Lecture 5 Flashcards

Question

law of mass action

Answer 1

- Ag AND ab complexes are reversible -rate of chemical reaction is directly proportional to the concentration of substances reacting -equilibrium is when the number of bonds being broken are equal to those being formed

Answer 2

a)immunocompromised - * Serum collected too early in the infection – usually it takes 10- 14 days for Ab to show up * Antibodies may not rise until months after acute infection -Lyme disease, Legionnaire’s b)* Production of cross-reacting antibody or heterophile antibody * Reactivation of latent organism due to infection by a different pathogen → Influenza A may cause reactivation of CMV * Passive transfer of multiple antibodies -Patient receiving intravenous immunoglobulin

Answer 3

* Test that detects the presence of and the amount of Abs within a person’s serum *Early infection more AG than AB -Abs dont come in until 2-3 weeks after infection and can be delayed depending on the infection * Seroconversion = the time during which a specific Ab develops and rises to detectable levels in blood *in order to determine when you got the infection you test paired samples, acute and convalescent sera * One sample from ACUTE-phase (within 1 week of infection) and one sample from CONVALESCENT-phase (2-3 weeks later - recovery period) -make doubling dilutions of pt serum -AB DILUTION and then add constant AG to each tube -see how many times you can add the same AG and still dilute * Run test in parallel → acute serum and convalescent serum

Answer 4

* Look for the highest dilution(MOST DILUTED) of a serum sample in both the acute and the convalescent set that causes a positive test reaction * Lack of Ab in either set = no exposure or exposure is too recent *having no Ab present in the acute sera to Ab present in convalescent sera = the development of antibodies in response to immunization or infection * A four-fold rise in titer between acute and convalescent serum = an active infection * If titer is the same in both tubes indicates the Abs were already in the serum and not recently produced = an old infection or vaccination

Answer 5

sensitization * Single Abs bind with antigen * Happens by accident -Ab must "bump" into the cell at the corresponding antigen site * forces that hold Ab and Ag together are weak * Reaction is rapid and reversible & not visible- no agglutination 2. Lattice formation * Most Ags are multivalent- have several epitopes to where Abs can bind * Abs have at least 2 antigenic binding sites -IgM ~10 *SLIDE PIC- the Fab portion of Ig molecule attaches to antigens on 2 adjacent cells (Y-Y-Y) * Large lattices of Ag-Ab complexes are formed * Agglutination becomes visible result * If Ag & Ab are soluble the visible reaction is called precipitation

Answer 6

DIRECT (Active) * Antigen is the natural carrier particle * Epitope on the organism reacts with the Ab and becomes visible as agglutination Example: Serology for Salmonella DIRECT (Active) Hemagglutination * Antigen is intrinsic part of red blood cell * RBC reacts directly with the Ab and becomes visible as agglutination Example: ABO grouping INDIRECT (Passive) * Ag is artificially attached to a latex bead * Detects Ab in sample * Ag-Ab complex made visible by latex Hemagglutination (INDIRECT Passive) * Ag attached to red cells * Detects Ab in sample * Ag-Ab complex made visible by RBC REVERSE INDIRECT (Passive) * Ab affixed to latex or RBC * If attached to a RBC then called Reverse Hemagglutination * Detects Ag (like bacteria) in sample

Answer 7

* Salmonella, Shigella or Escherichia coli O157:H7 require serological testing to confirm the tube biochemical identification * Serological tests for enteric pathogens are considered direct agglutination tests * No latex beads or carrier molecules involved cant use it right away because it can cause cross reactivity

Answer 8

Intact formalin-killed Staph aureus is the carrier * Protein A in cell wall of Staphylococcus aureus, can bind to Fc region of most IgG antibodies * Fab region of IgG free to interact with antigens present in the specimen * Visible agglutination of S. aureus particles = antigen-antibody reaction

Answer 9

*Serological classification of Streptococci as per the polysaccharide antigenic nature of cell wall * BH strep can be serogroups A, B, C, F, and G *NH strep is group D * AG extracted form streptococci * Reverse indirect agglutination test since blue latex particles pre-coated with group-specific antibodies react with antigen

Answer 10

* Use of Latex particles coated with plasma containing IgG and fibrinogen to detect clumping factor and Protein A on Staph aureus cells * Protein A reacts with the IgG clumping factor reacts with fibrinogen * Since there are latex particles coated with antibody (IgG) & you are detecting Ag (the Staph) then this is a reverse indirect agglutination test

Answer 11

*if organism can make coagulase enzyme that when mixed with rabbit plasma produces clumping or a clot. Two types of coagulase produced, bound & free Bound coagulase * clumping factor, found on cell wall of S. aureus * Reacts with fibrinogen and causes agglutination type clumping * Detected by slide and tube coagulase tests Free coagulase * Extracellular coagulase excreted by cell * Produces a thrombin-like substance that converts fibrinogen to fibrin (clot formed) * Detected by tube coagulase only

Answer 12

* viruses like Rubella have surface Ags that can agglutinate RBC *reaction can be inhibited by Abs found in a person’s immune or convalescent sera * Virus can be bound by the antibodies present in the serum therefore cannot agglutinate the RBC Method: * Serial dilutions of patient sera (Ab) are made * A standard amount of virus (Ag) is added * Add red cell suspension & incubate at room temp for 60min * Highest dilution of serum that inhibits hemaglutination is the hemagglutination titre a pellet is not a reaction the whole cell will be a hazy red * Sera + virus +Ab (bound virus) + RBC = No agglutination or HI - LOOKS LIKE PELLET *The highest dilution of serum that prevents hemagglutination is called the HI titer of the serum * Sera + virus - NO AB (free virus) + RBC = Agglutination * Then hemagglutination will be observed in all wells =- HAZY ALL RED

Answer 13

* Soluble Ag and Ab combine to form lattice (a 3d structure formed by the reaction of polyvalent antigens with antibodies) * WHEN concentration is optimum lattice falls out of solution & forms a visible insoluble precipitate * Precipitate that remains suspended as floccules = Flocculation -can take place in liquid media or in gels such as agar, agarose or polyacrylamide * Precipitation curve= you can plot how much a constant amount of Ab is precipitated when you add increasing amounts of Ag ON THE CURVE Prozone: * Ab excess * Saturation of antigenic sites with antibody * The Abs do not bridge between antigens, no lattice occurs Zone of Equivalence: * Ag and Ab in equivalent proportions * Produces lattice formation Postzone: * Antigen excess allows a number of antigens to bind to a single antibody (excess antigen binding sites) * Number of crosslinks decrease, prevents lattice formation- Can cause false negatives

Answer 14

1. If trying to detect whether a patient has a certain Ab or Ag -corresponding Ag in one well & patient sera in an adjacent well (or a known Ab in one well and patient specimen in the other) * Both diffuse out towards each other in gel (double diffusion) * If Ag-Ab reaction happens -get a line of precipitation at the optimum concentration 2. If you want to test whether a patient has a specific Ag you can do a test of relatedness * Place a known Ab to the Ag you are testing for in a center well *a known Ag in another well (positive control) * Place patient specimen in another well adjacent to the positive control * Compare patient result line to result line of know Ag for relatedness * Called the pattern or line of identity

Answer 15

quantitative * Single diffusion test since 1 of the reactants known Ab is evenly distributed throughout the gel * Sample you are testing for is added to wells cut into the agar * If Ag present it will diffuse into gel & form a precipitin band in a radial pattern, where equivalence occurs * Diameter of ring is measured- amount of Ag is proportional to area of precipitation ring * Standards of known Ag are also tested & a standard curve plotted * Unknown sample diameters also plotted on graph * Based on where they fall on graph can determine the concentration of Ag in each sample = quantitative AS THE SIZE OF RING INCREASES SO DOES THE ANTIGEN

Answer 16

uses of the binding between an Ag and its homologous Ab in order to identify and quantify specific Ag or Ab in a sample Label: * Chemically links Ab or AG with label to make the reaction detectable * Labels emit radiation, produce a color change in a solution or fluoresce under light * Two main types of Labeled Immunoassay are: * Immunofluorescence * Enzyme Immunoassay

Answer 17

* Uses one Ab to identify target Ag- can be on a slide * Antigen-specific fluorescent-labeled Ab is applied, incubated, washed and examined with fluorescent microscope * Can detect infectious agent inside cells or cell cultures

Answer 18

To detect target Ag in patient specimen * Patient sample on slide * First add unlabeled (primary) Ab that specifically binds the target Ag if it is present in the sample * Then add the secondary Ab (usually an anti species Ab like anti-human Ab), which is labeled to the fluorophore * The secondary Ab recognizes the primary Ab and binds to it To detect Ab in patient serum * Bacteria or virus fixed on slide – add patient sera * If Ab present in sera will attach – add Anti human Ab with fluorescent dye

Answer 19

* Uses a second, unlabeled antihuman Ab to bind to the microbial specific Ab in the patient serum * Then a third fluorescent labeled Ab directed to the second (antihuman Ab) is added

Answer 20

* Uses enzymes (alkaline phosphatase or horseradish peroxidase) conjugated to Ab or Ag * When substrate added if there is an Ag-Ab complex a color will be produced that can be quantified spectrophotometrically - wells in a microtitre tray or spherical plastic or magnetic beads *can be used to detect AB OR Ag in pt samples for direct or indirect tests Direct ELISA for Ag DETECTION * Patient specimen is absorbed to bottom of well * An enzyme LABELED Ab specific to the target Ag is added * If patient sample has Ag then will bind to Ab * Add substrate and a color change indicates positive result Indirect ELISA for Ag DETECTION * Patient specimen is absorbed to bottom of well * An UNLABELED Ab specific to the target Ag is added * If patient sample has Ag then will bind to Ab * Add second enzyme labeled species specific Ab (antimouse Ab) which will bind to the first Ab * Add substrate and a color change indicates positive result

Answer 21

Direct Sandwich Antigen detection: * Known Ab captured or bound to matrix * Add patient sample – if Ag is present will bind to Ab * Add secondary Ab to that Ag labeled with enzyme * Substrate added and a color change indicates Ag-Ab reaction Indirect Sandwich Antigen detection: * Known Ab captured or bound to matrix * Add patient sample – if Ag is present will bind to Ab * Add secondary Ab to that Ag * Add third Ab (anti human Ab) labeled with enzyme, that binds to primary Ab * Substrate added and a color change indicates Ag – Ab reaction

Answer 22

Only Indirect ELISA for Ab detection: * KNOWN Ag fixed to bottom of well or tube * Add patient sample – if Ab present will bind * Add second Ab (anti-human) labeled with enzyme * Add substrate and color change indicates positive reaction WESTERN BLOTTING

Answer 23

* Demonstrates presence of antibody in a patients serum * Consists of mixing antigen suspected of causing patients disease + patients serum * Limiting amount of complement is added * If serum has antibody to antigen these complexes will bind all the complement * Sheep RBC + hemolysin (indicator) added * Only if complement has not been bound by Ab-An complex will it be available to bind to sheep cell hemolysin complex and cause lysis Positive: failure of RBC to lyse in final test system Negative: lysis of indicator cells = lack of Ab TWO SYSTEMS Test system 1 - nonimmune pt serum WITHOUT SPECIFIC AB, test AG + complement , complement NOT fixed Test system 2 - IMMUNE pt serum with SPECIFIC AB, test AG + complement, complement FIXED both go into the INDICATOR SYSTEM - sheep rbcs, guinea pic complement, rabbit hemolysin TEST SYSTEM 1 -hemolysis negative CF AB test (pale) TEST SYSTEM 2 - NO hemolysis - Pos CF antibody test