Lecture 17: Introduction to Diagnostic Immunology

Question 1

What does nuclei acid amplification tests like PCR detect

Answer 1

Infectious agents, neoplastic mutations, musuclar dystrophy cystic fibrosis

Question 2

What is PCR cycle for nuclei acid amplification

Answer 2

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

Question 3

What does antibody-antigen complexing (serology) detect

Answer 3

Infectious agents, histocompatibility/blood typing, auto-immune disorders

Question 4

Serology

Answer 4

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

Question 5

How do antibodies work to bind antigens

Answer 5

Antibody specificity- conformational qualities, molecular interactions, antibody affinity, antibody avidity

Question 6

What forces are used in an antigen-antibody complex

Answer 6

Hydrogen bonds, van der waals, and electrostatic forces

Question 7

What does antibody specificity depend on

Answer 7

Conformational qualities and various molecular interactions

Question 8

How is antibody specificity refined

Answer 8

Clonal selection (antigens activating lymphocytes already programmed to act against specific antigen)

Question 9

What is antibody affinity

Answer 9

Strength of binding between antibody recognition site and antigenic epitope

Question 10

What is antibody avidity

Answer 10

Sum of all binding affinities between an antibody and antigen

Question 11

What is the most often used antibody is diagnostic tests

Answer 11

IgG
(Some tests target IgM)

Question 12

What is the main antibody responding/circulating during early stages of primary response and then what does it switch to

Answer 12

IgM (early) to IgG (later)

Question 13

What are the levels of circulating antigen during secondary response

Answer 13

very little IgM and lots of IgG

Question 14

When might an antibody test give bad information (false negative and false positives)

Answer 14

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)

Question 15

How are antibodies created from B cells

Answer 15

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

Question 16

How are polyclonal antibodies formed

Answer 16

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

Question 17

Are polyclonal antibodies sensitive or specific

Answer 17

Very sensitive because there are many of them recognizing a range of epitopes

Question 18

How are monoclonal antibodies made

Answer 18

Inject animal with virus, antigen binds B cell, create plasma B cells and specific cells (ex: immortalized tumor cells) and then create a hybridoma

Question 19

Are monoclonal antibodies sensitive or specific

Answer 19

Specific because they recognize a single/conserved epitope

Question 20

Compare and contrast polyclonal vs monoclonal antibodies

Answer 20

Polyclonal
- relatively inexpensive
- easier to develop
- multiple epitopes
-very sensitive

Monoclonal
- relatively expensive
- easy to mass produce
- single epitope
- very specific

Question 21

What is an antigen

Answer 21

Anything capable of eliciting an immune response or otherwise being recognized by antibodies

Question 22

What are some examples of antigens

Answer 22

Non-host proteins, host proteins, antibodies, nuclei acids, hormones and other biochemicals

Question 23

How do we “see” the antibody-antigen complexing

Answer 23

Conjugation

Question 24

What is horseradish perioxidase used for

Answer 24

Enzyme used to conjugate to antigen binding antibody to make visible on western blot

Question 25

What is alkaline phosphatase used for

Answer 25

Enzyme used to bind antigen binding to antibody to make antigen appear red in immunohistochemistry

Question 26

Fluorescent molecules

Answer 26

Used as a conjugate to antigen binding to antibody to detect via immunofluorescence

Question 27

Is western blot qualitative, semi-quantitative or quantitative

Answer 27

Qualitative and semi-quantitative

Question 28

Is immunofluorescence qualitative, semi-quantitative or quantitative

Answer 28

Qualitative, semi-quantitative

Question 29

Is enzyme-linked immunosorbent assay (ELISA) qualitative, semi-quantitative or quantitative

Answer 29

Semi-quantitative and quantitative

Question 30

How does enzyme linked immunosorbent assay (ELISA) measure antibody-antigen complex

Answer 30

Light absorbance measured directly using a spectrophotometer

Question 31

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

Answer 31

Virus neutralization

Question 32

Diagnostic tests often make use of _____ to identify disease states

Answer 32

Natural processes

Question 33

Antibodies must be ____ to allow for visualization

Answer 33

Conjugated to something

Question 34

How do we choose one test over another

Answer 34

Disease stage, cost, reported sensitivity, specificity, and turnaround time

Question 35

Sensitivity

Answer 35

1. 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)

Question 36

Specificity

Answer 36

1. Detection of “X” in the midst of A-Z 2. Ability to correctly identify animals without a specific disease (% truly - so if + can rule in)

Question 37

What method of antibody-antigen complexing is a good example of analytical sensitivity

Answer 37

Virus neutralization via serum dilutions because detects increasingly small concentrations of X

Question 38

What is a method of anti-body antigen complexing that is a good example of analytical specificity

Answer 38

Antibody binding specific epitope on specific antigen

Question 39

Equation for sensitivity

Answer 39

= (true positives)/ (true positives + false negatives) (A/a+c)

Question 40

Equation for specificity

Answer 40

= (true negatives)/(true negatives + false positives) = d/d+b

Question 41

How is an assays reported sensitivity and specificity calculated

Answer 41

Using blind samples with a known diagnosis to determine true positive, false positives, true negative and false negatives