Seminars - Some Basic First Few Weeks Stuff (Dustin)

Question 1

Polyclonal vs Monoclonal Antibody Definitions

Answer 1

• Polyclonal: product of many different B cell clones. Antibodies recognize different epitopes of same antigen
• Monoclonal: only 1 type of B cell products it, and antibodies are specific for only one epitope

Question 2

What is avidity in terms of antibodies?

Answer 2

Combined strength of many different bonds between antibodies and antigens.

IgM has high avidity because it’s a pentamer with 10 binding sites, whereas IgG only has 2 binding sites and thus lower avidity

Question 3

What are some good uses for immunodiffusion?

Answer 3

Detection of solule antigens or antibodies from body fluids

e.b. bacterial, viral antigens, transferrin, Ig classes

Question 4

What are some things that can be detected by serum electrophoresis?

Answer 4

• Multiple myeloma: most important. Shows narrow M spike in the gamma range (monoclonal gammopathy)
• Chronic Liver Failure
• Chronic inflammation
• Polyclonal gammopathies: many things with inflammation like hepatitis, AIDS, RA, endocarditis etc show broad increases in the gamma range

Question 5

Immuno electrophoresis can be used to help diagnose:

Answer 5

• Multiple myeloma
• Rheumatoid Arthritis
• Chronic Inflammation

Question 6

Direct and indirect agglutation are used to test for:

Answer 6

Direct: ABO blood group, Widal test, typhoid fever

Indirect for both ABO and Rh (Rh does not produce direct response) - used in Coombs test

Question 7

What is the direct and indirect Coombs test and what do they test for?

Answer 7

• Direct Coombs: Fetal red blood cells are coated to see if they’re Rh+ coated with maternal anti-Rh IgG. Just one step: add anti-human antibodies to see if they agglutinate
• Indirect Coombs: take maternal serum and test if it has anti-Rh antibodies by first adding Rh+ RBCs, then adding the anti-human antibodies to see if they agglutinate

Question 8

What lab test is more commonly used for ABO blood groups besides the two-sided ABO test?

Answer 8

Micro-column Agglutination Test

Tube has antibodies against IgG, IgM, and C3d. Add patient blood and centrifuge.

Direct and/or indirect agglutination occurs.

Question 9

What are some diagnostic uses of passive agglutination?

Answer 9

• Rheumatoid factor latex test: IgM antibodies that attack Fc receptor of IgG
• hCG latex test (pregnancy)
• CRP latex test: test for inflammation

Question 10

How do we generate monoclonal antibodies in the lab?

Answer 10

Inject a mouse with antigen, wait for it to produce antibodies to that antigen, isolate the mouse’s B cells

Now fuse those B cells with an immortal myeloma cell

Get hybridoma cell that can produce those antibodies indefinitely

Question 11

When doing immunohistochemistry, are the primary antibodies monoclonal or polyclonal?

What about the secondary antibodies?

Answer 11

Primary are monoclonal: only recognizes very specific antigen

Secondary are polyclonal: recognize many epitopes of the first Ab, are better able to give a strong signal

Question 12

What is the difference in method between indirect ELISA and sandwhich ELISA?

Answer 12

Indirect ELISA: starts with antigen bound to the test container. Test to see if you have antibodies to that antigen in your sample. Add secondary antibodies that label with an enzyme -> color product.

Sandwhich ELISA: starts with antibodies bound to the test container. Captures any antigens you want to test. Then add secondary antibodies to form “sandwhich” and also have enzyme -> color product

Question 13

How does ELISPOT work?

What is an example of something it could be used to diagnose?

Answer 13

ELISPOT is similar to Sandwich ELISA, but tests the products secreted by living cells. Has Ab against specific products of cells. Shows up as dark spots when colored enzyme added.

Example: test for latent TB by having living T-cells exposed to TB and seeing if they activate and begin secreting IFN gamma (ELISPOT has anti-IFN Abs).

Question 14

What is the difference between RIA and IRMA?

Answer 14

RIA: Radioimmuno Assay: Similar to Competitive ELISA, but with radioactive labeling instead of enzyme. Radiolabelled antigen competes for an unlabelled Ag binding site. More radiosignal means weaker competition (negative result)

IRMA: Immunoradiometric Assay: Similar to Sandwich ELISA. Stronger signal = more antigen

Question 15

What is the advantage of confocal microscopy vs traditional light microscopy?

Answer 15

Confocal microscopy allows 3D imaging.

The light is focused on specific points and a laser-scanning microscope can read those with multiple images that combine to show the object in 3 dimensions

Question 16

What type of specimens are immunofluorescence ideal for?

Answer 16

Sections / microscope slides with cells

IF uses immunohistochemistry (normally indirect but potentially also direct) to label antigens, light them up or color them to be visible with microscopy. For example, can identify B cell lymphoma with anti-CD20 Ab

Question 17

How does Western Blot work?

Answer 17

1. SDS PAGE: Take bio sample of cell lysate, separate the proteins from it with gel electrophoresis. Use SDS to neutralize their charges
2. Blotting: Transfer to a blot/membrane to prevent the pattern from changing via diffusion
3. Labeling: Use primary and secondary antibodies to label

Question 18

How does a lateral flow test work?

Answer 18

1. Get antibodies for the antigen you want to target, attach those antibodies to a colored latex bead (passive agglutination)
2. On a strip, have one “control” antibody section that targets the antibodies themselves to see if your latex-Ab thing works.
3. Also put your “test” antibody section that binds the Ag-Ab mix
4. If you see both the “control” and “test” sections as colored, then you have a positive result on a working test

Question 19

What can the lateral flow test be used for?

(many things, and this was an MCQ question on my midterm.. lot of things)

Answer 19

• H. pylori IgG - Rapid test
• Pregnancy test (hCG)
• Rotavirus
• Adenovirus
• Hemoglobin – Fecal occult blood
• Helicobacter pylori stool antigen
• Morphine (300 ng) Test
• E. Coli O antigen
• HIV

Question 20

What (generally, like what types of things), can the following lab techniques measure:

• ELISA, RIA, IRMA, lateral flow
• ELISPOT
• Western Blot
• Immunocytochemistry

Answer 20

• ELISA, RIA, IRMA, lateral flow: soluble antigens from body fluids (urine, blood etc)
• ELISPOT: products of secretory cells
• Western Blot: soluble proteins from lysed cells
• Immunocytochemistry: cell/tissue antigens in situ

Question 21

What are two absolute indications to use flow cytometry for routine clinical measurements?

Answer 21

1. Hematological disorders: diagnosis of Leukemia and recurrence, multidrug resistance, minimal residual disease (MRD), monitoring bone marrow transplantation
2. Immunodeficiencies: like AIDS

Question 22

What are the 2 most important things about a cell that flow cytometry tells us?

(gonna list the 2 minor things on the answer card too but dont worry about it)

Answer 22

1. Size: How big the cell is relatively. Comes out as forward scatter
2. Granularity: deflects light, comes out as side scatter. Distinguishes granulocytes

(other two minor things: relative light intensity and time)

Question 23

Scatter plot: try to identify which cells are where

Answer 23

Question 24

Which CD markers are used in flow cytometry to identify:

NKT

NK

γδT

B

B1

Answer 24

NKT: CD3+ CD56+

NK: CD3-, CD56+

γδT: CD3+, γδTCR+, CD4 and 8 -

B: CD19

B1: CD5+

Question 25

Which markers are used in flow cytometry to identify: Th1 Th2 Th17 Treg (this one is a lot harder)

Answer 25

_Th1_: IFNγ+, IL-12+, TNFα, β + _Th2_: IL-4,5,10,13 + _Th17_: IL-17 +, TGF β+ _Treg_: CD25+, CD4+, Foxp3+

Question 26

What type of graph/test is this? What is the purpose of this particular graph? If the CD3+ section shifts upwards (higher peak), what does that mean? What if the CD3+ peak is the same, but it all shifts to the left?

Answer 26

**_FCM histogram_** * Shows difference between T cells and others that are CD3-, with the ratio of antigen presenting cells. * Higher peak = more T cells expressing CD3+ * Shifts to the left = less antigen is expressed per T cell

Question 27

Which part of cells does immunophenotyping use to identify them?

Answer 27

Their protein pattern

Question 28

CD3 is X axis, CD4 is Y axis So which box represents Th cells and which represents Tc cells?

Answer 28

Th is top right box Tc is bottom right box, also maybe some γδT Others are various cells that are negative for both, or a few CD4+ non-T cells (some monocytes, macrophages, etc have CD4)

Question 29

What is this graph a representation of, and what technique is done to make this graph? What important information does it give you?

Answer 29

**_Cell Cycle Analysis**_ via _**Propidium Iodide (PI) Staining_** Stains DNA, shows # of DNA molecules. The normal 46 are in G0G1 stage. Some are replicating and have 2x. Very few are between, in S phase. Others have less than 46 and are in apoptosis. The below graph shows cancer with S phase and G2/M elevation. Particularly diagnostic for bone marrow cancer, acute lymphoblastic leukemias.

Question 30

How can a _multidrug resistance protein_ (for example, one that expels antibiotics from bacteria) be tested with FCM analysis? (2 ways)

Answer 30

1. Detection by **immunophenotyping**: antibodies that target the protein 2. Provide **immuofluorescent substrates** to see how well the protein is able to expell them. It will be brighter if it does not expell the substrate.

Question 31

If you want to check for HIV infection with ELISA, what would you check with the indirect ELISA method? What would you check with the sandwich ELISA method?

Answer 31

_Indirect ELISA:_ check for **anti-HIV IgG antibodies**. Lets you know if the infection is *older* _Sandwich ELISA_: detect **HIV virus protein** (antigen) from serum. Will be positive earlier in infection If sandwich ELISA is positive but indirect is negative, then you have a more recent infection

Question 32

How do we check for the _presence of complement system_ via lysis of red blood cells?

Answer 32

1. Sheep RBCs + anti-RBC antibodies -\> agglutination of those RBCs, which condense to bottom of tube 2. **Add human (test) serum: the RBCs are then lysed via the classical pathway of complement activation, turning the tube red** 3. If you do the same at a higher temperature, it doesnt work because complement proteins are denatured

Question 33

The _complement fixation (consumption) test_ tests for what? How does it work?

Answer 33

Tests if there are serum *antibodies* for a particular antigen 1. Add the antigen and complement into test tube, then add serum. If the person has antibodies to that antigen, the complement will become "fixed" to the Ab-Ag complex 2. Add Sheep RBCs plus anti-sheep RBC antibodies. If complement was fixed to the original Ab-Ag complex, then there will be no complement to lyse the cells (*postive* result). If the there was cell lysis (tube turns red), then the serum lacked that antibody and complement attacked and lysed the sheep RBCs (*negative* result)

Question 34

What does the _CH50_ test check for?

Answer 34

Tests the *concentration of complement proteins* by which testing serum dilution lyses 50% of sheep RBCs. All complement proteins (C1-C9) need to be present to form MAC. Typical value is 142-279

Question 35

When would the _CH50_ be **high**? When would it be **low**?

Answer 35

**_CH50 high_**: acute inflammation only. C1, C3, CRP etc are acute phase proteins. Chronic inflammation is normalized **_CH50 low_**: genetic defects, liver insufficiency, autoimmune diseases or others that use up complement

Question 36

What is the major complication of complement defects that impair MAC? What about defects of C3b that impair phagocytosis?

Answer 36

_Impaired MAC_: only causes recurrent Neisseria infections _Impaired C3b_: recurrent bacterial infections in general (Other complement defects can cause rheumatological disorders, glumerulonephritis, and generalized edema)

Question 37

If C3 level and C4 level are reduced, what does that indicate? What if only C3 is reduced, while C4 is normal?

Answer 37

_Both C3 and C4 reduced_: the **classical pathway** has been activated _Only C3 reduced_: the **alternative pathway** has been activated (doesnt use C4)

Question 38

What is defective with **HANE**?

Answer 38

**_C1 inhibitor_** is defective in **hereditary angioedema (HANE)** C1 complex continuously made, but more importantly C1 inhibitor also inhibits kallikrein. Without it, **bradykinin** is overproduced, causing similar edema effects to histamine. They receive normal serum as treatmnet because it has C1 inhibitor.

Question 39

**Syncitiotrophoblasts, fibroblasts, keratinocytes, endothelial cells,** and **mesangial cells** are not typically considered part of the immune system. However, they all can secrete which interleukin?

Answer 39

**_IL-6_** The main acute phase reaction cytokine (IL-1 can replace its function if it's deficient)

Question 40

Chemokines: the names below are based on their structure. What are the other names? CXCL8 CCL5

Answer 40

CXCL8: **IL-8** (IL-8 is most important chemokine to know. Released by macrophages at first-line defense, attracts neutrophils) CCL5: **RANTES**

Question 41

_Lymphocyte extravasation_: a naive T cell will keep rolling along HEV with weak connections until it encounters what?

Answer 41

Until it encounters an **activating** **chemokine** that activates lymphycte and begins having conformational change of integrin molecules. This will begin _adhesion_, and then _transendothelial migration_ will follow

Question 42

Why don't HeLa cells form a monolayer when doing cell cultures with them?

Answer 42

Because they lack **contact inhibition**