molecular probes Flashcards
define molecular probes
- molecules whcih bind very specifically to other “target” molecules
- used to track, locate, purify and siable
- natural and synthetic antibodies, Carbohydrate binding modules (CAB),
discuss antibodies and describe their limitations
- a protein from the immune system which binds very specifically to antigens (something which elicits an immune response)
- binding to antigens involved the Fab region
- binding occurs at the antigens epitope (3-8AAs long)
- monoclonal (identical antibodies which bind a single epitope) and polyclonal antibodies exist (different antibodies which bind the same antigen)
- polyclonal antibodies are cheaper and easier to make and have higher overall binding strength due to binding to multiple sites but lack specificity and bind to multiple epitopes.
- polyclonal antibodies are made by injecting the target molecule into an animal and extracting and purifying the blood; once the anitbodies are used the same antibodies be available, only slightly different ones
- monoclonal antibodies are made by immunising the animal and then isolating the B cells from the spleen which are fused with cancerous myelonia cells so they can be kept alive. A single “hybridoma” cell can be grown and replicated, all of which willl secrete identical antibodies, whcih can be frozen and used forever
- use of animals has ethical and economic considerations
- targte molecule must be immunogenic (some arent)
- toxic target molecules cant be used for immunization
- some antibodies are recognised as foreign so are removed by immune system
- timely to get antibodies from animals
solution
1) bypassing the immune system and transferring the cellular machinery for antibody production to a phages (viruses) like M13 phage
- target molecule doent have to be immunogenic and can even be toxic.
- more rapid production (1 month rather than 6)
2) synthetic phage display systems; make a library of antibodies with different binding domains (SCFVs) by muation whicha re then incorporated into phages and applied to microarrays containing the target molecules. after washing step only the enatibody with a compatible SCFV is left behind
- low cost, high speed and no ethical restrictions
example: a plant cell wall polysaccaride (homoglacturonan) was not immunogenic so an antibody (PAM1) was developed using phage system. PAM1 was florescently taggegd and used to localise the polysaccaride
humanised antibodies
discuss the use of antibodies in
a) research
b) medicinal diagnostics
c) medicinal therapy
research
1) molecule localisation via florescent antibodies and microscope; alpha-tubulin in cytoskeleton
2) molecule quantification using enzymatic tagged antibodies. western blots, ELISAS and microarrays
diagnostics
1)diagnosing pregnant women (carry hCG) with pregnancy tests which have anti-hCG antibodies.
lateral flow system: sample moves from absorbent pad to another pad passed immobilised antibodies. hcg binds to antibodies whcih are enzyme tagged, which then moves to other enzymes which have the enzyme substrate attached. other antibodies light up under any urine sample (ensure test worked properly)
3)therapy: antibody tagged with a toxin (gemtuzumab system) binds to cells which are to be killed (leukaemia cells)
describe the use of traditional, humanised and chimeric antibodies
- antibodies are often from rodents so are recognised as foreign and eliminated by the human immmune system
- antibodies can be made to be more human
- chimeric: combination of varibale domain from rodents with human constant domain
- humanised: tiny part of chimeric antibody and a large proportion of human antibody
discuss carbohydrate binding modules
- carbohydrates have limited use in terms of immunisation of animals to produce antibodies, because most antigens are protein
- antibody-like protein molecular probes which act against carbohydrates rather than proteins
- bacterial origin; attached to bacterial enzymes needed to break down cell walls
- many differeent CBMs already exist in nature
describe microarrays
- surface with target molecules attached (DNA, protein etc) so that information about samples can be obtained
- very high throughput
- presence of a protein
- amount of protein
describe microarray creation
microarray surface may be glass slide or nitrocellulose
1) in situ synthesis; biomolecules are made by adding things by layer (enzymatic/chemical synthesis between molecules); targets are created on the microarray
- Genechip by affymetrix which has probes for all 45,000 human genes
2) using a microarray robot to print target molecules which have already been created/obtained.
- print head used to deposit molecules from source plate (384 well plate)
a) pin based robots; pin dips into liquid, deposits liquid then cleans itself
b) peizo robots; print head scans across membrane in a non contact way. overprinting over the same spot can be done to increase molecule concentration. electrical current used to expell sample from chamber of print head.
- peizo is faster, more consistent in terms of spot morphology and therefore more commonly used
during probing, various washing steps are done to remove excess biomolecules
target biomolecules are attached to microarray surface non-covalently (simple, quick but not quantitative and the oritentation of the molecule cant be defined which can be a problem if a certain molecule side is needed for binding) or covalently (chemical links, orientation can be defined and probing is quantitative but the process is more complex and time consuming, and cannot be done with all biomolecules)
how are microarrays analysed
- software used to remove bad spots which have been incorrectly printed
- degree of florescene from spots of biomolecules indicates degree of binding
- data represented using heat maps and scatterplots if two samples are being directly compared
what are the applications of microarrays
molecular profiling; identify molecules present
- comparative analysis within populations
a) glycomics
b) transcriptomics; cDNA from healthy and unhealthy people is florescently tagged different colours and is mixed together and bathed over microarray; the colour the spot floresces indicates which person has increased/decreased expression for that gene
intraction studies; identify new binding molecules for target molecules (cancer cells have the galectin-3 receptor on their surfaces, which when bound causes cancer cell death. other molecules apart from beta-gelactin were identified in order to be fed to cancer patients as treatment
2 examples of antibodies being used theraputically
Antibodies tagged sirh gemtuzumab bind to and eliminate leukaemia
Antibdies ind and disable targets which are in excess(imfliximab binds TNF-alpha during crohns disease treatment)
