Lecture 17: Introduction to Diagnostic Immunology Flashcards
What does nuclei acid amplification tests like PCR detect
Infectious agents, neoplastic mutations, musuclar dystrophy cystic fibrosis
What is PCR cycle for nuclei acid amplification
Start with components of DNA, DNA primer, and nucleotide
Denature protein (94-98 *C)
Annealing (add DNA primer) at 50-68 *C
Elongation (add nucleotides) at 72 *C
What does antibody-antigen complexing (serology) detect
Infectious agents, histocompatibility/blood typing, auto-immune disorders
Serology
Used to describe a variety of antibody-antigen dependent diagnostic tests
- study of molecules in blood/serum
- utilization of serum to detect antibodies, antigen or other blood components to characterize a disease state
How do antibodies work to bind antigens
Antibody specificity- conformational qualities, molecular interactions, antibody affinity, antibody avidity
What forces are used in an antigen-antibody complex
Hydrogen bonds, van der waals, and electrostatic forces
What does antibody specificity depend on
Conformational qualities and various molecular interactions
How is antibody specificity refined
Clonal selection (antigens activating lymphocytes already programmed to act against specific antigen)
What is antibody affinity
Strength of binding between antibody recognition site and antigenic epitope
What is antibody avidity
Sum of all binding affinities between an antibody and antigen
What is the most often used antibody is diagnostic tests
IgG
(Some tests target IgM)
What is the main antibody responding/circulating during early stages of primary response and then what does it switch to
IgM (early) to IgG (later)
What are the levels of circulating antigen during secondary response
very little IgM and lots of IgG
When might an antibody test give bad information (false negative and false positives)
Early in primary antigen exposure can get false negative before antibodies start circulating
Late/after secondary response can get false positive results due to drawn out circulating IgG (or other antibodies)
How are antibodies created from B cells
B cell binds to antigen, receives chemical signal from Helper T cell for the B cell to become a plasma cell and then release antibodies
How are polyclonal antibodies formed
Inject virus into animal, bind to B cells, create memory and plasma B cells and secrete antibodies. Then redraw blood and spin down to get serum
Are polyclonal antibodies sensitive or specific
Very sensitive because there are many of them recognizing a range of epitopes
How are monoclonal antibodies made
Inject animal with virus, antigen binds B cell, create plasma B cells and specific cells (ex: immortalized tumor cells) and then create a hybridoma
Are monoclonal antibodies sensitive or specific
Specific because they recognize a single/conserved epitope
Compare and contrast polyclonal vs monoclonal antibodies
Polyclonal
- relatively inexpensive
- easier to develop
- multiple epitopes
-very sensitive
Monoclonal
- relatively expensive
- easy to mass produce
- single epitope
- very specific
What is an antigen
Anything capable of eliciting an immune response or otherwise being recognized by antibodies
What are some examples of antigens
Non-host proteins, host proteins, antibodies, nuclei acids, hormones and other biochemicals
How do we “see” the antibody-antigen complexing
Conjugation
What is horseradish perioxidase used for
Enzyme used to conjugate to antigen binding antibody to make visible on western blot
What is alkaline phosphatase used for
Enzyme used to bind antigen binding to antibody to make antigen appear red in immunohistochemistry
Fluorescent molecules
Used as a conjugate to antigen binding to antibody to detect via immunofluorescence
Is western blot qualitative, semi-quantitative or quantitative
Qualitative and semi-quantitative
Is immunofluorescence qualitative, semi-quantitative or quantitative
Qualitative, semi-quantitative
Is enzyme-linked immunosorbent assay (ELISA) qualitative, semi-quantitative or quantitative
Semi-quantitative and quantitative
How does enzyme linked immunosorbent assay (ELISA) measure antibody-antigen complex
Light absorbance measured directly using a spectrophotometer
What method of antibody-antigen performs sample (ex: serum) dilutions to determine the lowest level necessary for an affect (preventing cell death)
Measures cytoplasmic effects to determine normal vs virus infected cells
Virus neutralization
Diagnostic tests often make use of _____ to identify disease states
Natural processes
Antibodies must be ____ to allow for visualization
Conjugated to something
How do we choose one test over another
Disease stage, cost, reported sensitivity, specificity, and turnaround time
Sensitivity
- Detection of increasingly small concentrations of “X”
2.ability to correctly identify animals with a specific disease
(% truly + so if test is negative then can rule out)
Specificity
- Detection of “X” in the midst of A-Z
- Ability to correctly identify animals without a specific disease
(% truly - so if + can rule in)
What method of antibody-antigen complexing is a good example of analytical sensitivity
Virus neutralization via serum dilutions because detects increasingly small concentrations of X
What is a method of anti-body antigen complexing that is a good example of analytical specificity
Antibody binding specific epitope on specific antigen
Equation for sensitivity
= (true positives)/ (true positives + false negatives)
(A/a+c)
Equation for specificity
= (true negatives)/(true negatives + false positives)
= d/d+b
How is an assays reported sensitivity and specificity calculated
Using blind samples with a known diagnosis to determine true positive, false positives, true negative and false negatives
