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123 Cards in this Set

  • Front
  • Back
The two proteins that IgG binds to that IgM does not
Staph protein A and Strep protein G
Polyclonal antiserum is found in ____, where antigens have multiple _____ on their surface.
nature, epitopes
Monoclonal antibodies are found in the _____. Monoclonal antibodies react with only one _____. This is known as _______.
When an anibody produced in response to one antigen also reacts with another, different antigen, this is known as ______
cross reactivity
-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
To create clumping in a test, need ______ amount of ______ and _____. Is this reaction reversible?
equal, antibody, antigen

Yes, it is reversible
Why are controls used for immunoassays?
-Make sure protocol is working correctly
-Serum factors may interfere
What are the serum factors that may interfere with immunoassays?
-Rheumatoid factor (autoanibody)
-Cross reacting antibody
-Antibodies to tissue in which antigen was made
-Non-specific factors (lipids, fibrin)
Serological assay that clumps particulate matter, such as cells or synthetic material (latex, charcoal)
Agglutination Assay
Assay that prevents agglutination in the presence of antibody to agglutinin antigen
Inhibition assay
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?
Allgutination (clumping occurs)

Uniform dispersal of particles in solution (no clumping)
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
Allugitination Assay where cells or particles are coated with antigen or antibody to cause clumping
Indirect Agglutination Assay (passive)
What is used to attach antigens to cells in an agglutination assay?
tannic acid
chromic chloride
Streptococcus pneumoniae & Neisseria meningitidis, Rubacell, Rubascan, Syphilis, Cryptococcus, and Rickettsia are all what types of Agglutination Assays?
Indirect Passive Agglutintion Assays
Agglutination with RBC as indicator
Receptors on RBC surface bind to antigens, such as in ABO blood typing.
Direct hemagglutination
If RBCs fall to the bottom of the well ___________
no agglutination has occured
Anti-globulic assay test which detects antibody on one's own Red Blood Cells, leading to agglutination of cells by anti-human immunoglobulin
Coombs Test
Agglutination of cells via the Coombs test means a person is positive for:
hemolytic disease - autoimmune antibody or antibody from incompatible RBC transfusion ---> anemia
In agglutination Assays, to detect recent infection to specific agents:
4 fold change (rise) in titer between acute & convalescent sera when tested in the SAME run
Acute serum is collected :
Convalescent serum is collected:
at onset to 10 days post onset
10-28 days after acute
Inhibitiion of RBC agglutination by virus indicated the presence of _____
specific anti-viral antibody
In a Hemagglutinin inhibition assay, if agglutination of RBCs occurs, then _________. If no agglutination occurs, ______ are present, and the RBC in the well, ________.
-non-specific antibodies are present
- specific antibodies are present
-RBCs fall to bottom of the well
Non-treponmal flocculation card test that is an agent of syphillis
Rapid Plasma Reagin Assay
***False positives for other disease
***False negatives - prozone
2 Disadvantages of Rapid Plasma Reagin Assay
False positives from other diseases and false negatives from the prozone
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
Advantages of Complement Fixation Test
- Same reagents to detect and identify antigen
- One protocol
- Not species specific
- more specific than HAI
Disadvantages of Complement Fixation Test
- less sensitive than ELISA, HAI
- Technically demanding
- Interference by lipids
- Limited quality reagents available
What are the two steps of the Complement fixation test?
1. Addition a known and unknown (antigen and serum) in the presence of complement
2. Addition hemolysin to sheep red blood cells
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?
No Lysis --- All complement bound by original complex
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?
Lysis of RBC due to unbound/free complement
Advantages of Immunofluorescence
-quck and cheap
-one protocol for many agents
-detect tissue and cellular antigens
-multiplex - flurochromes detect >1 antigen
Disadvantages of immunofluorescence
-antibody often neither sensitive nor specific
-requires conjugate antibodies
-need fluorescent microscope
Uses fluorescent labeled monocolonal antibodies to identify specific markers on cells and sort them by lineage and type
Cell Sorter
Sorts and separates cell types using a laser
Flow Cytometry / FACS (fluorescent activated cell sorter)
Immunofluorescent assay where a cell with membrane antigen binds directly to fluorochrome-tagged antibody
Direct Immunofluorescence
immunofluorescent assay that uses to two antibodies to detect antigen or antibody in serum
Indirect Immunofluorescence
Two antibodies used in Indirect Immunofluorescence to detect or identify antigen?
- primary - IgG made in animal to antigen
- secondary: anti-animal IgG conugated to fluorochrome
**Test is reliable, sensitive, and specific
Two antibodies used in Indirect Immunofluorescence to detect antibody?
-Primary: test serum (human)
-Secondary: anti-human IgG conjugated to fluorochrome
**Low sensitivity, specificy, false positives and negatives
In indirect immunofluorescence, ____ in serum and _______ can interfere with detection of IgM
IgG & rheumatoid factor
An immunofluorescence method for detecting antigen using an antibody attached to magnetic bead and conjugated with fluorescence. Read under an FE microscope
Immunomagnetic Separation
ELISA Advantages
-Sensitive, Specific, Reliable
-Low cost
-small volume of sample
-can use serum, CSF, stool
ELISA Disadvantages
-Need plate washer and reader
-Species specific antisera
-Many protocols with standardized reagent
ELISA Assay:
-Coated with antigen
-Addition of primary antibody
Addition of secondary antibody conjugate
-Addition of enzyme substrate
Indirect ELISA
ELISA assay:
-Coated with capture antibody
-Addition of antigen
-Addition of secondary conjugate antibody
Addition of enzyme substrate for color
Sandwich ELISA
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
-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
-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
In an ELISA protocol, this must be added so that antibody or antigen do not bind to open binding sites
Blocking Reagent
Reading enzymes in a spectrophotometer determines ________.
Optical Density
Fluorescence is read in a _____________.
This amplifies the signal in ELISA
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 __________
>2x the P/N of the negative control serum & >2x the P/N of the control antigen
What is the P/N ratio formula?
Optical Density of Test serum + Antigen / Optical Density of negative control +antigen
Assay that enumerates cells secreting antibody to specific antigen
-Uses agar overlay that shows dots representing a cell that has secreted the correct antibody
-Using similar protocols to ELISA, this test merges ELISA & Flow cytometry, but uses Luminex beads instead of plates.
Microsphere-based Immunoassay
Coupled to sepharose matrix, this bacterial protein binds IgG only, NOT IgM
Protein G
-Measures loss of infectivity or toxicity after interaction with specific antibody
-identifies etiological agents
-detects/quantifies microbial toxins
-detects specific antibodies
Neutralization Test
Advantages of the Neutralization Test
-most specific and very sensitive
-indicate presence of protecting antibody
Disadvantages of the Neutralization Test
-requires LIVE infectious agents
-requires cell culture/animal host
-high level staffing
In neutralization, the concentration of agent that kills of 50% of test animals
50% of tissue culture of infective dose
reciprocal of highest dilution of serum protecting against agent
Virus LD50 in negative serum - Virus LD50 in presence of test serum
-Significant >1.7
Neutralization Index
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.....
neutralizing antibodies
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 ________
virus or antibody sera that are low titered or diluted
-specific neutralizing antibodies to that virus
When a neutralizing test uses constant virus and constant serum, it is trying to determine
-what the virus is by identifying what serum neutralizes it
Rate of a passive diffusion gel assay affected by:
-molecular size
-viscosity and hydration of gel
-matrix interactions
Passive diffusion gel assay where a precipitation line forms where the antigen and antibody meet while diffusing through the gel
Ouchterlay double diffusion
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)
Radial immunodiffusion
The addition of electric current to a gel-based assay increases ______ and _______
speed and sensitivity
Immuno-double diffusion + electric current
Countercurrent electrophoresis (CIEP)
Single radial immunodiffusion + electric current =
Rocket electrophoresis
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
Western Blot
Why Serological Testing??
-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
-Retrospective studies
-Chemical elements required by living organisms other than carbon, hydrogen, nitrogen, and oxygen
Minerals that have a catalytic role in enzymes
Minerals that provide structure and electrolytes
How many vitamins are there and which one is NOT essential?
Vitamin D (synthesized by skin)
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)
Insufficient, excessive, or imbalanced consumption of nutrients
-Primary cause of immunodeficiency worldwide
Macronutrients include
-Carbs, Fats, Protein, Water
Micronutrients include:
Minerals and vitamins
Regulate mineral metabolism, cell tissue growth and differentiation, are antioxidants, and are precursors for enzyme cofactors
The Vicious Cycle of Malnutrition and Infection
-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
-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
Protects neutrophils from apoptic death
-Protein that regulates energy intake and expenditure (appetite and metabolism)
-Circulates at levels proportional to body fat
-Obese may be resistant
-Regulates inflammatory response
Cytokine responsible for
-Macrophage Activation
-Natural Killer cytotoxicity
-Th1 stimulation
-Th2 inhibition
-Dendritic cell activation
Two types of protein-energy malnutrition
Kwashiorkor and Marasmus
Reasons for malnutrition in Kwashiorkor and Marasmus
K: Diet high in starch and low in protein
M: Deficiency in both carbohydrate and protein nutrition
Differences between Kwashiorkor and Marasmus?
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
Immunogenic problems from Protein-Energy Malnutrition
-Atrophy of thymus
-Reduced phagocytosis
-Th cell proliferation halted
-Reduction in IgA, lysozyme, complement
Needed by the retina in the form of retinal for low light and color vision
-Storage form is ______
Vitamin A
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
Vitamin involved in macronutrient metabolism
Vitamin B
Vitamin that stimulates chemotaxis and phagocytosis (primarily neutrophils)
Vitamin C
-Antioxidant vitamin where deficiency may result in immunosenescence
Vitamin E
Gradual deterioration of the immune system with increasing age
-Most prominent in T Cells
In immunosenescence, increasing the level of vitamin E enhances cell dividing and interleukin production in ________, but not ________
naive T-cells, but not memory cells
Vitamin that inhibits inflammatory response
Vitamin K
Vitamin that regulates the concentration of calcium and phosphate in bloodstream and is involved in regulation of proliferation, differentiation and apoptosis of cells
Vitamin D
When retinoic acid stimulates B Cells stimulates IL-5/IL-6 producing IgA, which uses _____ to cross the gut
Effect of Vitamin D3 on myeloid cells
Macrophage Differentiation, Inhibit Dendritic Cell differentiation
Effect of D3 on lymphoid cells
Increase T Cells differentiation, decrease T Helper Cell differentiation, Decrease plasma B cell and memory B cell differentiation
Mineral that recruits transcription factors and is increased by complement
-Mineral that is a cofactor for Ig synthesis
-Is increased during apoptosis and atrophy of thymus
mineral that activates proteases in phagocytic vacuole of neutrophils
-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
If deficient of this mineral, need to eat twice as much food in order to attain the same weight gain
Most common trace element involved in oxygen transportation and is used in the active site of some enzymes
-Mineral that is used to synthesize thyroid hormones
-Improves phagocytosis
- increase movement of granulocytes into areas of inflammation
Ways to combat malnutrition in the short term? Long term?
-supplements, fortify food, distribute food/money
-Education and food security
Adding micronutrients to food
Food fortification
Live microorganisms in food that provide competitive inhibition, increase phagocytosis and regulate cytokine function
Non-digestible food ingredients that stimulate the growth and/or activity bacteria in the digestive system
-Dietary fiber
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)
Screening assays should be _____
very sensitive
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)
Confirmatory assays should be -________
very specific
Probability of disease in a patient with a positive test result (TP/TP+FP)
Predictive Value Positive
probability of not having a disease when the test result is negative (TN/TN+FN)
Predictive Value Negative