Antibodies as Diagnostic Tools Flashcards
How can Abs be used as diagnostic tools?
Abs can be raised against almost ANY antigen!
The Fc-region is CONSTANT
o this is where you attach molecules (REPORTERS/DRUGS)
o it does NOT affect the specificity of the Ab
What types of Reporters/Drugs can you attach to the Ab?
o Enzymes
e. g. peroxidase, ALP
- Ags washed over Abs w. enzymes
- colourless substrate then added which turns colour
o Fluorescent probes
e.g. dyes, beads to different sizes
o Magentic beads
e. g. purification of cell types
- magnet-Ab attaches to receptor
- then runs sample over a magnet
- only linked cells bind
o Drugs
e.g. Kadcula - anti-HER2 linked to emtansine
What is an anti-antibody?
Used to detect existing Abs in the body
2 ways Abs can be produced?
- Produced by PATIENT
o for autoimmune disease
o for defence against infection
- Produced ARTIFICIALLY
o ANTISERA from immunised animals (polyclonal so multiple specifities BUT limited amount)
o MONOCLONAL Abs
o GENETICALLY ENGINEERED Abs
How are monoclonal Abs generated?
- Take normal B-lymphocytes from spleen
- producing Ab of INTEREST
- have LIMITED cell division - FUSE it with myeloma cells
- these cells do NOT produce own Ab - This gives you HYBRIDOMA
- these can produce Ab on interest indefinitely (as fused w. tumour cell)
How can you produce Abs using recombinant DNA technology?
- Isolate all V-segments of the needed Abs
- Display all V-segments
- on protein
- OR on bacteriophage so each one display different specificity V segments - Use the phage-display library to screen plates for Ags
- complimentary phage will STICK to plate
- other phages will WASH off
2 broad uses of Manufactured Abs?
Therapeutic
&
Diagnostic
Therapeutic uses of manufactured Abs?
- Prophylactic - against microbial infection
e. g. Synagic (anti-RSV) - Anti-cancer therapy
e. g. anti-HER2 - Removal of T-cells from bone marrow graft (avoid attacking own cells)
e. g. anti-CD3 - Block cytokine activity
e. g. anti-TNFalpha
Diagnostic uses of manufactured Abs?
- Blood group serology
- Quantitative immunoassays
e. g. hormones, Abs, Ags - Immunodiagnosis
e. g. infectious disease, autoimmunity, Allergy (IgE), Malignancy (myeloma)
ELISA?
Enzyme Linked Immuno-Sorbent Assay
How does ELISA work?
- Two samples are used to coat the walls
- Anti-A Ab is covalently linked to a reporter (enzyme)
- When it binds, colourless substrate is added
- If Ab is still present, it turns it into a colour in the well after washing
- Absorbance of the light can then be measured
Lateral Flow Assay?
LITERALLY the hCG protein in pregnancy test kits!!
How can you diagnose HIV?
HIV-Ab test
When may you have anti-HIV Abs but NOT have HIV?
o Maternal Abs
o Volunteers in clinical trial
What can cause ‘vague aches and pains’?
Immune complexes!
- Inflammation / complement activation
- Serum sickness (I.Cs in circulation)
- I.C glomerulonephritis
- I.C deposits at other sites (skin, joints, lungs)
