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Immunology > Lecture 17: Introduction to Diagnostic Immunology > Flashcards

Lecture 17: Introduction to Diagnostic Immunology Flashcards

1
Q

What does nuclei acid amplification tests like PCR detect

A

Infectious agents, neoplastic mutations, musuclar dystrophy cystic fibrosis

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2
Q

What is PCR cycle for nuclei acid amplification

A

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

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3
Q

What does antibody-antigen complexing (serology) detect

A

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

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4
Q

Serology

A

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
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5
Q

How do antibodies work to bind antigens

A

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

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6
Q

What forces are used in an antigen-antibody complex

A

Hydrogen bonds, van der waals, and electrostatic forces

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7
Q

What does antibody specificity depend on

A

Conformational qualities and various molecular interactions

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8
Q

How is antibody specificity refined

A

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

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9
Q

What is antibody affinity

A

Strength of binding between antibody recognition site and antigenic epitope

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10
Q

What is antibody avidity

A

Sum of all binding affinities between an antibody and antigen

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11
Q

What is the most often used antibody is diagnostic tests

A

IgG
(Some tests target IgM)

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12
Q

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

A

IgM (early) to IgG (later)

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13
Q

What are the levels of circulating antigen during secondary response

A

very little IgM and lots of IgG

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14
Q

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

A

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)

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15
Q

How are antibodies created from B cells

A

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

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16
Q

How are polyclonal antibodies formed

A

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

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17
Q

Are polyclonal antibodies sensitive or specific

A

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

18
Q

How are monoclonal antibodies made

A

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

19
Q

Are monoclonal antibodies sensitive or specific

A

Specific because they recognize a single/conserved epitope

20
Q

Compare and contrast polyclonal vs monoclonal antibodies

A

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

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

21
Q

What is an antigen

A

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

22
Q

What are some examples of antigens

A

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

23
Q

How do we “see” the antibody-antigen complexing

A

Conjugation

24
Q

What is horseradish perioxidase used for

A

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

25
Q

What is alkaline phosphatase used for

A

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

26
Q

Fluorescent molecules

A

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

27
Q

Is western blot qualitative, semi-quantitative or quantitative

A

Qualitative and semi-quantitative

28
Q

Is immunofluorescence qualitative, semi-quantitative or quantitative

A

Qualitative, semi-quantitative

29
Q

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

A

Semi-quantitative and quantitative

30
Q

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

A

Light absorbance measured directly using a spectrophotometer

31
Q

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

A

Virus neutralization

32
Q

Diagnostic tests often make use of _____ to identify disease states

A

Natural processes

33
Q

Antibodies must be ____ to allow for visualization

A

Conjugated to something

34
Q

How do we choose one test over another

A

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

35
Q

Sensitivity

A
  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)

36
Q

Specificity

A
  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)

37
Q

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

A

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

38
Q

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

A

Antibody binding specific epitope on specific antigen

39
Q

Equation for sensitivity

A

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

(A/a+c)

40
Q

Equation for specificity

A

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

= d/d+b

41
Q

How is an assays reported sensitivity and specificity calculated

A

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

Immunology (22 decks)

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