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55 Cards in this Set
- Front
- Back
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-the proportion of people with the disease who have a positive test
-how good is the test as detecting infection in those who have the disease -test will rarely miss people who have the disease -few false negatives -importance in screening assay |
Sensitivity
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-proportion of people without the disease who have a negative test
-how good is the test at calling uninfected people negative -test will rarely misclassify people without without the disease as infected -few false positives -importance in confirmatory assay |
Specificity
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-the probability of disease in a given patient with a positive test result
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Predictive Value Positive
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-the probability of not having disease when the test result is negative
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Predictive Value Negative
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Antibody:
-means serum concentration of 13.5 mg/mL -sedimentation cosntant 7s -molecular weight 146 amu -binds to staph protein A -bind to strep protein G |
IgG
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Antibody:
-means serum concentration of 1.5 mg/mL -sedimentation cosntant 19s -molecular weight 970 amu |
IgM
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Response Type:
-5-10 days lag after immunization -smaller peak response -usually greater IgM than IgG -lower average affinity, more variable |
Primary Response
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Response Type:
-1-3 days lag after immunization -larger peak response -Relative increase in IgG and, under certain situations, in IgA or IgE (heavy chain class switching) -higher average affinity, (affinity maturation) |
Secondary Response
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-antigens have multiple epitopes on their surface
-found in nature |
Polyclonal Antiserum
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-an antibody reacts with only one epitope (specificity)
-developed in the lab |
Monoclonal Antibody
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Factors that affect antibody specificity:
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1. Shared epitopes on different cells or pathogens (closely related organisms)
2. Unrelated antigens with similar epitopes (different affinity) 3. unrelated epitopes with similar 3D shape |
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Serum factors that may interfere with immunoassays:
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1. Rheumatoid factor (too much lipids)
2. Cross reacting antibodies (serum antibodies are polyclonal in origin) 3. Antibodies to the tissue in which antigen was made (e.g., egg, brain) 4. non-specific factors (lipids, fibrin) 5. unless appropriate test controls are utilized, the effect of these factors will not be recognized |
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Type of assay:
-clumping of particulate matter, such as cells or synthetic material (latex, charcoal) by agglutinin |
Agglutination Assays
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-for agglutination assays, adding antigen to a particle to serum
-if antibody is present, ___________ |
reaction agglutinates the antigen-particle (clumping)
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-for agglutination assays, adding antigen to a particle to serum
-if antibody is absent, ___________ |
-no clumping, particles uniformly dispersed in solution
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-agglutination with red blood cells as the indicator
-receptors on the RBC bind to antigens (agglutinins) added to the RBCs -ex. Direct method, ABO blood typing (clumping), some viruses (arboviruses, influenza) (network) -antigens may be added to surface of red blood cell- indirect method -RBCs are heavy and do not stay suspended on their own; they fall to the bottom of the well |
Hemagglutination
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Type of Assay:
-Coombs test -to detect antibody on patient's erythrocytes -if antibody is present on RBC, anti-human immunoglobin will agglutinate the cells -positive in hemolytic disease secondary to either autoimmune antibody or antibody from incompatible RBC transfusion |
Agglutination Assays- Anti-globulin Assays
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Type of Assays:
-may be performed in tubes, in microplates, on slides, on cards; microscopic or macroscopic -manual or automated -to detect antibody to specific agents -to detect recent infection to specific agents: a) 4 fold change (rise) in titer between acute and convalescent era when tested in the same run b) acute serum: collect at onset to ~10days p.o. c) convalescent serum: 10-28 days after acute -to identify etiologic agents using specific anti-sera |
Agglutination Assays
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Inhibition of agglutination of RBCs by the virus indicates ____________
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the presence of "specific" anti-viral antibody
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Inhibition of viral agglutination of RBCs by specific known anti-viral antisera, indicates ____________
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the identity of the virus
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Type of Assay:
-detects reagin antibody in blood by binding to an antigen composed of a complex of cardiolipin-lecithin-cholesterol bound to activated charcoal, resulting in macroscopic flocculation -quick, cheap, easy, no major equipment |
Rapid Plasma Reagin (RPR) Assay
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Type of Assay:
-cells infected with some viruses extrude hemaglutinin protein on their surface -RBC will adsorb to these infected cells -adsorption is inhibited in the presence of antibody to the viruses -identification of influenza and parainfluenza viral isolates made in cell culutre -detection of serum antibody to influenza and parainfluenza (HAdI) |
Agglutination Assay- Hemadsoprtion
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Type of Assay:
-major advantage is its ability to screen against a battery of antigens (or antisera) with a single test format and minimal variety of reagents |
Complement Fixation
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2 sequential antigen-antibody reactions for Complement Fixation Test
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1. a specific reaction between a known antigen and unknown (patient) serum in the presence of added complement (if there is a specific antigen-antibody reaction, complement is bound up in it- fixed)
2. a hemolytic system is used to detect fixation of the complement |
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Type of Assay:
-assay to detect/identify antigen is generally reliable, sensitive and specific -assay to detect serum antibody is poor -assay used to detect IgM (use anti-human IgM conjugate to fluorescein) prone to interference from IgG in serum and from rheumatoid factor |
Indirect Immunofluorescence Assay
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Type of Assay:
-antibody is attached to a magnetic bead -bead is mixed with solution containing suspect antigen -a magnet is used to trap the beads while rest of solution is discarded -beads are washed to remove unbound target -fluorescent antibody conjugate is added and after incubation, decanted. Bead washed again. -measure fluorescence or treat to remove bead from antigen-antibody complex, spot on slide and examine by FA microscope |
Immunoflourescence Immunomagentic Seperation
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Type of Assay:
-either antigen or antibody is attached to a solid phase, allowing for separation from unreacted material during the test -unreacted material is washed away -antibody or antigen is added and homologous binding to the surface phase occurs -unreacted material is washed away -an enzyme conjugated to detection antibody is added -unreacted material is washed away -enzyme substrate is added; reaction is detected |
Enzyme Linked Immunosorben Assay (ELISA)
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Type of Assay:
-sequence order of ELISA: 1. Antigen 2. Blocking Reagent 3. Binding Antibody 4. Adds conjugate 5. Add substrate |
Indirect ELISA
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Type of Assay:
-sequence order of ELISA: 1. Add capture antibody 2. Blocking Reagent 3. Add antigen 4. Add serum with antibody 5. Add conjugate 6. add substrate |
Sandwich ELISA
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Type of Assay:
-sequence order of ELISA: 1. Add anti-human IgM 2. Blocking Reagent 3. Add serum with IgM to virus 4. Add antigen 5. Add conjugate 6. Add substrate for enzyme |
IgM capture (MAC- ELISA)
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-a naturally occuring IgM antibody against Fc region of IgG
-binds IgG and may give a false positive reaction in a test for pathogen specific IgM if IgG that reacts with the antigen is also present in the serum |
Rheumatoid Factor
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Type of Assay:
-sequence order of ELISA: 1. Add Monoclonal-Antivirus A antibody 2. Blocking Reagent 3. Add antigen 4. Add substrate |
Competitive ELISA
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-amplifies signal for ELISA
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-detecting antibody conjugated to biotin
-add avidin conjugated with enzyme -multiplies signal |
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Type of Assay:
-the assay components are attached to microspheres -results are read using a modified flow cytometer |
Microsphere-based Immunoassays
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Type of Assay:
-oldest serological assay -measures loss of infectivity or toxicity after interaction with specific antibody -performed only is specialized laboratories -to identify etiological agent -to detect/quantify microbial toxins -to detect specific antibodies: 4 fold rise in titer (acute-convalescent sera) indicative of recent infection |
Serum Neutralization
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-the concentration of agent that kills 50% of the test animals
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LD50
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-50% infected tissue culture
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TCID 50
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-the reciprocal of the highest dilution of serum protecting against the agent
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Titer
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-measure lethal dose
(virus LD 50) - (the virus LD 50 in presence of the test serum) |
Neutralization Index
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when is the neutralizing index significant?
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significant NI > 1.7 logs (about 50%)
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During a serum neutralization with a constant virus and varying serum dilution from 1:10 to 1:640, all the mice specimen had died:
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No neutralization
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During a serum neutralization with a constant virus and varying serum dilution from 1:10 to 1:640, all the mice specimen from 1:10 to 1:80 survived:
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Serum has antibodies, protection
>4 fold, recent infection |
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Type of Assay:
-used to identify viruses, or for antibody when sera are low titred or already diluted -use multiple LD50 of virus as challenge dilutions -add each virus dilution to each seru |
Serum Neutralization
(constant virus, constant serum) |
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Type of Assay:
-to identify (serotype) virus -usually use 100 (10^2) TCID50 or LD50 of virus in challenge ("working dilution") -add same amount of virus to each standardized serum containing a type of specific antibody -serum dilutions preventing death of host contain neutralizing antibody -the virus type is that of the serum neutralizing the virus |
Serum Neutralization
(constant virus, constant serum) |
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Type of Assay:
-some viruses form plaques in cell culture when gelled media is used 1. each plaque is an area of cells killed by the virus 2. each plaque originates from 1 virus particle- the plaque forming unit (pfu) -antibody specific to the virus neutralizes the virus, preventing plaque formation -mix virus (100 TCID50) with equal volume test serum, incubate, inoculate cell cultures -count plaques in the presence and absence of test serum, 95% reduction is signficant |
Serum Neutralization
(plaque reduction assay) |
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Type of Assay:
-antigen-antibody complexes precipitate from solution when combined at optimal proportions -used since the 1940s -most of these assays are qualitative -used for detection of antibody to fungi, parasites, viruses, bacteria -used for separating antibody classes, quantifying and characterizing specific immunoglobins (e.g. myeloma, leukemia) |
Gel-based Assays
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How to quantify antigen via radial immunodiffusion:
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1. high affinity monoclonal antisera is added to the agar before the gel is poured
2. standard volume of antigen is added to the well |
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How to quantify antibody via radial immunodiffusion:
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1. antigen is added to the agar before the gel is poured
2. standard volume of antibody is added to the well |
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For gel-based assay, the standard curve for known reactants is the ________ vs _________
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concentration vs. area within precipitin ring
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Type of Assay:
-Rate of diffusion through gel is affected by: 1. molecular size 2. temperature 3. gel viscosity and hydration 4. matrix interactions -slow, insensitive, easy -ouchterlony double-diffusion -older method -precipitin line forms where antigen and antibody meet while diffusing throught the gel if they are homologous -qualitative |
Gel-based assays: passive diffusion
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Type of Assay:
-Rate of diffusion through gel is enhanced by electrophoresis-increase sensitivity and speed 1. proteins are charged molecules and move in an electric field 2. the charge is related to pH, matrix composition 3. the size of the molecule is also important for mobility |
Gel-based assays: active diffusion
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Type of Assay:
Immuno-double diffusion + electric current |
Gel-based Assays
Counter-immuno electrophoresis (CIEP) |
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Type of Assay:
Single radial immunodiffusion + electric current |
Gel-based Assays
Rocket electrophoresis |
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Type of Assay:
-two staged method 1. antigen mixture (e.g. serum) is separated by electrophoresis 2. antibody is allowed to diffuse perpendicular to the separated antigens -used for detection of myeloma proteins, screening for antibody classes |
Gel-based Assays
Combination (Immunoelectrophoresis) |
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Type of Assay:
-may use an enzyme conjugate followed by substrate addition to stain protein and visualize bands instead of radioactivity |
Gel-based Assay
Western Blot |
