. This indicates the patient was not exposed to the virus. However if the serum+virus mixture when added to the cells growing in culture has no effect then the virus has been neutralized and it is likely that the patient has been previously exposed to the virus.

9 Question No. 9 of Which of the following statements are true about direct fluorescent antibody test? Question #9 (A) They can identify the presence of antigen in tissue. (B) It is not a quantitative test. (C) A fluorescently labeled antibody is required for the test. (D) All of the above This answer is partially correct but not the best answer. This answer is partially correct but not the best answer. This answer is partially correct but not the best answer. All of the answers are correct. Fluorescent dyes are attached as labels for several serologic tests to antibodies. The most important of these dyes fluorescein. When exposed to a fluorescent light, as in a microscope, fluorescein glows bright green. Fluorescein labeled antibodies is used in direct and indirect fluorescent antibody tests. Direct fluorescent tests are not quantitative. The amount of fluorescence is not directly related to the amount of antigen present. These are straight forward assays that be used to detect antigen in tissue and fluids.

10 Question No. 10 of Which answer is false with respect to immunochromatographic assays? Question #10 (A) They are faster then an ELISA assay. (B) They require a lot of time but are very sensitive. (C) They involve detection of an antigen by observing color changes. (D) These assays are used for pregnancy testing, pathogen detection and other applications. These assays are faster then an ELISA assay so the answer is incorrect. You are looking for a false answer. B. Correct! These assays are sensitive but do not require a lot of time. Immunochromatographic assay does involve color changes. D. Incorrect! The applications for the Immunochromatographic assays are many including pathogen detection and a diverse set of antigen detection. Immunochromatographic assays are even faster than immunofiltration assays and easier to read thean ELISAs. In this system an antigen solution (such as urine sample) flows through a porous strip and encounters antibody labeled with either pink colloidal gold or blue colloidal selenium. Where antigen and antibody bind a colored immune complex form in the fluid. The fluid then flows through a region where the complexes encounter antibody against the complex. This binding results in a clearly visible pink or blue line.