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Immunology Lab > Exam 1 > Flashcards

Exam 1 Flashcards

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1
Q

1:9 Dilution

A

ImL reagent and 8mL water

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2
Q

Initial concentration

A

number of colonies / (dilution of sample (amount plated))

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3
Q

2 mL reagent and 13 mL water

A

2:15 dilution

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4
Q

1:4 dilution x 2 dilutions

A

1:16

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5
Q

1:4 x 4 dilutions

A

1:4^4

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6
Q

1:4 x 5 dilutions

A

1:4^5

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7
Q

Find original concentration if the 5th 1:4 dilution is 10ug

A

1:4^5 dilution factor
10ug x 4^5 = 10240 g

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8
Q

What does an antigen do

A

illicits an immune response

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9
Q

What does an antibody do

A

binds to the epitope of an antigen

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10
Q

What produces IgG

A

CD8 cells

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11
Q

What surface proteins do red blood cells have

A

A, B, and Rh factors

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12
Q

What is an Rh factor

A

Rho factor (+/-)
positive if protein is present, negative if not

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13
Q

Dilution buffer used in blood precipitation lab

A

1XTAE

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14
Q

Double diffusion setup

A

Agarose solution
Antibody dilution wells in circle
Antigen in middle

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15
Q

Radial immunodiffusion setup

A

Agarose + antigen solution
Antibody standard wells, blank (dilution buffer), unknown
in a row

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16
Q

What do you plot for immunodiffusion expt

A

Diameter of precipitation over antibody concentration (increasing dilutions of antibodies)

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17
Q

Indirect results for blood typing

A

agglutination is positive reaction for that protein

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18
Q

What happens if you receive incompatible blood?

A

Immune response begins, hemolysis, and then death may follow

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19
Q

What does precipitation mean in immunodiffusion experiments

A

agglutination - antigen interacting with an antibody and forming a insuluble complex

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20
Q

3 forms of innate immunity

A

Neutralization - Ab binds to pathogen and prevents it from binding to a cell and inactivates it
Opsonization - coating of pathogen in antibodies to stimulate phagocytosis
Complement - type of opsonization, makes it quicker to phagocytose a pathogen

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21
Q

Purpose of antibody standards in diffusion experiment

A

to know the concentration of antibodies versus the antigen (double diffusion) and versus the unknown (radial diffusion)???

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22
Q

Radial immunodiffusion is used for what

A

to determine antigen concentration by finding diameter of precipitation ring

Antibody diffuses outwards to develop a circle of precipitation

increase in diameter = increase in [Ab]

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23
Q

What is double diffusion used for

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A

testing of how similar the antibodies are in reacting to 1 antigen

both antibodies and antigen diffuse outward - come into contact at one spot

24
Q

What is bradfords assay used for

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A

to determine protein concentration

25
colors of the bradfords reagent
Red with no protein (cationic, protonated) Blue with protein (anionic, deprotonated) Green at neutralization
26
What causes the color change in coomassie dye?
comassie blue is deprotonated and suspended in acid/protein solution and turns from red -> blue
27
How is protein concentration measured
measuring intensity of blue color at 595nm - absorbance is directly related to [protein]
28
How to determine the concentration of an unknown sample
use standard curve of absorbance/concentration for known concentrations and can determine the unknown on that standard line
29
What is extrapolation and why dont we do it
Only consider unknown absorbances that fall within the linear portion of the standard curve no assumptions outside the trendline
30
What is R^2
correlation/coefficient of determination - how confident your results are low R2 means that the change in concentration is not explaining the variation outside of the trendline
31
What R^2 value is sufficient for linear determination
R2 >= .98
32
Max absorbance of green blue and red
Red < Blue (595) < green
33
Analyzing pipette skills uses what
precision
34
C values
C < 1 = good pipette skills C > 10 = bad pipette skills via a log and residual for error
35
when to not use pipettes
when needing under 10% of the max: 100 in p1000 2 in p20 10 in p100
36
binding order of ELISA
Antigen --> primary antibody --> secondary antibody -->fluorophore
37
antigen name
chicken gamma globulin (from egg yolk)
38
primary antibody
rabbit anti chicken antibody (inject antigen into rabbit and harvest)
39
secondary antibody
goat anti rabbit antibody (injected primary antibody into goat and harvest)
40
Substrate for ELISA
3,3,5,5 tetramethylbenzidine
41
What oxidizes the TMB to turn it blue
horseradish peroxidase
42
Where is the horseradish peroxidase that oxidizes TMB to turn it blue
on secondary antibody
43
What is in the wash buffer for ELISA
PBS has tween 20, a detergent that binds to any unbound antibody or antigen to avoid a false positive result with non specific binding
44
ANA
antinuclear antibodies - are auto antibodies that attack nuclear antibodies within the cell
45
What diseases can be diagnosed with ANA
MS, mixed CT disease, SLE (lupus), or other autoimmune disorders
46
What causes the blue color in TMB
enzyme horseradish peroxidase linked to the secondary antibody
47
Dark spots in ANA
due to flourophores bleaching
48
What makes the color in ANA
antibody
49
IFA
indirect fluorescent antibody
50
Epithelial cell line
HEP 2 (greater sensitivity than tissue sections)
51
what is the conjugate in ANA and what is it labeled with
goat anti-human antibodies, labeled with FitC
52
What do you rinse with in ANA
PBS
53
Homogenous ANA
attacks all nuclear components all green
54
Speckled ANA
green border and small dots in the cell
55
peripheral ANA
only green border around cell
56
Nucleolear ANA
large green spots within cell, no green ring like in specked
57
Centromere ANA
small green spots within cell, no green ring like in speckled

Immunology Lab (2 decks)

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