Techniques Flashcards
Quiz 1 (Lecture 2)
What are Vaccines?
Injection of foreign substate (antigen) to generate an immune response.
What is in a Vaccine?
The immunogen/antigen, adjuvant, preservatives/ stabilizers, and residuals
What does the antigen in a vaccine do?
Creates an immune response.
What is the Adjuvant do in a vaccine?
Increases immune response, but will not get the immune response on its own. Import since antigen alone is (usually) insufficient.
How does SARS-CoV2 mRNA vaccines work?
Uses mRNA to have host cell make spike protein (antigen). The mRNA is recognized as ‘foreign’ and acts as its own adjuvant. mRNA is too immunogenic so it was modified to turn down response.
How do you generate polyclonal antibodies?
- Inject antigen into rabbit
- The antigen activates multiple B-cells which recognize different antigens
- Paslma B-cells produce polyclonal antibodies and the antibodies are secreted.
- Obtain antiserum from rabbit containing polyclonal antibodies
what is serum
liquid part of the blood after coagulation
what is antiserum
liquid part of the blood of an immunized individual
what is polyclonal
mixture of antibodes from different B-cell lineages
How do you purify the antibodies against a specific protein?
immobolize protein, soak in antiserum, wash, elute antibody with high salt, antibodies are extremely stable
What are some issues with polyclonal antibodies
- The antibodies are completely different from animal to animal (this is only an issue for lab research)
- Micture of antibodies will bind different epitopes with different affinities (problems with westernblots)
- can never recreate perfectly
What are monoclonal antibodies?
Generated from a single Bcell lineage, ony generates one type of antibody, immortal (can generate antibody forever)
How do you generate monoclonal antibodies?
- Immunize mouse or other organism
- Isolate spleen cell (b/c biggest organ)
- Fuse with myeloma with polyethylene glycol
- Move to HAT medium: spleen cells die after so many decisions, myeloma cells lack HGPRT and die, Hybridomas are immortal and have HGPRT
- Select the right hybrid cell
- Grow and isolate
How does a Western Blot work?
- Perform gel electrophoresis. Blot proteins from the gel onto nitrocellulose
- Block sites with nitrocellulose with casein
- Incubate with antibody to the protein of interest
- Wash and incubate with an enzyme-linked secondary antibody
- Assay the linked enzyme with a colorimetric reaction
Why would you use a Western blot?
Useful for mixtures of proteins and can quantify but it needs to be compared to another band.
How do you do immunoprecipitation
- Affinity ligands are covalently attached to a chromatography support and packed in a column
- Run the sample through the affinity column in the binding buffer.
- Target molecules bind to affinity ligands
- The sample is washed through the column to remove unbound components
- elution buffer is applied to disrupt the interaction between the affinity ligand and the target molecule
- Purified target molecule is eluted from the column
Why would you do immunoprecipitation
great for purification or enrichment, can add an antibody-recognized tag to a protein
What is the difference between immunoprecipitation and co-immunoprecipitation (Co-IP)?
The procedure is the same, but in Co-IP, you are trying to examine what your protein interacts with. In other words, purify protein with antibody and then examine what else purifies with it/interacts with
What are the three different types of ELISA
- Direct ELISA
- Indirect ELISA
- Sandwich ELISA
What does ELISA stand for
Enzyme-Linked ImmunoSorbent Assay
What is direct ELISA
the antigen is attached to the surface and the primary antibody conjugate directly attaches to the antigen
What is indirect ELISA
can be used to measure antibody levels, the antibody already present in the body binds to the antigen attached to the surface. The conjugate antibody is the secondary antibody.
What is sandwich ELISA
The capture antibody (against the virus) is attached to the surface. The antigen is attached to the capture antibody and the primary and secondary antibodies are attached to that.
How is ELISA used for diagnostic tests?
For example, pregnancy test: The urine contains HCG which will attach the mobile antibodies which have an enzyme attached to them. Immbolized antibodies in the test zone bind to HCG
What is Immunoelectron microscopy (EM)
EM uses electrons instead of light, uses gold-linked antibodies, and show up as black spots
How does flow cytometry
use fluorescent dyes linked to antibodies that bind surface proteins without killing the cells, however, the cells still need to be isolated
How are the cells separated after flow cytometry
the cells are put through a small that (single-cell width) and they are hit with a laser individually, the fluorescence of a single cell can be measured/counted.
What does forward scatter measure for flow cytometry?
the cell size
What does side scatter measure for flow cytometry?
Cell density and used to measure of granularity
Where are lymphocytes located on a SSC vs. FSC
low FSC and low SSC
Where are monocytes located on an SSC vs. FSC
high FSC and low SSC
Where are neutrophils located on an SSC vs. FSC
High FSC and high SSC
Why does the lab use Transgenic Mice?
Experiments on humans are expensive, genetic manipulation is immoral, unethical, and illegal. Also mice are similar to humans and they breed fast.
How do you make a transgenic mouse?
-Stimulate ovulation and mating by injecting hormones
-fertilize eggs are injected with DNA
-Eggs are added to new female
-Mate correct offspring to get the desired outcome
What is the basic genetic process of a knockout mouse?
-The target gene is added to DNA
-The target gene is interrupted by insertion of neomycin-resistance gene
-The resistance gene is surrounded by flanking region matching your target gene
-Homologous recombination will swap-out your resistance gene into the gene in the chromosome (the gene no longer works).
Why is the HSC-tk gene used in knockout protocol?
The sequence can be inserted incorrectly, HSV-tk acts as a sensitizer gene
What protocol would you follow to express something?
-Insert a target into a non-coding region of DNA (or unimportant gene
-This target has your gene of interest on a promoter
-that promoter can be constitutive or inducible
What is an agglutination test?
Clinically-focused immunology technique. Antibodies bind to large antigens, this can lead to clumping (agglutination), can measure the presence of antigen (direct) or antibody (indirect)
When would you use an agglutination test?
One example is the Strep test, signs of agglutination would indicate strep-positive
What is the Hemagglutination test?
It is an Agglutination test and will test for blood compatibility, which is important for organ transplants. Agglutination means incompatibility
What is the purpose of Human Leukocyte antigen test (HLA)
How to match donors to prevent rejection
How does HLA work
Testing a few loci to make sure there is a near match, a perfect match is very rare due to the multitude of alleles
Describe Hematopoietic stem-cell transfers (HSC’s)
HSC’a are purified (they can be from the same person or donor. They are injected into the bloodstream (for bone marrow transplants, they move to the bone and regenerate marrow)
