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123 Cards in this Set
- Front
- Back
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The two proteins that IgG binds to that IgM does not
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Staph protein A and Strep protein G
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Polyclonal antiserum is found in ____, where antigens have multiple _____ on their surface.
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nature, epitopes
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Monoclonal antibodies are found in the _____. Monoclonal antibodies react with only one _____. This is known as _______.
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lab
epitope specificity |
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When an anibody produced in response to one antigen also reacts with another, different antigen, this is known as ______
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cross reactivity
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-Not absolute
-Cross reactivity occurs -shared epitopes on different cells or pathogens -Unrelated antigens with similar epitopes -Unrelated epitopes with similar 3D shape |
Antibody Specificity
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To create clumping in a test, need ______ amount of ______ and _____. Is this reaction reversible?
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equal, antibody, antigen
Yes, it is reversible |
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Why are controls used for immunoassays?
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-Make sure protocol is working correctly
-Serum factors may interfere |
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What are the serum factors that may interfere with immunoassays?
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-Rheumatoid factor (autoanibody)
-Cross reacting antibody -Antibodies to tissue in which antigen was made -Non-specific factors (lipids, fibrin) |
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Serological assay that clumps particulate matter, such as cells or synthetic material (latex, charcoal)
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Agglutination Assay
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Assay that prevents agglutination in the presence of antibody to agglutinin antigen
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Inhibition assay
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In an agglutination assay, if antigen on a particle is added to serum and the antibody is resent for the antiger, what happens? What if no antibodies are present?
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Allgutination (clumping occurs)
Uniform dispersal of particles in solution (no clumping) |
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Agglutination assay twhere antigen particles or cells with surface antigen clumped by cross linking antibody
-Receptors on cell surface can be agglutinated by cross linking antigen |
Direct (Classic) Agglutination Assay
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Allugitination Assay where cells or particles are coated with antigen or antibody to cause clumping
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Indirect Agglutination Assay (passive)
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What is used to attach antigens to cells in an agglutination assay?
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tannic acid
glutaraldehye chromic chloride |
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Streptococcus pneumoniae & Neisseria meningitidis, Rubacell, Rubascan, Syphilis, Cryptococcus, and Rickettsia are all what types of Agglutination Assays?
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Indirect Passive Agglutintion Assays
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Agglutination with RBC as indicator
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hemagglutination
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Receptors on RBC surface bind to antigens, such as in ABO blood typing.
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Direct hemagglutination
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If RBCs fall to the bottom of the well ___________
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no agglutination has occured
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Anti-globulic assay test which detects antibody on one's own Red Blood Cells, leading to agglutination of cells by anti-human immunoglobulin
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Coombs Test
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Agglutination of cells via the Coombs test means a person is positive for:
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hemolytic disease - autoimmune antibody or antibody from incompatible RBC transfusion ---> anemia
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In agglutination Assays, to detect recent infection to specific agents:
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4 fold change (rise) in titer between acute & convalescent sera when tested in the SAME run
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Acute serum is collected :
Convalescent serum is collected: |
at onset to 10 days post onset
10-28 days after acute |
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Inhibitiion of RBC agglutination by virus indicated the presence of _____
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specific anti-viral antibody
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In a Hemagglutinin inhibition assay, if agglutination of RBCs occurs, then _________. If no agglutination occurs, ______ are present, and the RBC in the well, ________.
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-non-specific antibodies are present
- specific antibodies are present -RBCs fall to bottom of the well |
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Non-treponmal flocculation card test that is an agent of syphillis
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Rapid Plasma Reagin Assay
***False positives for other disease ***False negatives - prozone |
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2 Disadvantages of Rapid Plasma Reagin Assay
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False positives from other diseases and false negatives from the prozone
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Agglutination assay that uses RBC to absorb virus infected cells that extrude hemagglutinin protein on their surface
-used to identify influenza and parainfluenza viral isolates |
Hemadsorption
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Advantages of Complement Fixation Test
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- Same reagents to detect and identify antigen
- One protocol - Not species specific - more specific than HAI |
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Disadvantages of Complement Fixation Test
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- less sensitive than ELISA, HAI
- Technically demanding - Interference by lipids - Limited quality reagents available |
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What are the two steps of the Complement fixation test?
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1. Addition a known and unknown (antigen and serum) in the presence of complement
2. Addition hemolysin to sheep red blood cells |
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If an antigen-antibody complex is formed in step one of Complement Fixation, what will happen to the sheep red blood cells in step two?
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No Lysis --- All complement bound by original complex
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If an antigen-antibody complex is not formed in step one of Complement Fixation, what will happen to sheep red blood cells in step 2?
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Lysis of RBC due to unbound/free complement
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Advantages of Immunofluorescence
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-quck and cheap
-one protocol for many agents -detect tissue and cellular antigens -multiplex - flurochromes detect >1 antigen |
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Disadvantages of immunofluorescence
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-antibody often neither sensitive nor specific
-requires conjugate antibodies -need fluorescent microscope |
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Uses fluorescent labeled monocolonal antibodies to identify specific markers on cells and sort them by lineage and type
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Cell Sorter
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Sorts and separates cell types using a laser
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Flow Cytometry / FACS (fluorescent activated cell sorter)
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Immunofluorescent assay where a cell with membrane antigen binds directly to fluorochrome-tagged antibody
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Direct Immunofluorescence
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immunofluorescent assay that uses to two antibodies to detect antigen or antibody in serum
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Indirect Immunofluorescence
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Two antibodies used in Indirect Immunofluorescence to detect or identify antigen?
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- primary - IgG made in animal to antigen
- secondary: anti-animal IgG conugated to fluorochrome **Test is reliable, sensitive, and specific |
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Two antibodies used in Indirect Immunofluorescence to detect antibody?
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-Primary: test serum (human)
-Secondary: anti-human IgG conjugated to fluorochrome **Low sensitivity, specificy, false positives and negatives |
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In indirect immunofluorescence, ____ in serum and _______ can interfere with detection of IgM
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IgG & rheumatoid factor
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An immunofluorescence method for detecting antigen using an antibody attached to magnetic bead and conjugated with fluorescence. Read under an FE microscope
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Immunomagnetic Separation
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ELISA Advantages
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-Sensitive, Specific, Reliable
-Low cost -small volume of sample -can use serum, CSF, stool |
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ELISA Disadvantages
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-Need plate washer and reader
-Species specific antisera -Many protocols with standardized reagent |
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ELISA Assay:
-Coated with antigen -Addition of primary antibody Addition of secondary antibody conjugate -Addition of enzyme substrate |
Indirect ELISA
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ELISA assay:
-Coated with capture antibody -Addition of antigen -Addition of secondary conjugate antibody Addition of enzyme substrate for color |
Sandwich ELISA
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ELISA Assay:
-antihuman IgM coats plate -Test serum w/IgM added (bound to antihuman IgM) -Virus antigen added (bound by test serum w/IgM to virus) -Monoclonal antibody to virus conjugated to enzyme (specific) -Enzyme substrate ****Only captures IgM |
IgM Capture MAC ELISA
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-An naturally occuring IgM antibody against Fc region of IgG
-This binds the IgG, which can bind to a specific antigen, resulting in a false positive (Want IgM to pick up antigen) |
Rheumatoid Factor
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ELISA Assay
-Incubated Ag-Ab complex added to antigen coated wall -Plate washing and unbound antibody washes away -Enzyme linked secondary antibody added -Substrate added eliciting color signal ***Secondary enzyme linked competing with test serum IgG) |
IgG Competitive ELISA
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In an ELISA protocol, this must be added so that antibody or antigen do not bind to open binding sites
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Blocking Reagent
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Reading enzymes in a spectrophotometer determines ________.
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Optical Density
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Fluorescence is read in a _____________.
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Fluorometer
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This amplifies the signal in ELISA
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Avidin-Biotin
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A positive sample by ELISA must have a specimen P/N ratio that is greater than _#__ times the P/N of _________ AND greater than __#___ the P/N of __________
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>2x the P/N of the negative control serum & >2x the P/N of the control antigen
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What is the P/N ratio formula?
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Optical Density of Test serum + Antigen / Optical Density of negative control +antigen
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Assay that enumerates cells secreting antibody to specific antigen
-Uses agar overlay that shows dots representing a cell that has secreted the correct antibody |
Elispot
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-Using similar protocols to ELISA, this test merges ELISA & Flow cytometry, but uses Luminex beads instead of plates.
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Microsphere-based Immunoassay
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Coupled to sepharose matrix, this bacterial protein binds IgG only, NOT IgM
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Protein G
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Assay
-Measures loss of infectivity or toxicity after interaction with specific antibody -identifies etiological agents -detects/quantifies microbial toxins -detects specific antibodies |
Neutralization Test
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Advantages of the Neutralization Test
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-most specific and very sensitive
-indicate presence of protecting antibody |
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Disadvantages of the Neutralization Test
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-requires LIVE infectious agents
-requires cell culture/animal host -expensive -slow -high level staffing |
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In neutralization, the concentration of agent that kills of 50% of test animals
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LD50
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50% of tissue culture of infective dose
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TCID50
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reciprocal of highest dilution of serum protecting against agent
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Titer
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Virus LD50 in negative serum - Virus LD50 in presence of test serum
-Significant >1.7 |
Neutralization Index
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When a neutralizing agent uses constant virus and varying serum dilution, it is looking to detect specific _______. Serum dilutions that prevent death of test animal have.....
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antibodies
neutralizing antibodies |
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When a neutralization test uses constant serum and varying virus dilution, it is being used to identify ______ or _______. If the host does not die the serum must contain ________
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virus or antibody sera that are low titered or diluted
-specific neutralizing antibodies to that virus |
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When a neutralizing test uses constant virus and constant serum, it is trying to determine
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-what the virus is by identifying what serum neutralizes it
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Rate of a passive diffusion gel assay affected by:
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-molecular size
-temperature -viscosity and hydration of gel -matrix interactions |
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Passive diffusion gel assay where a precipitation line forms where the antigen and antibody meet while diffusing through the gel
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Ouchterlay double diffusion
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Ring shaped Passive diffusion gel assay that quantifies the antigen (monoclonal antisera added to agar before gel poured) and quantifies antibody (antigen added to agar before gel is poured)
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Radial immunodiffusion
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The addition of electric current to a gel-based assay increases ______ and _______
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speed and sensitivity
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Immuno-double diffusion + electric current
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Countercurrent electrophoresis (CIEP)
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Single radial immunodiffusion + electric current =
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Rocket electrophoresis
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Gel-based assay that allows you to detect specific proteins in a sample using electrophoresis to separate and transferring proteins to membrane by electroblotting to create visual bands
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Western Blot
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Why Serological Testing??
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-Antibody present at onset of illness
-Infectious agent not always presesnt at onset of illness -Antibody may bind agent -Determine etiology after acute illness -Some agents not culturable -Surveillance -Retrospective studies |
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-Chemical elements required by living organisms other than carbon, hydrogen, nitrogen, and oxygen
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Minerals
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Minerals that have a catalytic role in enzymes
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Microminerals
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Minerals that provide structure and electrolytes
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Macrominerals
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How many vitamins are there and which one is NOT essential?
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13
Vitamin D (synthesized by skin) |
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One of a number of chemical compounds that have similar structure, each of which shows vitamin activity defined by its different biological properties
-Vitamin A has 6 (retinol, retinal, and 4 carotenoids) |
Vitamer
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Insufficient, excessive, or imbalanced consumption of nutrients
-Primary cause of immunodeficiency worldwide |
Malnutrition
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Macronutrients include
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-Carbs, Fats, Protein, Water
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Micronutrients include:
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Minerals and vitamins
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Regulate mineral metabolism, cell tissue growth and differentiation, are antioxidants, and are precursors for enzyme cofactors
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Vitamins
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The Vicious Cycle of Malnutrition and Infection
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-Inadequate dietary intake --> Weight loss, growth faltering, lowered immunity, mucosal damage -->
Disease: Incidence, duration, severity --> Appetite loss, nutrient loss, malabsorption, altered metabolism ---> Improper dietary intake |
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-secretes cytokines and chemokines
-obesity associated with inflammation of ________ from chronic activation of the immune system -Decreased T- and B-Cell function, increased monocyte and granulocyte phagocytosis, and increased leukocytes |
Adipose Tissue
-White Adispose Tissue |
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Protects neutrophils from apoptic death
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Glucose
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-Protein that regulates energy intake and expenditure (appetite and metabolism)
-Circulates at levels proportional to body fat -Obese may be resistant -Regulates inflammatory response |
Leptin
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Cytokine responsible for
-Macrophage Activation -Natural Killer cytotoxicity -Th1 stimulation -Th2 inhibition -Dendritic cell activation |
Leptin
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Two types of protein-energy malnutrition
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Kwashiorkor and Marasmus
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Reasons for malnutrition in Kwashiorkor and Marasmus
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K: Diet high in starch and low in protein
M: Deficiency in both carbohydrate and protein nutrition |
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Differences between Kwashiorkor and Marasmus?
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K:-Occurence incease after 18 months old, humid climate, swelling in feet, belly
M: before 1 year old, dry climate, Tissue and muscle wasting ***Both cause stunted growth |
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Immunogenic problems from Protein-Energy Malnutrition
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-Atrophy of thymus
-Reduced phagocytosis -Th cell proliferation halted -Reduction in IgA, lysozyme, complement |
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Needed by the retina in the form of retinal for low light and color vision
-Storage form is ______ |
Vitamin A
-Retinol |
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Important hormone-like growth factor for epithelial cells, produced by gut dendritic cells ---provided to T cells and B cells by activated dendritic cells
-Involved in gene transcription |
Retinoic Acid
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Vitamin involved in macronutrient metabolism
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Vitamin B
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Vitamin that stimulates chemotaxis and phagocytosis (primarily neutrophils)
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Vitamin C
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-Antioxidant vitamin where deficiency may result in immunosenescence
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Vitamin E
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Gradual deterioration of the immune system with increasing age
-Most prominent in T Cells |
Immunosenescence
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In immunosenescence, increasing the level of vitamin E enhances cell dividing and interleukin production in ________, but not ________
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naive T-cells, but not memory cells
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Vitamin that inhibits inflammatory response
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Vitamin K
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Vitamin that regulates the concentration of calcium and phosphate in bloodstream and is involved in regulation of proliferation, differentiation and apoptosis of cells
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Vitamin D
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When retinoic acid stimulates B Cells stimulates IL-5/IL-6 producing IgA, which uses _____ to cross the gut
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pIgR
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Effect of Vitamin D3 on myeloid cells
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Macrophage Differentiation, Inhibit Dendritic Cell differentiation
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Effect of D3 on lymphoid cells
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Increase T Cells differentiation, decrease T Helper Cell differentiation, Decrease plasma B cell and memory B cell differentiation
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Mineral that recruits transcription factors and is increased by complement
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Calcium
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-Mineral that is a cofactor for Ig synthesis
-Is increased during apoptosis and atrophy of thymus |
Magnesium
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mineral that activates proteases in phagocytic vacuole of neutrophils
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Potassium
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-Is found in over 300 enzymes
-Has a role in signal transduction and gene expression -Regulates apoptosis -Prevents normal release of vitamin A from liver -Decreases function of neutorophil, NK cell, complement, T and B lymphocytes...whatever... fuck |
Zinc
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If deficient of this mineral, need to eat twice as much food in order to attain the same weight gain
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Zinc
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Most common trace element involved in oxygen transportation and is used in the active site of some enzymes
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Iron
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-Mineral that is used to synthesize thyroid hormones
-Improves phagocytosis - increase movement of granulocytes into areas of inflammation |
Iodine
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Ways to combat malnutrition in the short term? Long term?
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-supplements, fortify food, distribute food/money
-Education and food security |
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Adding micronutrients to food
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Food fortification
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Live microorganisms in food that provide competitive inhibition, increase phagocytosis and regulate cytokine function
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Probiotics
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Non-digestible food ingredients that stimulate the growth and/or activity bacteria in the digestive system
-Dietary fiber |
Prebiotics
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The proportion of people with the disease who have a positive test (TP/TP+FN)
-Rarely miss people who have the disease (few false negatives) |
Sensitivity
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Screening assays should be _____
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very sensitive
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The proportion of people without the disease who have a negative test (TN/TN+FP)
-Rarely misclassify people without the diseases as infected (few false positives) |
Specificity
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Confirmatory assays should be -________
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very specific
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Probability of disease in a patient with a positive test result (TP/TP+FP)
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Predictive Value Positive
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probability of not having a disease when the test result is negative (TN/TN+FN)
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Predictive Value Negative
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