Diagnostics 5: Antibodies as Diagnostic Tools Flashcards Preview

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Flashcards in Diagnostics 5: Antibodies as Diagnostic Tools Deck (8)
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1
Q

Attach things (reporter, drug) to antibody constant region –doesn’t affect antigen binding:

A
  1. Enzymes: e.g. peroxidase, alkaline phosphatase (produces blue from colourless substrate)
  2. Fluorescent probes: dyes, beads of different sizes
  3. Magnetic beads: e.g. purification/isolation of cell types – use magnet to pull away antibodies conjugated with beads (others washed away)
  4. Drugs: e.g. Kadcyla, an anti-HER2 antibody linked to emtansine (for breast cancer, HER2 overexpressed commonly)
2
Q

Antibodies in Diagnostics:

A
  • The unique specificity of antibodies for target antigens is basis of many diagnostic tests
  • Antibodies can be raised against almost any antigen (often, but not always proteins)
    • Including immunoglobulins from other species = anti-antibodies
  • Can perform indirect labelling using anti-antibody (diagram)
3
Q

Source of Antibodies:

A
  • Produced by patient:
    • In autoimmune disease
    • For defence against infection (in Billy’s case, used to diagnose his infection)
  • Manufactured antibodies
    • Antisera from immunised animals (polyclonal antiserum)
    • Monoclonal antibodies – many antibodies with the same specificity
    • “genetically engineered” antibodies = produce antibodies using recombinant DNA technology, no need of animal

Producing Monoclonal Antibodies:

  • Start with 2 different cell types à produce hybridoma
    • B cells produce desired antibodies for specific antigen, and have necessary enzyme
    • Fuse with myeloma cells, B cell tumours, which grow indefinitely and don’t produce own antibodies and doesn’t have particular enzyme
  • Hybridomas are immortal, have enzyme, and produce desired antibodies
  • Use certain medium to selectively grow hybridoma cells with the enzyme
  • Clone by limiting dilution: put single cells in wells à screen clones for those that produce antibody

Antibody Production using recombinant DNA tech:

  • Certain phage will bind specific antigen à wash others away
  • Clone DNA segments that code for the variable region attached to that phage à put onto whole antibody again, specific to given antigen
4
Q

Uses of Manufactured Antibodies:

A
  • Therapeutic
    1. Prophylactic protection against microbial infection e.g. IV-IG (for patients that can’t make own antibodies), synagis (anti-RSV)
    2. Anti-cancer therapy e.g. anti-HER2 (breast cancer)
    3. Removal of T-cells from bone marrow grafts e.g. anti-CD3
    4. Block cytokine activity e.g. anti-TNF-α antibody e.g. in rheumatoid arthritis
  • Diagnostic:
  1. Determine blood groups
  2. Immunoassays to measure hormones, antibodies, antigens
  3. Immunodiagnosis
    1. Infectious diseases
    2. Autoimmunity
    3. Allergy (look for IgE against agent e.g. penicillin allergy)
    4. Malignancy (myeloma)
  • ELISA = enzyme-linked immunosorbent assay
    • Antigen (or antibody) immobilised in well à antibody-reporter added à wash away ones that don’t bind à add substrate à strength of colour indicates amount of antigen
    • (Sandwich ELISA’s: immobilise antibody, add antigen, then 2nd antibody with reporter à double bind antigen)
  • Rapid Testing: use strip containing antibodies (conjugated to gold nanoparticles)
    • Add sample/antigens à move down by capillary flow à antibody binds antigen à complex move down and binds 2nd antibody on test line (control line further down where only 1st antibodies bind, show sample has moved all way down)

Used for rapid diagnosis of e.g. infections

5
Q

Therapeutic Monoclonal Antibody Nomenclature:

A
  • “-omab” = mouse monoclonal e.g. Muronomab = anti-CD3, transplant immunosuppression
  • “-imab” = chimeric or partly humanised e.g. Infliximab (Remicade) = anti-TNFa, Rituximab = anti-CD20
  • “-umab” = fully human antibody e.g. Palivizumab, anti-RSV (Synagis)
    • But patient may still have immune response to fully human antibody
6
Q

Immunological Concerns – Tests done if suspect immunodeficiency:

A
  1. Serum Immunoglobulin levels (MAG)
    1. IgG (all sub-types), IgM, IgA
    2. (Serum electrophoresis/ELISA/Nephelometry) – measure levels
      1. In serum electrophoresis, if large band in gamma region à active immune response à not immunodeficient
      2. If sharp band à shows monoclonal B cell expansion (not immunodeficient), possible B cell malignancy à investigate for myeloma
  2. Test for Specific Antibodies (ELISA) e.g. after given vaccine, should see antibodies
    1. If immunodeficient, check for protein antigens (Tetanus & Haemophilus)
    2. Or polysaccharides antigens (Pneumococcus)
  3. Lymphocyte subsets quantified by Flow Cytometry detects fluorescence emitted from antibody-reporter when bound to marker, use antibodies against markers:
  4. CD3+ found on all T cells
  5. CD4+ = T helper cells
  6. CD8+ = cytotoxic T cells
  7. CD19+ = B cells
  8. CD56+ = NK Cells
7
Q

ART Treatment:

A
  • HIV patients get CD4 count + viral load measured, before + after receiving ART
  • CD4 T cell count defines extent of immune damage à predicts short-term outlook in HIV-1 patients w/o ART à more susceptible to infections e.g. opportunistic infections or Kaposi’s sarcoma
  • Billy’s diagnosis:
    • HIV antibody test positive
    • Monitoring
      • CD4 lymphocyte count = 64 cells per ul
      • HIV viral load = 155,000 copies per ml
8
Q

Natural History of HIV Infection:

A
  • CD4 T cell count eventually goes down and down during latent period