Micro/Immuno 6

Question 1

What does ELISA stand for?

Answer 1

Enzyme linked immunosorbent assay

Question 2

What is ELISA?

Answer 2

Enzyme linked antibody can covalently bind to antibody molecules bound to a pathogen

Question 3

Where is ELISA performed (in what medium)?

Answer 3

Micro titer dishes

Question 4

What component of ELISA is used so we can see antibodies?

Answer 4

Colorless substrate

Question 5

What component of ELISA causes the colorless substrate to become colorful?

Answer 5

Enzyme on the second antibody

Question 6

What is a direct ELISA test?

Answer 6

Looking for pathogen
Add antibodies for pathogen
Add enzyme linked antibody and colorless substrate
If color appears, the pathogen was present

Question 7

What is an indirect ELISA?

Answer 7

Looking for antibodies
Add antigen of pathogen
Add enzyme linked antibody and colorless substrate
If color appears, the antibody was present

Question 8

What does it mean in an ELISA test if no color appears?

Answer 8

Pathogen is not present

Question 9

What are the steps of ELISA?

Answer 9

Antibodies to virus bound to wells of micro titer
Add patient sample suspected of containing virus particles
Wash wells with buffer
Add enzyme linked antibody
Wash with buffer
Add colorless substrate
Look for colored product

Question 10

What does washing the well with buffer do?

Answer 10

After washing, if antibody/antigen are not bound, then they wash away and enzyme linked antibody has nothing to bind to

Question 11

What is electrophoresis?

Answer 11

Moving a molecule with a charge through a filed with an electromagnet

Question 12

What is another name for Immunoblotting?

Answer 12

Western blotting

Question 13

What is western blotting?

Answer 13

Separation fo proteins in a gel by electrophoresis, followed by transfer to a membrane and detection by addition of specifically labeled antibodies

Question 14

What two substances does western blotting detect?

Answer 14

Detects specific antigens or antibodies to the antigens

Question 15

What substances are used to label antibodies?

Answer 15

Radioisotopes or chemiluminescent chemicals

Question 16

How are protein bands visualized in western blotting once it is transferred to another membrane?

Answer 16

Visualized using enzyme tagged antibodies

Question 17

Is western blotting specific?

Answer 17

It is very specific, but it takes a longer time to complete

Question 18

What do molecular diagnostic tests look for?

Answer 18

Look for nucleic acids

Question 19

Are molecular diagnostic tests direct or indirect?

Answer 19

Direct only

Question 20

What are three types of molecular diagnostic tests?

Answer 20

Ribotyping
Nucleic acid hybridization and probing
Polymerase chain reaction (PCR)

Question 21

What is ribotyping?

Answer 21

It compares the nucleotide sequence of amplified DNA to a database to see if it matches a known pathogen

Question 22

What segment of DNA is used for ribotyping?

Answer 22

Based on the high level of 16S rRNA gene conservation among bacteria

Question 23

What test is used to amplify the rRNA encoding genes so that ribotyping can be used?

Answer 23

Polymerase chain reaction

Question 24

What components are important in nucleic acid hybridization?

Answer 24

Complementary nucleotide bases

Question 25

What are the complementary nucleotide bases?

Answer 25

A-T
C-G
A-U (RNA)

Question 26

What is a probe?

Answer 26

A short segment of DNA

Question 27

What is nucleic acid hybridization?

Answer 27

Nucleic acid probes hybridize with complementary specimen DNA or RNA

Question 28

How are probes distinguished from the pathogen sequences?

Answer 28

They are labeled with radioisotope or fluorescent dye

Question 29

What is nucleic acid hybridization not dependent on?

Answer 29

Pathogen isolation or growth

Detection of an immune response to pathogen (antibodies)

Question 30

What are homologous strands in nucleic acid hybridization?

Answer 30

Separated DNA strands from pathogen source and stock that reconnect with one another

Question 31

What are the steps to nucleic acid hybridization?

Answer 31

DNA is denatured - heat to separate the strands
DNA from the pathogen is placed on a filter
Probe is added
If the two are homologous, strands will bind
If non homologous, probe will settle out

Question 32

What is PCR?

Answer 32

Polymerase chain reaction - enzymatic amplification of a segment of DNA or RNA

Question 33

What are the components of a PCR?

Answer 33

Template
Primer
Nucleotides
Buffers
Thermal stable enzyme

Question 34

What is an example of a common thermal stable enzyme used in PCR?

Answer 34

Taq polymerase

Question 35

Which molecular diagnostic test is the most specific?

Answer 35

PCR - can detect small amounts of DNA

Question 36

What result of PCR confirms the presence of a pathogen?

Answer 36

Presence of amplified gene segment confirms presence of pathogen

Question 37

What is amplification?

Answer 37

Making many copies of DNA

Question 38

What is amplification used for?

Answer 38

We need many copies of the DNA in order to get a probe

Question 39

Why is a thermal stable enzyme needed for PCR?

Answer 39

Taq polymerase can handle very high temperatures which is needed because the process involves many denaturations at increasingly higher temperatures

Question 40

What is reverse transcriptase PCR?

Answer 40

Uses pathogen specific RNA to make cDNA (used for RNA genomes) - mRNA is converted to DNA before the process begins

Question 41

What are the steps to PCR?

Answer 41

Denaturation of strands
Primers connect to separated strands to start the writing process
Taq polymerase continues to comprise the strand copy out of stock nucleotides
Process repeats over and over, at increasingly higher temperatures