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Patho > Immuno 3 Immunoassays > Flashcards

Immuno 3 Immunoassays Flashcards

1
Q
  1. Specificty?
  2. Affinity?
  3. Function?
A

1-ability to descriminate between different molecules
2-monovalent - 10^13 is strong
-10^4-10^5 weak
-usually 10^7-10^10
multivalent have more epitopes so stronger binding too
3. Different antibody isotypes have different fucntions, mediated by the FC portion of the Ig

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2
Q

Most native antigens are?

A

multivalent, effects how they can be detected

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3
Q

Two types of Epitopes?

A
  • Linear

- Conformational/ Discontinous

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4
Q

Monoclonal Antibody Tech?

A
  • Spleen cells producing antibody from mouse immunized with antigen A
  • Myeloma cells lacking antibody secretion and the enzyme HGPRT
  • Mix and fuse cells with PEG
  • Select hybridoma that makes antibody specific for antigen A
  • Clone selected hybridoma
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5
Q

Detection of specific antibody

-Types?

A
  1. Antibody alone can be detected by precipiation or agglutination
  2. Enzymes can be linked to antibody - Horseradish peroxidase - Alkaline phosphatase
  3. Radioisotoeps can be linked to antibdoy
  4. Solid matrix can be linked to antibody
  5. Fluorophor can be linked to antibody
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6
Q

Affinity Purification of Protein Antigens

A
  1. Antibody to antigen A bound to beads?
  2. Add a mixture of molecules
  3. Wash away unbound molecules
  4. Elute specifically bound molecules
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7
Q

Immunoprecipitation

A
  1. Normal cells and radiolabel are added together
  2. Labelled cells are washed
  3. Cells lysed in detergent
  4. Antibodies on beads added
  5. Wash away everything else but beads
  6. SDS-page seperates proteins
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8
Q

Flow Cytometry

A

-cells labeled with one fluorphore can be sepeareted from cells with another fluorphore

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9
Q

Magnetic separtion

A
  1. Heterogenous population of lymphocytes are mixed with antibodies coupled to paramagnetic particles or beads
  2. Magnetic field is applied
    - coupled cells stick to the iron wool
    - unlabeled cells are washed out
  3. Magnetic field is removed releaseing the coupled cells
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10
Q

Protein samples

-what are they

A
  • Serum, Urine, ect
  • Whole cell extracts
  • Cellular fractions
  • chromatography
  • in vitro expressed proteins
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11
Q

Cellular samples

-what are they

A
  • Cell surface molecules

- Intracellular molecules

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12
Q

Immunofluorescence Microscopy

A
  • FITC-labeled antibody specific for GAD stains the B cells of the pancreatic islets
  • Rhodamine-labeled antibody specific for glucagon stains the alpha cells
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13
Q

Agglutination assays

A

-based on maximized matrix formation which occurs when [antibody] = [antigen]

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14
Q

Blood types

A

anti A- A and AB
anti B- B and AB
anti A and B - A, B, AB

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15
Q

Elisa

A
  1. Anti-A antibody covalent linked to enzyme
  2. Wash away unbound antibody
  3. Enzyme makes colored product from added colorless substrate
  4. Measure absorbance of light by colored product
  5. sensitize plate antigen
  6. wash
  7. add test antibody
  8. wash
  9. add ligand
  10. wash
  11. add chromgren
  12. develop plate
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16
Q

Western blotting

A
  1. SDS page
  2. Transfer to nitrocellulose and overlay with antiserum
  3. Detect bound antibody with enzyme-linked anti-IgG
  • detection of proteins in cell lysates
  • different types of epitotes require differnet detection antibodies
17
Q

Enhancing sensitivity/Flexibility

A
  • Do indirect rather than direct assays using secondary detection antibodies
  • Amplify the signal
  • set up a competitve inhibition Elisa
18
Q

Immunization with an antigen and the detection of antigen specifc antibodies

A
  • In Vivo measurement of immune repsonses
  • Skin tests
  • Protection from infectious organisms
19
Q

Commercial immunodiagnostic Kits

A
  • Enzyme immunoassay
    1. Low in toxicity
    2. high in sensitivity
    3. Easy to use with no Training
    4. Simpe and reliable
    5. home prego test
20
Q

Immunodiagnostic kits

  • advantages?
  • disadvantages?
A
  1. Advantages
    - high in sensitivty
    - low in toxicity
    - easy to use
    - simple and reliable
  2. Limitations
    - Cross-reactivity
    - Presence of inhibitors
    - Storage failure
    - Human error

Patho (5 decks)

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