Antibodies as diagnostic tools Flashcards
(37 cards)
Describe the immunology department
88 tests listed
57 of these detect specific antibodies
Describe the structure of antibodies
Fc: constant part of antigens don’t change so can attach other groups without affecting ability to bind to antigen
Fab: is variable and produces specificity to antigen
What can be added to the Fc region of the antibody
antibodies can be raised against almost ANY antigen.
The Fc-region is constant – this is where you attach molecules (reporters) and you don’t affect the specificity of the antibody.
Reporters or drugs:
o Enzymes – e.g. peroxidase, alkaline phosphatase.
Antigens washed over antibodies with enzyme, colourless substrate is then added which turns colour.
o Fluorescent probes – e.g. dyes and beads of various size.
o Magnetic beads – e.g. purification of cell types.
Magnet-antibody attaches to receptor and then run sample over a magnet and only linked-cells bind.
o Drugs – e.g. Kadcyla, anti-HER2 linked to emtansine.
What colour does peroxidase stain
Brown
What is the purpose of using fluorescent probes of different sizes
Fluorecent probes- muitiplexing- different sizes
screen for different antibodies
Summarise the use of antibodies in diagnostics
The unique specificity of antibodies for their target antigens is the 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
Antibodies can be made against DNA or phospholipid
Describe indirect labelling using an anti anti-body
Use of an anti-antibody to detect existing antibodies in the body.
Antibodies:
o Produced by patient – for autoimmune disease OR defence against infection.
o Produced artificially – antisera from immunised animals (polyclonal – have many specificities), monoclonal antibodies and genetically engineered antibodies.
Secondary Ab with reporter molecule attached
secondary Ab bound to primary Ab- which is bound to antigen
Single secondary Ab tod etect many bound primary antibodies
Where do the antibodies come from
Produced by the patient
in autoimmune disease
for defence against infection
and in Billy’s case, used to diagnose his infection
Manufactured antibodies
antisera from immunised animals (polyclonal)
monoclonal antibodies
“genetically engineered” antibodies
Describe the use of genetic engineering
G.E- DNA sequence to make libraries of antibodies
Describe antisera
Natural- polyclonal – anti-serum with a mix of different antibodies – tetanus to immunize horses- anti-sera to treat patients with tetanus- but only a limited supply of antibody- can’t be used for vast majorirty of diagnostic tests- use mAbs- can make a large amount and of single specificity
Describe the process of generating monoclonal antibodies
Challenge mouse with antigen you want to make antibody against
Harvest B cells from mice which produce Ab
Fuse with immortal myeloma cells
This forms a hybridoma
Culture in HAT medium (ultra-medium which only allows hybridomas to grow)
Select for positive cell
Clone by limiting dilution
Limiting dilution- one cell in each well –let expand- screen supernatants for antibodies
Harvest monoclonal antibodies
Grow antibody infedinitley
Describe the production of antibodies using recombinant DNA technology
Population of genes encoding Ab variable regions isolated
Variable region gene fused with bacteriophage coat protein gene
Random population of variable region bacteriophages are cloned - a phage display library
Phages that bind to specific antigen are selected
Phage display library- expressed on surface
Wash overs other- multiply cycles ot enrich phage
Can be expanded massively by growing in bacteria
Describe the therapeutic use of manufactured antibodies
Therapeutic
Prophylactic protection against microbial infection -e.g. IVIG, synagis (anti-RSV)- IVIG- polyclonal- purified from many different patients
RSV- babies at risk
Anti-cancer therapy- anti-HER2
Removal of T-cells from bone marrow grafts - ANTI-cd3
Block cytokine activity - anti-TNFa- used in rheumatoid arthritis
Anti-calcitonin gene-related peptide (CGRP) for migraine
recently against myeloid protein
What is the issue with the therapeutic use of mAbs
Very expensive- volatile and need rigorous quality control
Name the therapeutic monoclonal antibodies
Therapeutic:
o Prophylactic against microbial infection – e.g. Synagis (anti-RSV).
Suffix “-umab” – human – e.g. Synagis (anti-RSV).
o Anti-cancer therapy – e.g. anti-HER2.
o Removal of T-cells (bone marrow transplant) – e.g. anti-CD3.
Suffix “-omab” – mouse monoclonal – e.g. anti-CD3.
o Block cytokine activity – e.g. anti-TNF-alpha.
Suffix “imab” – chimeric/partly humanised – e.g. anti-TNF-alpha
Describe the diagnostic use of monoclonal antibodies
Diagnostic Blood group serology Quantitative immunoassays hormones antibodies antigens Immunodiagnosis Infectious diseases Autoimmunity Allergy (IgE) Malignancy (myeloma)
Describe the process of ELISA in HIV
Partially purified, inactivated HIV antigens pre-coated onto an ELISA plate
Patient serum which contains antibodies. If the patient is HIV+, then this serum will contain antibodies to HIV, and those antibodies will bind to the HIV antigens on the plate.
Anti-human immunoglobulin coupled to an enzyme. This is the second antibody, and it binds to human antibodies.
Chromogen or substrate which changes color when cleaved by the enzyme attached to the second antibody.
Describe the general process of ELISA
ELISA: Enzyme Linked ImmunoSorbant Assay
Capture antibody fixed to plate
Test sample containing target antigen added
Target antigen binds to capture antibody and excess washed away
Detection antibody with enzyme on Fc added to dish
Detection antibody binds to target antigen and excess washed
Clear solution added and enzyme on Fc converts to coloured
Binds to different place to first antibody
Signal proportional to amount of antigen in sample-quantitative
Inflamamtory markers in patients
Describe some potential errors in ELISA
It is entirely possible that an individual not infected with HIV has antibodies which may give a positive result in the HIV ELISA. This is called a false positive. One reason for this is that people (especially women who have had multiple pregnancies) may possess antibodies directed against human leukocyte antigens (HLA) which are present on the host cells used to propagate HIV. As HIV buds from the surface of the host cell, it incorporates some of the host cell HLA into its envelope. False negatives can occur during the window between infection and an antibody response to the virus (seroconversion).
Describe rapid testing
Test sample added to sample pad, and runs over antibodies conjugated to gold nanoparticles
Ag-Ab complexes run along pad, binding to test line if positive and control line in all cases to ensure validity
How can an individual have anti-HIV antibodies without being infected with HIV
Mother hIV psotivie- IgG antibodies will cross placenta to fetus
To diagnose HIV, you can have an HIV-antibody test. You may have anti-HIV antibodies but NOT HIV if:
o Maternal antibodies.
o Volunteers in clinical trials.
Describe Billy’s symptoms
Signs & Symptoms
Vague aches and pains
Loss of appetite
Weight loss
“Glands” up in his neck
Fever, rash, small red patches, some lumpy
Describe the immunological concerns we have for Billy
Immune complexes
Effect of poor nutrition on bone marrow cells
Immune activation
Acute phase, activation, immune complexes
What is causing Billy’s symptoms
Signs and symptoms – Immunological concerns Immune complexes Inflammation / complement activation Serum sickness (immune complexes in circulation) Immune complex glomerulonephritis Immune complex deposition at other sites Skin Joints Lungs
