Techniques

Question 1

What are Vaccines?

Answer 1

Injection of foreign substate (antigen) to generate an immune response.

Question 2

What is in a Vaccine?

Answer 2

The immunogen/antigen, adjuvant, preservatives/ stabilizers, and residuals

Question 3

What does the antigen in a vaccine do?

Answer 3

Creates an immune response.

Question 4

What is the Adjuvant do in a vaccine?

Answer 4

Increases immune response, but will not get the immune response on its own. Import since antigen alone is (usually) insufficient.

Question 5

How does SARS-CoV2 mRNA vaccines work?

Answer 5

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.

Question 6

How do you generate polyclonal antibodies?

Answer 6

1. Inject antigen into rabbit
2. The antigen activates multiple B-cells which recognize different antigens
3. Paslma B-cells produce polyclonal antibodies and the antibodies are secreted.
4. Obtain antiserum from rabbit containing polyclonal antibodies

Question 7

what is serum

Answer 7

liquid part of the blood after coagulation

Question 8

what is antiserum

Answer 8

liquid part of the blood of an immunized individual

Question 9

what is polyclonal

Answer 9

mixture of antibodes from different B-cell lineages

Question 10

How do you purify the antibodies against a specific protein?

Answer 10

immobolize protein, soak in antiserum, wash, elute antibody with high salt, antibodies are extremely stable

Question 11

What are some issues with polyclonal antibodies

Answer 11

1. The antibodies are completely different from animal to animal (this is only an issue for lab research)
2. Micture of antibodies will bind different epitopes with different affinities (problems with westernblots)
3. can never recreate perfectly

Question 12

What are monoclonal antibodies?

Answer 12

Generated from a single Bcell lineage, ony generates one type of antibody, immortal (can generate antibody forever)

Question 13

How do you generate monoclonal antibodies?

Answer 13

1. Immunize mouse or other organism
2. Isolate spleen cell (b/c biggest organ)
3. Fuse with myeloma with polyethylene glycol
4. Move to HAT medium: spleen cells die after so many decisions, myeloma cells lack HGPRT and die, Hybridomas are immortal and have HGPRT
5. Select the right hybrid cell
6. Grow and isolate

Question 14

How does a Western Blot work?

Answer 14

1. Perform gel electrophoresis. Blot proteins from the gel onto nitrocellulose
2. Block sites with nitrocellulose with casein
3. Incubate with antibody to the protein of interest
4. Wash and incubate with an enzyme-linked secondary antibody
5. Assay the linked enzyme with a colorimetric reaction

Question 15

Why would you use a Western blot?

Answer 15

Useful for mixtures of proteins and can quantify but it needs to be compared to another band.

Question 16

How do you do immunoprecipitation

Answer 16

1. Affinity ligands are covalently attached to a chromatography support and packed in a column
2. Run the sample through the affinity column in the binding buffer.
3. Target molecules bind to affinity ligands
4. The sample is washed through the column to remove unbound components
5. elution buffer is applied to disrupt the interaction between the affinity ligand and the target molecule
6. Purified target molecule is eluted from the column

Question 17

Why would you do immunoprecipitation

Answer 17

great for purification or enrichment, can add an antibody-recognized tag to a protein

Question 18

What is the difference between immunoprecipitation and co-immunoprecipitation (Co-IP)?

Answer 18

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

Question 19

What are the three different types of ELISA

Answer 19

1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA

Question 20

What does ELISA stand for

Answer 20

Enzyme-Linked ImmunoSorbent Assay

Question 21

What is direct ELISA

Answer 21

the antigen is attached to the surface and the primary antibody conjugate directly attaches to the antigen

Question 22

What is indirect ELISA

Answer 22

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.

Question 23

What is sandwich ELISA

Answer 23

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.

Question 24

How is ELISA used for diagnostic tests?

Answer 24

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

Question 24

What is Immunoelectron microscopy (EM)

Answer 24

EM uses electrons instead of light, uses gold-linked antibodies, and show up as black spots

Question 24

How does flow cytometry

Answer 24

use fluorescent dyes linked to antibodies that bind surface proteins without killing the cells, however, the cells still need to be isolated

Question 25

How are the cells separated after flow cytometry

Answer 25

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.

Question 25

What does forward scatter measure for flow cytometry?

Answer 25

the cell size

Question 26

What does side scatter measure for flow cytometry?

Answer 26

Cell density and used to measure of granularity

Question 27

Where are lymphocytes located on a SSC vs. FSC

Answer 27

low FSC and low SSC

Question 28

Where are monocytes located on an SSC vs. FSC

Answer 28

high FSC and low SSC

Question 29

Where are neutrophils located on an SSC vs. FSC

Answer 29

High FSC and high SSC

Question 30

Why does the lab use Transgenic Mice?

Answer 30

Experiments on humans are expensive, genetic manipulation is immoral, unethical, and illegal. Also mice are similar to humans and they breed fast.

Question 31

How do you make a transgenic mouse?

Answer 31

-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

Question 32

What is the basic genetic process of a knockout mouse?

Answer 32

-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).

Question 33

Why is the HSC-tk gene used in knockout protocol?

Answer 33

The sequence can be inserted incorrectly, HSV-tk acts as a sensitizer gene

Question 34

What protocol would you follow to express something?

Answer 34

-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

Question 35

What is an agglutination test?

Answer 35

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)

Question 36

When would you use an agglutination test?

Answer 36

One example is the Strep test, signs of agglutination would indicate strep-positive

Question 37

What is the Hemagglutination test?

Answer 37

It is an Agglutination test and will test for blood compatibility, which is important for organ transplants. Agglutination means incompatibility

Question 38

What is the purpose of Human Leukocyte antigen test (HLA)

Answer 38

How to match donors to prevent rejection

Question 39

How does HLA work

Answer 39

Testing a few loci to make sure there is a near match, a perfect match is very rare due to the multitude of alleles

Question 40

Describe Hematopoietic stem-cell transfers (HSC's)

Answer 40

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)