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IHO Week 6 > Immunologic Tests > Flashcards

Immunologic Tests Flashcards

1
Q

What do you use ELISPOT assays for?

A
  • Commonly used to see if person has cytokine deficiency
  • Like direct ELISA
  • Growing cells over plate coated with specific cytokines antibodies
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2
Q

How should you think of direct agglutination?

A

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

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3
Q

How should you think of indirect (passive) agglutination?

A

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.

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4
Q

What do you see in direct agglutination?

A

Clumped cells

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5
Q

What happens in direct agglutination?

A
  • 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
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6
Q

What is an example of direct agglutination?

A

Blood typing

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7
Q

What do you see in indirect (passive) agglutination?

A

See clumped latex beads

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8
Q

What do you see in a negative indirect agglutination?

A

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

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9
Q

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

A
  • Very common to use small, latex beads for this

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

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10
Q

How does indirect (passive) agglutination work?

A
  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.
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11
Q

What are the three types of ELISAs?

A
  • Direct
  • Indirect
  • Competition
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12
Q

What are you detecting in direct ELISA?

A

You’re detecting the antigen/viral protein

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13
Q

Why don’t we use direct ELISAs often?

A

It is more expensive.

It costs more to make a specific antibody.

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14
Q

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

A

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

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15
Q

What are the 5 steps of direct ELISA?

A
  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
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16
Q

What results of a direct ELISA indicate a positive tests?

A

Colored product

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17
Q

How do you quantitate a direct ELISA?

A

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

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18
Q

What does the test plate of a direct ELISA show?

A

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

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19
Q

What are you looking for in an indirect ELISA?

A

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

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20
Q

Why do we use indirect ELISA more often?

A

It’s cheaper

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21
Q

What stars out on the plate in an indirect ELISA?

A

Antigen

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22
Q

What are the 6 steps of an indirect ELISA?

A
  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
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23
Q

What is a positive result of an indirect ELISA?

A

Colored product

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24
Q

How do you quantify an indirect ELISA?

A

Amount of colored product is proportional to antibody concentration.

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25
Q

What can’t we find for competition ELISA?

A

A clinical application :(

26
Q

What are the six steps involved in a competition ELISA?

A
  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.
27
Q

How do you quantify a competition ELISA?

A

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

28
Q

What sera is needed for an IgM test?

A

Single, acute (collected at onset of illness)

29
Q

What is the interpretation of a positive IgM test?

A

Newborn, Positive: in utero (congenital) infection

Adult, Positive: primary or current infection

30
Q

What is the interpretation of a negative IgM test?

A

Adult, negative: no infection or past infection

31
Q

What diseases can the IgM tests be applied to?

A

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

32
Q

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

A

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

33
Q

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

A

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

34
Q

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

A

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.)

35
Q

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

A

Any infectious agent

36
Q

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

A

Single specimen collected between onset and convalescence.

37
Q

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

A

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.)

38
Q

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

A

Newborn, positive: maternal antibodies that crossed the placenta

39
Q

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

A

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.)

40
Q

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

A

Any infectious agent

41
Q

What sera is needed for an immune status evaluation?

A

Single specimen collected at any time

42
Q

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

A

Positive: previous exposure
Negative: no exposure

43
Q

How is an immune status evaluation applied?

A

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
44
Q

What does Hemagglutination detect?

A

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)

45
Q

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

A
  1. Antibodies that bind surface of RBCs

2. Viruses

46
Q

What happens with the antibody hemagglutination test?

A
  • 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
47
Q

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

A

Positive result for antibodies

48
Q

What happens in the virus hemagglutination test?

A
  • 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
49
Q

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

A
  • 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]
50
Q

What is a neutralization assay?

A
  • 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.
51
Q

What is a complement fixation assay?

A

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.
52
Q

What is immunohistochemical microscopy?

A

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

53
Q

What are hemagglutination inhibition reactions most useful for?

A

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.

54
Q

How is the hemagglutination reaction used to detect influenza?

A
  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.
55
Q

What are the three components of HAT media?

A

Hypoxoxanthine, Aminoptenin, Thymidine

56
Q

What is important to know about HAT media?

A

This is where you grow myeloma cells!!

57
Q

What is the function of hypoxoxanthine?

A

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

58
Q

What is the function of Aminoptenin?

A

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

59
Q

What is the function of Thymidine?

A

Rescues cells

60
Q

What is the use of making hybridomas and monoclonal Abs?

A

Coming up with a way to produce antibody to specific antigen

61
Q

How do you make hybridomas and monoclonal Abs?

A
  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.]

IHO Week 6 (16 decks)

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