LAB FINAL

Question 1

What are the diagnostic methods used for?

Answer 1

identify the pathology of a particular patient

Question 2

What is an immunoassay?

Answer 2

analytical methods using antigens/antibodies to determine the presence of their corresponding antigen/antibody in different media

Question 3

What are immunoassays used for?

Answer 3

find the presence of specific antibodies that are produced in the response to a non-self protein (antigen)

Question 4

Why are immunoassays useful in the diagnostic field?

Answer 4

they are rapid, cost-effective, sensitive and specific for particular antigens

Question 5

What are the 3 main parts of a Western Blot assay?

Answer 5

separate antigens by weight | transfer (blot) to a membrane | test for antigen via enzyme-substrate reaction

Question 6

What is the weakness of Western Blot assays?

Answer 6

less sensitive compared to others

Question 7

What is an ELISA?

Answer 7

enzyme linked immunosorbent assay (ELISA) 96-microtiter plate capture specific antibody/antigen in sample

Question 8

What are the 3 main steps of ELISA?

Answer 8

absorb protein-interest into wells (solid-phase support system) | use antibody-specific to protein | develop using enzyme-substrate reaction

Question 9

What are the 3 most common ELISAs?

Answer 9

direct, indirect, sandwich

Question 10

What is a strength of ELISAs?

Answer 10

fast, cheap, easy to perform, more sensitive

Question 11

What is a weakness of ELISAs?

Answer 11

false positives

Question 12

What are ELISAs used to determine?

Answer 12

ability of an antigen to bind to extracellular matrix proteins

Question 13

What is an agglutination assay?

Answer 13

ability of antibody to attach to specific antigen = create a lattice = clumping of particles results in visible conglomerate (aggluntination)

Question 14

What is the agglutination assay commonly used to test for?

Answer 14

Syphilis in diagnostic laboratories (VDRL assay)

Question 15

What is a significant step of the agglutination assay? Why?

Answer 15

dilute antigen and antibody bc agglutination can only occur when the concentration of antibody and antigen is equal = zone of equivalence

Question 16

What happens if there is an excess of antigen in the agglutination assay?

Answer 16

agglutination will not occur = leads to false positives

Question 17

What is the precipitation assay?

Answer 17

uses insoluble complex formed between antigen/antibody

Question 18

How are results observed in a precipitation assay?

Answer 18

aggregate precipitates out of solution = visualization of complex at base of tube

Question 19

What is a weakness of precipitation assays?

Answer 19

false-negatives

Question 20

What is an immunofluorescence assay (IFA)?

Answer 20

detect presence of antibody/antigen in serum samples

Question 21

What does the immunofluorescence assay involve?

Answer 21

binding of a specific antibody to an antigen in the sample

Question 22

What modification is done to the antibody(ies) used in the IFA assays?

Answer 22

chemically tagged with fluorescent dye = allows pathogen to be detected by fluorescent microscope

Question 23

What are the 2 types of IFA techniques?

Answer 23

direct and indirect

Question 24

What is the Direct IFA Antibody assay?

Answer 24

uses 1 antibody | detects antigens in fecal, blood, tissue samples (not serum)

Question 25

What is Indirect IFA Antibody assay?

Answer 25

uses 2 antibodies, label with fluorochrome | detects antigens in serum samples

Question 26

Define molecular techniques?

Answer 26

study of molecular basis of physiological processes that occur in microbes - such as translation and transcription

Question 27

Why are molecular techniques useful?

Answer 27

can detect slow-growing/unculturable pathogens | identify particular bacterial species present in sample via nucleic acid sequence

Question 28

What are molecular techniques commonly used as by medical professionals?

Answer 28

identify disease prognosis = allows doctors to decide on potential therapies based on specific characteristics og the pathogen

Question 29

What are the 3 molecular techniques discussed?

Answer 29

PCR | RT-PCR | electrophoretic mobility shift assay

Question 30

What is PCR?

Answer 30

polymerase chain reaction | identifies particular pathogen in a sample by a targeted nucleic acid sequence

Question 31

What is the concept behind PCR?

Answer 31

use of one DNA template strand (or many from a sample) and use of polymerase’s high efficiency to make many copies of the targeted nucleic acid

Question 32

How can you identify a particular pathogen using the PCR technique?

Answer 32

must use primer specific to a particular bacterial species

Question 33

What is PCR commonly used for?

Answer 33

identify unknown bacterial isolates and uncultivable pathogens

Question 34

What makes PCR highly usable?

Answer 34

cheap, fast, specific, uses very small sample

Question 35

What is RT-PCR?

Answer 35

reverse transcriptase-PCR | determine which genes are expressed in a particular pathogen

Question 36

What are the main steps of RT-PCR?

Answer 36

reverse transcriptase amplifies mRNA &raquo_space;> makes cDNA (complimentary DNA) &raquo_space;> PCR amplifies cDNA

Question 37

What is RT-PCR useful for?

Answer 37

determine various characteristics of a particular pathogen (ie: toxins)

Question 38

What is a weakness of RT-PCR?

Answer 38

unstable nature of RNA | RNase can degrade product

Question 39

What are the results of most immunoassays expressed as?

Answer 39

positive = presence of Ab/antigen | negative = absence of Ab/antigen