Immunologic Tests

Question 1

What do you use ELISPOT assays for?

Answer 1

• Commonly used to see if person has cytokine deficiency
• Like direct ELISA
• Growing cells over plate coated with specific cytokines antibodies

Question 2

How should you think of direct agglutination?

Answer 2

Thinks of it in terms of being able to visualize a reaction after the Fab portion of the antibody has bound an antigen.

Question 3

How should you think of indirect (passive) agglutination?

Answer 3

Think of it in terms of being able to visualize a reaction because the Fc portion of the antibody was bound to the colored latex bead so that when the Fab portion of the antibody binds to the antigen the beads clump together - but what you see are the beads bound to the Fc portion of the antibody.

Question 4

What do you see in direct agglutination?

Answer 4

Clumped cells

Question 5

What happens in direct agglutination?

Answer 5

• Put a droplet of antibody into blood
• RBC is large enough to see agglutination with naked eye
• Antibody + sample directly interact and you can see result

Question 6

What is an example of direct agglutination?

Answer 6

Blood typing

Question 7

What do you see in indirect (passive) agglutination?

Answer 7

See clumped latex beads

Question 8

What do you see in a negative indirect agglutination?

Answer 8

If you don’t have antibodies present, you just get a smear of color = negative result!!

Question 9

What do you use for indirect agglutination and what can’t you see with the naked eye?

Answer 9

• Very common to use small, latex beads for this

- You don’t see antigen-antibody reaction with the naked eye

Question 10

How does indirect (passive) agglutination work?

Answer 10

1. Add Colored Latex beads (scaffold) to + Specific Antibody
2. Then add antigen (ex: Viral particles, bacterial cells)
3. You’ll see particle agglutination in a Positive test.

Question 11

What are the three types of ELISAs?

Answer 11

• Direct
• Indirect
• Competition

Question 12

What are you detecting in direct ELISA?

Answer 12

You’re detecting the antigen/viral protein

Question 13

Why don’t we use direct ELISAs often?

Answer 13

It is more expensive.

It costs more to make a specific antibody.

Question 14

What starts out on the surface of the direct ELISA micrometer wells?

Answer 14

Antibody to the specific antigen we’re looking for in the patient’s serum.

Question 15

What are the 5 steps of direct ELISA?

Answer 15

1. Antibodies to the virus start off bound to the wells of the micrometer plate
2. Add patient sample (secretions, serum, etc.) suspected of containing virus particles or virus antigens and wash wells with buffer.
3. Add antivirus antibody containing conjugated enzyme.
4. Wash with buffer (wash away extra antivirus antibody)
5. Add substrate for the enzyme and measure amount of colored product

Question 16

What results of a direct ELISA indicate a positive tests?

Answer 16

Colored product

Question 17

How do you quantitate a direct ELISA?

Answer 17

Amount of colored product produced is proportional to amount of antigen.

Question 18

What does the test plate of a direct ELISA show?

Answer 18

Shows many reciprocal serum dilutions from 2-1024, also shows positive and negative controls.

Question 19

What are you looking for in an indirect ELISA?

Answer 19

Looking for patient’s immune response/antibodies to pathogen.

Question 20

Why do we use indirect ELISA more often?

Answer 20

It’s cheaper

Question 21

What stars out on the plate in an indirect ELISA?

Answer 21

Antigen

Question 22

What are the 6 steps of an indirect ELISA?

Answer 22

1. Coat microtiter wells with antigen preparation from disrupted HIV particles.
2. Add patient serum sample. HIV-specific antibodies bind to HIV antigen. Other antibodies do not bind.
3. Wash with buffer (Wash away extra antibodies)
4. Add anti-IgG antibodies conjugated to enzyme
5. Wash with buffer (wash away unbound anti-IgG antibodies-enzyme)
6. Add substrate for enzyme and measure amount of colored product

Question 23

What is a positive result of an indirect ELISA?

Answer 23

Colored product

Question 24

How do you quantify an indirect ELISA?

Answer 24

Amount of colored product is proportional to antibody concentration.

Question 25

What can't we find for competition ELISA?

Answer 25

A clinical application :(

Question 26

What are the six steps involved in a competition ELISA?

Answer 26

1. Mix patient specimen containing antigen with known amount of antigen-specific antibody. 2. Add specimen-antibody complex to antigen-coated well. 3. Wash to remove unbound antibody 4. Add anti-IgG antibodies conjugated to enzyme. 5. Wash to remove unbound anti-Ig. 6. Add substrate for enzyme and measure amount of colored product.

Question 27

How do you quantify a competition ELISA?

Answer 27

Colored product is inversely proportional to antigen concentration in patient serum.

Question 28

What sera is needed for an IgM test?

Answer 28

Single, acute (collected at onset of illness)

Question 29

What is the interpretation of a positive IgM test?

Answer 29

Newborn, Positive: in utero (congenital) infection | Adult, Positive: primary or current infection

Question 30

What is the interpretation of a negative IgM test?

Answer 30

Adult, negative: no infection or past infection

Question 31

What diseases can the IgM tests be applied to?

Answer 31

Newborn: STORCH (syphilis, toxoplasma, rubella virus, cytomegalovirus, herpes simplex virus) agents; other organisms Adult: any infectious agent

Question 32

What sera is needed for an IgG test (first type) ?

Answer 32

Acute and convalescent (collected 2-6 weeks after onset)

Question 33

What is the interpretation of a positive IgG test (first type)?

Answer 33

Positive: fourfold rise or fall in titer between acute and convalescent sera tested at the same time in the same test system.

Question 34

What is the interpretation of a negative IgG test (first type)?

Answer 34

Negative: no current infection or past infection, or patient is immunocompromised and cannot mount a humoral antibody response, or convalescent specimen collected before increase in IgG (Lyme disease, Legionella sp.)

Question 35

What can IgG tests be applied to detect (first type)?

Answer 35

Any infectious agent

Question 36

What sera is needed for an IgG test (second type)?

Answer 36

Single specimen collected between onset and convalescence.

Question 37

What does a positive IgG test (second type) in an adult indicate?

Answer 37

Adult, positive: adult evidence of infection at some unknown time except in certain cases in which a single high titer is diagnostic (rabies, Legionella, Ehrlichia spp.)

Question 38

What does a positive IgG test (second type) in a newborn indicate?

Answer 38

Newborn, positive: maternal antibodies that crossed the placenta

Question 39

What does a negative IgG test (second type) in newborn indicate?

Answer 39

Newborn, negative: patient has not been exposed to microorganism or patient has a congenital or acquired immune deficiency or specimen collected before increase in IgG (Lyme disease or Legionella sp.)

Question 40

What can IgG tests be used to detect (second type)?

Answer 40

Any infectious agent

Question 41

What sera is needed for an immune status evaluation?

Answer 41

Single specimen collected at any time

Question 42

What does a positive and negative result on an immune status evaluation indicate?

Answer 42

Positive: previous exposure Negative: no exposure

Question 43

How is an immune status evaluation applied?

Answer 43

It is used for: - Rubella testing for woman of childbearing age - Syphilis testing may be required in some states to obtain a marriage license - Cytomegalovirus testing for transplant donor and recipient

Question 44

What does Hemagglutination detect?

Answer 44

Are individuals producing antibodies that can bind red blood cells -OR- Is an individual infected with something that can bind to antibodies that normally bind to RBCs (how we detect flu in patient serum)

Question 45

What are you looking for in the two main hemagglutination tests?

Answer 45

1. Antibodies that bind surface of RBCs | 2. Viruses

Question 46

What happens with the antibody hemagglutination test?

Answer 46

- Same number of RBC in each well, but a decreasing amount of serum in each well - If an individual produces Ab that reacts with RBC, it forms a lattice and the RBC floats on the surface of the solution. - At hight amounts of serum, RBCs float on the surface - As soon as you have too few RBC to form a lattice, the RBCs pool at the bottom of the wells and form a button

Question 47

What does a NON-BUTTON indicate in an antibody hemagglutination test?

Answer 47

Positive result for antibodies

Question 48

What happens in the virus hemagglutination test?

Answer 48

- You have a constant amount of RBCs and you'll plate them across all the wells - Then, you add a constant amount of antibody with a decreasing amount of the patient virus - In a positive result, the cell surface proteins of the virus particles are soaking up the antibodies so they can't form a lattice with RBC so the RBCs sink to the bottom

Question 49

What does a positive result look like in the virus hemagglutination test?

Answer 49

- A positive result looks the opposite of the antibody agglutination test - Buttons will show on the left side and lattice (red) on the right side - Cell surface proteins of virus particle are soaking up the antibodies so they can't form lattice with RBCs so RBCs sink to the bottom [Ab mixed with high amount of virus gets all bound up and then RBCs sink to the bottom]

Question 50

What is a neutralization assay?

Answer 50

- Way to gauge whereto you have neutralizing antibodies against a particular toxin in a patient's serum. 1. First, you add the toxin molecules to cells and witness cell damage. 2. Then, you add toxin and antibodies toward that toxin together. 3. If the antibodies are neutralizing, they will bind the toxin. 4. Then neutralized toxin will be added in with cells and the cells with NOT be damaged.

Question 51

What is a complement fixation assay?

Answer 51

Checking to see if there is something wrong with the patient's formation of complement/fixation of complement. 1. Take RBC and antibodies to those red blood cells and add patients serum with complement. 2. If you have a combination of blood cell and antibody for blood cell and complement for the RBC, you would expect the cells to die/lyse.

Question 52

What is immunohistochemical microscopy?

Answer 52

Using fluorescently tagged antibodies, washing them over cells and looking for a microscopic signal under the scope. Blue stain = cells. Red stain = antibodies.

Question 53

What are hemagglutination inhibition reactions most useful for?

Answer 53

Detection of viruses and of antiviral antibodies. -Some viruses (most notably influenza) bear multivalent proteins or glycoproteins on their surfaces that interact with macromolecules on the RBC surface and induce agglutination of the RBCs.

Question 54

How is the hemagglutination reaction used to detect influenza?

Answer 54

1. To determine whether a patient has Abs to a particular stain of influenza virus, a technician would perform a serial dilution of the patient's antiserum in a micrometer plate. 2. Then, the technician would add the relevant virus and RBCs to each well, at concentrations known to induce hemagglutination. 3. If the patient's antiserum has anti-HA antibodies that bind the particular influenza strain being tested, the antibodies will attach to the HA molecules on the surface of the virus and prevent these molecules form inducing hemagglutination 4. Therefore, the hemagglutination reaction will be inhibited at several antibody concentrations.

Question 55

What are the three components of HAT media?

Answer 55

Hypoxoxanthine, Aminoptenin, Thymidine

Question 56

What is important to know about HAT media?

Answer 56

This is where you grow myeloma cells!!

Question 57

What is the function of hypoxoxanthine?

Answer 57

(rescues cells) --> requires the enzyme hypoxoxanthine phosphoribosyl transferase (HGPRT

Question 58

What is the function of Aminoptenin?

Answer 58

Poison to myeloma cells ---> blocks nucleotides --> blocks activity of DHFR --> Loss of classical pathway to make GTP, ATP

Question 59

What is the function of Thymidine?

Answer 59

Rescues cells

Question 60

What is the use of making hybridomas and monoclonal Abs?

Answer 60

Coming up with a way to produce antibody to specific antigen

Question 61

How do you make hybridomas and monoclonal Abs?

Answer 61

1. Challenge mouse with antigen you want to make Ab against 2. Isolate Antibody-producing B cells from spleen 3. Fuse myeloma cells with spleen B cells to make hybridomas 4. Grow cells in invitro culture system (to keep B cells alive in lab!); clone individuals hybridomase in microtiter wells [takes about a week or longer to "grow out" cells] 5. Test clones to identify desired antibody 6. Perpetuate clone in cell culture and/or mouse hybridoma tumor 7. Mouse hybridoma tumor gets injected back into mouse for testing. 8. Cell culture clones go on to produce monoclonal antibodies [These antibodies can be used for Western Blot, ELISA, etc.]