Immunologic Assay

Question 1

what are the parts of an immunoglobulin?

Answer 1

heavy chain and a light chain

variable region and constant region of each chain

Question 2

which probes can immunoglobulin molecules be conjugated with?

Answer 2

1. Fluorescence dyes for immunofluorescence (IF) staining
2. Gold particles for immunoelectron microscopy
3. Enzymes for immunohistochemistry, ELISA

the different probes are depending on the purpose

probes are usually conjugated to the contact region of immunoglobulin to preserve the antigen-binding property of the antibody

Question 3

what is the purpose of direct IF assay?

Answer 3

to visualize the localization of antigen “X”, anti-X antibody is conjugated with FITC

it’s to study the localization of something in the cell

Question 4

what is a cryostat section?

Answer 4

thin (6-10 mm) sections sliced from frozen tissue samples

many antibodies do not recognize the antigens in formalin-fixed samples due to conformational changes caused by fixation

cryostat sections are, thus, most frequently used for IF staining

Question 5

what are the steps in doing a direct IF assay?

Answer 5

1. cryostat sections are prepared from the tissue of interest
2. ehe sections are incubated with FITC-conjugated antibody
3. the antibody binds to antigen X expressed Cell Type A
4. the sections are washed to remove unbound antibody
5. the sections are observed under fluorescence microscope to visualize FITC-associated fluorescence signals
6. antigen X can be visualized with green fluorescence

Question 6

what does EBS stand for?

Answer 6

epidermolysis bullosa simplex

really nasty skin blisters

Question 7

what mutation leads to EBS?

Answer 7

mutation of the keratin 5 or keratin 14 gene

specifically, a homologous one base pair deletion in exon 1

this mutation results in a frame shift and a formation of a premature termination codon

Question 8

which test would help diagnose EBS?

Answer 8

IF staining

if you stain the tissue with anti-keratin 5 or anti-keratin 14 it would let you see if both proteins are present

if they’re present, they will show up bright green but if there’s no green showing up in the stain, you know the proteins are absent

Question 9

how does immunoelectron microscopy work?

Answer 9

1. an antibody against MHC class II molecule is labeled with gold particles, which appear as electron dense zones at the EM level
2. ultra-thin skin sections are incubated with gold particle-labeled anti-MHC class II antibody.
3. gold particles are found predominantly within multivesicular bodies, known as the MHC class II compartment (MIIC), where antigen processing and loading take place

Question 10

what findings would support a SLE diagnosis?

Answer 10

1. H&E staining of kidney biopsy shows a wire-loop lesion of glomerulus
2. IF staining with FITC-conjugated anti-human IgG antibody reveals IgG deposition along glomerular capillary loops
3. IF staining with FITC-conjugated anti-human C3 antibody reveals complement deposition as well

these changes indicate deposition of immune complex (IC) along glomerular capillary loops

Question 11

how does immunohistomchemistry work?

Answer 11

it’s a modified version of IF staining!

an antibody is conjugated with an enzyme that converts a colorless substrate into a colored product

peroxidase and alkaline phosphatase are most commonly used to label the antibodies for this application

Question 12

what kind of tissues can you do immunohistochemistry on?

Answer 12

although IF staining can be performed only with cryostat sections prepared from frozen tissues, immunohistochemistry may be applicable to conventional formalin-fixed tissues

Question 13

what would a kidney biopsy of an SLE patient show?

Answer 13

IgG deposition along glomerular capillary loops

Question 14

what’s the different between direct and indirect IF staining?

Answer 14

indirect IF staining enables the detection of auto-antibodies in SERUM samples

direct IF staining enables the detection of deposition of auto-antibodies in TISSUE biopsy samples = skin, kidney, etc.

Question 15

how does indirect IF staining work?

Answer 15

a serum sample from an SLE patient can be tested for anti-nuclear antibody (ANA)

1. human epithelial cell line (HEp-2) is incubated with serum to allow binding of ANA to the nuclei
2. the cells are then incubated with FITC-conjugated anti-human IgG to allow the detection of ANA
3. the IF image (green color) indicates the presence of ANA in the blood circulation
4. the cells are counter-stained to indicate cytoplasmic areas (red color)

Question 16

how can you estimate the relative antibody concentration in a serum sample?

Answer 16

by diluting a serum sample, one can estimate relative antibody concentration

1. serum from an SLE patient is serially diluted and then tested for ANA
2. if ANA is still detectable at 1:320 dilution and becomes undetectable at 1:640, it means the titer of 1:320

antibody titers are often used to follow the disease activity – a change from 1:40 to 1:160, for example, indicates an increase in circulating ANA concentration and, thus, implies exacerbation of the disease

by contrast, a change from 1:640 to 1:160 indicates a reduction in ANA concentration and, thus, implies improvement of the disease

Question 17

what do different staining patterns in indirect IF staining mean?

Answer 17

different staining patterns imply different distributions of antigens recognized by autoantibodies

ex. the auto-antigen recognized in one person could be located at the inter-cellular space between keratinocytes while another person may have auto-antigen recognized at the basement membrane and they would be two totally different diseases!

pemphigus vulgaris vs. bullous pemphigoid

Question 18

what are the steps in a Western blot?

Answer 18

it analyzes the biochemical properties of an antigen

1. tissue and cell extracts containing antigens are separated by SDS-PAGE based on the molecular weight
2. the proteins are transferred to a membrane
3. he membrane is incubated with a serum sample containing antibodies
4. he membrane is then incubated with enzyme-conjugated anti-IgG
5. he membrane is developed by addition of colorless substrate
6. colored band indicates the location (molecular weight ) of the antigen

Question 19

what is an example of a diagnostic application of immunoblotting? aka Western Blot

Answer 19

1. Human keratinocyte extracts are separated in SDS-PAGE
2. proteins are transferred onto a membrane
3. the membrane is stained with Coomassie blue for all proteins
4. the membrane is incubated with serum from Pt #1 (Pemphigus vulgaris), a new patient, or healthy volunteer
5. a band of about 130 kD (desmoglein-3) is detected with serum samples from Pt #1 and the new patient
6. the results imply that auto-antibodies in both patients recognize an antigen of the same molecular size

Question 20

what are the steps in an ELISA?

Answer 20

1. Test samples containing antigen “X” (red triangles) are immobilized to the bottoms of a 96-well plate
2. enzyme-conjugated anti-X antibody is added to the plate
3. colorless substrate is added to the plate
4. development of colored products indicates the presence of antigen X in the test samples

it’s those plates with a ton of holes you used to use to calibrate purple with in Sakima’s lab

Question 21

what is an ELISA used for?

Answer 21

1. to measure the amount of antigen

2. to measure the amount of antibodies

Question 22

how is an ELISA used to measure the amount of antigen?

Answer 22

1. to test the concentration of IL-1 in human serum samples, anti-IL-1 monoclonal antibody (produced in mice) is first immobilized onto the 96 well plate
2. graded amounts of recombinant IL-1 are added to individual wells to create a standard curve – IL-1 molecules (red triangles) are captured by anti-IL-1 antibody

3 .serum samples are also added to individual wells

4. nzyme-conjugated anti-IL-1 monoclonal antibody (which binds to a different epitope) is added to the plate
5. colorless substrate is then added to the plate
6. the amount of IL-1 in a test sample can now be calculated from the standard curve

Question 23

how can an ELISA be used to measure the amount of antibodies?

Answer 23

1. to test the concentration of antibody against demoglein-3, recombinant desmoglein-3 proteins (red triangles) are immobilized onto a 96 well plate.
2. serum samples from pemphigus vulgaris patients are added to individual wells
3. enzyme-conjugated anti-human IgG is added to the plate
4. colorless substrate is then added to the plate
5. one can estimate the amount of anti-desmoglein-3 antibody in the sample based on the standard curve

Question 24

what is an agglutination test used for?

Answer 24

1. it enables rapid detection of antigen-reactive antibodies

2. also enables rapid detection of antigens

Question 25

how does an agglutination test detect antigen-reactive antibodies?

Answer 25

1. to test antibodies against Toxoplasma gondii, latex beads (yellow circles) are coated with T. gondii-derived antigens (red triangles) 2. the beads are mixed with serum sample on slide glass 3. if the sample contains antibodies against T. gondii-derived antigens, it causes agglutination (clump), which can be detected visually 4. the assay can be easily performed without specific equipment The assay can be completed in a few minutes.

Question 26

how does an agglutination test detect antigens?

Answer 26

1. To identify Methicillin resistant Staphylococcus aureus (MRSA), latex beads (yellow circles) are coated with antibodies against penicillin-binding protein 2’ (PBP2’) 2. PBP2’ is expressed on the surface of MRSA 3. the beads are mixed with clinical isolates on slide glass 4. if the sample contains MRSA (red bacteria), it causes agglutination, which can be detected visually

Question 27

what is a hemagglutination test used for?

Answer 27

to determine blood type

Question 28

how does a hemagglutination test work?

Answer 28

1. to determine the ABO and Rh blood groups, antibodies against Type A, Type B, and Rh (D) antigens are mixed with peripheral blood on slide glass 2. erythrocytes (red circles) expressing Type A antigen (blue triangles) form agglutination with anti-A antibody. 3. the example shows agglutination with anti-A and with anti-D (Rh), but not with anti-B antibody 4. his indicates the blood type of A+

Question 29

what does IF staining of leukocytes do?

Answer 29

measures the numbers of different T cell subsets in blood samples CD3 is expressed by all T cell subsets. CD8 is expressed by cytotoxic T cell subset. CD4 is expressed by helper T cell subset. NK T cells and gd T cells express neither CD8 nor CD4 to identify different T cell subsets, antibodies against CD3, CD8, and CD4 are conjugated with different dyes blood samples are incubated with a mixture of the three antibodies there will be four populations that are distinguishable from each other by fluorescence profiles

Question 30

how does flow cytometry work?

Answer 30

1. flow cytometry is a technique for counting and examining thousands of cells per second 2. laser beam is directed onto a stream of cell suspensions 3. each cell passing through the beam is excited to emit fluorescence signals 4. a detector sees the signal 5. the information is sent to the computer it gives you that dot plot with 4 quadrants and the speckles in certain quadrants if the CD marker is present

Question 31

what is a dot plot?

Answer 31

it displays flow cytometry data! each dot represents an individual cell with the indicated levels of fluorescence intensities (FI)

Question 32

which cells only express CD3 in a CD3 vs CD8 dot plot?

Answer 32

helper T cell, NK T cell, and gd T cell subsets

Question 33

which cells express CD3 and CD8 in a CD3 vs CD8 dot plot?

Answer 33

cytotoxic T cells

Question 34

what is a cytotoxicity assay used to measure?

Answer 34

used to measure cytotoxic activities of leukocytes

Question 35

how does a cytotoxicity assay work?

Answer 35

1. to measure cytotoxicity T cell activities to kill a melanoma target, melanoma cells are labeled with 51Cr 2. CD3+/CD8+ T cells are purified by FACS from a melanoma patient before (blue symbols) or after vaccination (red) or from a healthy volunteer (green) 3. the target cells (T) and effector cells (E) are cultured together at different E:T ratios for 4-12 hours 4. 51Cr is released from dying cells into culture media. 5. after centrifugation, the pellets and supernatants are measured for radioactivity 6. the % lysis is calculated by dividing the radioactivity in the supernatants by the total radioactivity

Question 36

what's the full list of all the different immunological assays?

Answer 36

Direct immunofluorecence (IF) staining Indirect IF staining Immunohistochemistry Immunoelectron microscopy Immunoblotting (Western blotting) Enzyme-linked immunosorbent assay (ELISA) Agglutination test Fluorescence activated cell sorter (FACS) or flow cytometry Cytotoxicity assay