Diagnostics 5: Antibodies as Diagnostic Tools

Question 1

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

Answer 1

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)

Question 2

Antibodies in Diagnostics:

Answer 2

• 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)

Question 3

Source of Antibodies:

Answer 3

• 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

Question 4

Uses of Manufactured Antibodies:

Answer 4

• 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 2^nd 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 2^nd antibody on test line (control line further down where only 1^st antibodies bind, show sample has moved all way down)

Used for rapid diagnosis of e.g. infections

Question 5

Therapeutic Monoclonal Antibody Nomenclature:

Answer 5

• “-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

Question 6

Immunological Concerns – Tests done if suspect immunodeficiency:

Answer 6

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

Question 7

ART Treatment:

Answer 7

• 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

Question 8

Natural History of HIV Infection:

Answer 8

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