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Immunology > Exam 2 > Flashcards

Exam 2 Flashcards

1
Q

If the concentration of the dilution is 100mg/dL and a 1:5 dilution of the specimen was done, what is the concentration of the original specimen?

A

100 * 5 = 500mg/dL

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2
Q

How much diluent needs to be added to 0.2 mL of serum to make a 1:20 dilution?

A

3.8mL

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3
Q

A 1:2 dilution of patient serum is necessary to run a serological test. There is 0.1 mL of serum that can be used. What amount of diluent is necessary to make this dilution using all of the serum?

A

0.1 mL of diluent

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4
Q

Describe the principle of precipitation and list techniques used:

A

Precipitation: the crystallization of previously soluble products
Examples: Single and Double immunodiffusion, Immunofixation electrophoresis

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5
Q

Describe the principle of electrophoresis:

A

The separation of proteins through cellulose via an electrical field. The proteins are separated based on the size and charge.

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6
Q

Compare and contrast electrophoresis and immunofixation

A

Immunofixation: combines electrophoresis and immunodiffusion. After the separation of bands, allowed to act as antigens against Ab, separated also by Ab specificity

Electrophoresis: separates proteins by size and charge. Separates alb, A1, A2, Beta and gamma

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7
Q

State the clinical application of immunofixation

A

Used to detect concentration changes in immunoglobulins

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8
Q

Describe the principle, advantages, and disadvantages of nephelometry

A

Light scattering properties of antigen/antibody complex. The amount of scattered light is proportional to the number of insoluble complexes. It is used to measure complement, immunocomplexes and presence of antibody.

The disadvantages are the high cost of equipment and interference substances present frequently

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9
Q

End point nephelometry

A

The reaction is allowed to continue until completion (not as accurate)

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10
Q

Kinetic or rate nephelometry

A

The rate of scattering increase is measured immediately after the reagent is added. The rate changed is directly proportional to antigen concentration when the quantity of antibody remains the same

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11
Q

What is the primary use for nephelometry?

A

The measurement of antibodies

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12
Q

Describe complement fixation technique

A

Patient serum is added to known antigen. Complement is added. If the serum contains the antibody that matches the known antigen it will bind complement, using the entire supply available. When RBCs and hemolysins are added the complement cannot react with the RBC’s because it is tied up in the 1st stage of testing

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13
Q

What outcome of complement fixation indicates that patient 1 has the antibody in question and patient 2 doesn’t have the antibody.

A
  • No hemolysis means the patient antibody was there

* Hemolysis means there was no patient antibody

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14
Q

Explain neutralization procedures.

A

Patient serum is mixed with a suspension of infectious virus suspected in disease. If the patient has antibody present it will attack the virus and make it incapable of invading additional cells upon exposure of the mixtures to new cells. If the patient does not have the antibody, the virus is free to invade the new previously uninfected cells

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15
Q

Illustrate where the antigen and antibody placement are and examples of identify, partial, and nonidentity results in a double diffusion technique:

A

Antigen and Antibodies diffuse independently though a semisolid medium in two dimensions, horizontally and vertically; commonly used to compare similarities between two antigens. A line of precipitation indicates the antigen—antibody complex. The same antigens will be at the top where the precipitation lines meet, partial ID will have one line with a longer tail of precipitation above the area where the lines meet and non ID has crossing lines of precipitation.

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16
Q

What would be the precipitation results for an Ouchterlony plate depicting the following information: Multiple antigens in center well. In outside wells, A=antibody 1, B=antibody 1 & 2, C=antibody 3, D=antibody 4 & 5

A

Partial antibodies between wells A and B. Nonidentity between wells B and C, C and D

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17
Q

What is usually moving in a single diffusion technique, the antigen or the antibody? What is usually stationary:

A

The antigen moves and the antibody is stationary

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18
Q

How is radial immunodiffusion measured?

A

The amount of precipitate formed is in proportion to the antigen present in the sample. In the mancini endpoint method, concentration is proportional to the diameter squared

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19
Q

Where is the antigen and antibody and which is usually the unknown in radial (single) immunoassays?

A

Gel-antibody-known

Well-antigen-unknown

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20
Q

Radioimmunoassay (RIA)

A

Competitive binding-Ag is absorbed on the surface of a solid phase/pt. Ab combines with radioactively labeled purchased antibody for the Ag sites/extremely sensitive, this testing method has the ability to detect trace amount in a large number of tests in a short time, care and disposal of isotopes is cumbersome

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21
Q

Radioallergosorbent test (RAST)

A

Antigen specific IgE-bound to cellulose disk/highly sensitive to bind small amounts of IgE

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22
Q

Radioimmunosorbent (RIST)

A

Competitive binding—checking for total IgE/the more IgE in patient serum, the less radioactivity in the test result (inversely proportional)

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23
Q

Immunoradiometic assay (IRMA)

A

Sandwich assay uses antibody excess on solid phase, easily automated. Unknown patient antigen is added and binds if matches. A second antibody with the isotope tag is added and binds to the patient antigen. Free unbound molecules are washed away. Radioactivity is proportional to amount of unknown patient antigens.

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24
Q

Explain the complement fixation test and how to interpret results using rbc’s:

A

If an antigen and antibody combination has occurred, complement will be bound. This can be an indicator of the presence of either specific antigen or antibody.
If the antibody—antigen combination takes place it uses up the complement so no hemolysis. If there isn’t an antibody—antigen combination, there the complement is free to cause hemolysis, which would indicate a negative result

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25
Q

What is flocculation?

A

Downy precipitate that may occur in an antibody—antigen reaction when the antigen is soluble

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26
Q

Would a flocculation technique be considered precipitation or agglutination?

A

Precipitation—Because the antigen is soluble

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27
Q

Explain the principle of immunofixation electrophoresis testing:

A

Unknown patient antigen is placed on a gen and separated by electrophoresis. A purchased antibody is applied. Precipitation occurs when the antibody—antigen complex form. The precipitate is strained and interpreted

28
Q

What are we usually looking for in the patient using immunofixation electrophoresis?

A

Identity of monoclonal antibodies

29
Q

What is passive immuodiffusion?

A

No electrical current is used to accelerate diffusion through the gel

30
Q

What is the law of mass action?

A

Free reactants are equal to bound reactants

Ag + Ab ↔ AgAb

31
Q

Zone of equivalence

A

Ag and Ab combine in appropriate amount with no excess antigen or antibody in the supernatant. Optimal precipitation occurs

32
Q

Prozone

A

No complexing or latticing to form a visible reaction due to antibody excess, possible cause of false negative reaction

33
Q

Postzone

A

No complexing or latticing to form a visible reaction due to antigen excess, possible cause of false negative reactions

34
Q

Describe the principle of agglutination. What affects the agglutination reaction?

A

Aggregation of particulate, insoluble test antigen depends on:
• Time of incubation with antibody source
• Amount of avidity of an Ag conjugated to the carrier
• Condition of the test environment (pH and protein)
Direct agglutination: Ab-Ag reaction occurs with antigens naturally occurring on the particles
Indirect or Passive Agglutination: Ag-Ab reaction occurs, particles are labeled with antigens not naturally occurring

35
Q

What is sensitization?

A

Antibody-Antigen combination without lattice formation

36
Q

What is the purpose and procedure for passive or indirect hemagglutination (Hemagglutination inhibition)

A

Purpose: Used for detection of some viral antibodies in patients
Procedure: RBC’s have naturally occurring viral receptors and will spontaneously agglutination if a virus is present. The presence of a viral antibody in the patient’s serum will block this reaction

37
Q

In hemagglutination inhibition a passive reaction would be evident by what type of reaction?

A

No agglutination

38
Q

Describe the principle of coagglutination?

A

S. aureus has protein A on the surface that naturally attaches to the Fc portion of antibodies. The Fab region of the antibody is free to attach to a specific antigen and link the bacteria together in a agglutination rxn

39
Q

How do you inactivate complement in a patient’s serum if it interferes with the testing:

A

Heat the patient serum for 30 min at 56 C (if more than 4 hours since inactivation, then reheat at 56 C for an additional 10 min)

40
Q

Explain agglutination inhibition:

A

There is competition between particulate and soluble antigens for limited amounts of purchased antibody. If there is no agglutination, then there must have been patient antigen present

41
Q

List factors that can enhance agglutination:

A

Centrifugation, enzymes, AHG, Colloids, Proper pH, Specific time frame, concentration

42
Q

What type of immunotechnique would be inherently good at detecting viral antibodies?

A

Hemagglutination inhibition

43
Q

Define avidity and affinity in relation to antigen / antibody complexes:

A

Avidity: the overall strength of binding between an antibody and an antigen
Affinity: is the initial force of the attraction between and antibody and antigen

44
Q

Define direct agglutination

A

Antigens are found naturally on a particle—such as A antigens on RBC

45
Q

Define hemagglutination

A

Agglutination reaction involves RBCs

46
Q

Explain passive or indirect agglutination.

A

Antigens not normally on particle
Antigens artifially adsorbed onto carrier particle/cell
Used to detect certain antibodies

47
Q

What is the difference between passive agglutination and reverse passive agglutination

A

Passive agglutination = antigen is attached to carrier molecule, the unknown is the antibody

Reverse passive agglutination = antibody is attached to the carrier molecules, the unknown is a multiple determinant antigen

48
Q

What are some reasons a false positive agglutination reaction may occur?
How do you correct them

A
  • over centrifugation
  • contamination glassware/reagents
  • crossreactivity
  • drying of slide test
49
Q

What are some reasons a false negative agglutination reaction may occur?

A
  • under centrifugation
  • poor washing techniques
  • reagents expired
  • delays in testing
  • insufficient incubation time, temp
50
Q

List general categories of tags that are used to visualize, or detect by machine, antigen-antibody complexes:

A

Enzyme label—EIA, Fluorescent label—immunofluorescent, Colloidal—metal or insoluble—SPIA, RBC’s Latex, Charcoal, Chemiluminescence

51
Q

ELISA

EMIT

A

ELISA - use of solid phase and wash prep

EMIT - Enzyme multiplied immunoassay technique. It is homogenous test (no wash step). If patient’s antibody is present it combines with the quenching antibody to leave the color reaction available. More color = more patient antibody to stop the quenching of the color indicator molecule

52
Q

Discuss the principle of competitive binding and noncompetitive binding.

A

Competitive: Purchased labeled antibody is mixed with unknown antigen. They compete to bind with a controlled amount of antigen. The more patient antibody in the serum, the less labeled antibody is bound to the antigen. Indicator (tag) is inversely proportional to patient unknown concentration

Non competitive:

  • antibody (capture antibody) is connected to a solid phase
  • unknown patient antigen is allowed to react
  • wash
  • second antibody with a label is added
  • amount of label is directly proportional to unknown
53
Q

Direct fluorescent testing

Indirect fluorescent testing

A

Direct: A fluorescent tag is connected to the antibody to detect antigen-antibody reactions at microscopic levels. Used for tissue sections or smears. The antigen is part of the molecule itself and not a separate substance.

Indirect: Purchased antigen on solid phase, unknown patient antibody is added. Then a fluorescent tagged anti-human antibody is added. Washed. If an unknown antibody is present, it will allow the tagged antibody AHG to attach giving a signal that is present.

54
Q

Briefly discuss the principle and use of chemiluminescent immunoassays.

A

Certain chemicals will emit a flash of light, which can be used as a tag to indicate location and or amount present. Used to detect structural abnormalities and concentration changes in proteins. The amount of patient antibody is inversely proportional to the amount of color

55
Q

Competitive ELISA

Noncompetitive ELISA/Sandwich ELISA

A

Comp: Specific antibody on solid phase, the enzyme labeled antigen and the unknown antigen are added. Excess is washed away. The color is then activated. The color is inversely proportional to the concentration of the unknown patient antigen

Noncomp: Needs a bivalent or polyvalent antigen. A wash phase is done, followed by the addition of another with a color indicator. The color is directly proportional to the amount of patient antigen

56
Q

Discuss whether the color is directly proportional or inversely proportional to the amount of patient antigen in the ELISA and EMIT

A

ELISA - Competitive—inversely Sandwich—directly

EMIT - Most assays are directly proportional. T4 assay are inversely

57
Q

Define solid phase and list examples that are commonly used:

A

The stable structure that can either hold the antigen or antibody. Glass beads, cellulose membranes, microtiter plates, glass slides, test tubes

58
Q

List the five groups of chemiluminescence labels:

A

1) Luminal
2) Acridinium esters
3) Peroxyoxalates
4) Dioxetanes
5) Tris—ruthenium II

59
Q

List the commonly used enzyme tags:

A

Acetylcholinesterase, Alkaline phosphatase, β-galactosidase, glucose oxidase, G6PD, Lysozyme, Malate dehydrogenase, peroxidase, horseradish peroxidase

60
Q

List commonly used fluorescent tags in immunoassays.

A

Fluorescein and Rhodamine

61
Q

If an enzyme immunoassay was allowed to incubate longer than the maximum required in the testing procedure, what may be altered in the results?

A

The test would most likely be falsely elevated because additional substrate may be utilized

62
Q

What is the difference between a homogeneous enzyme immunoassay and a heterogeneous immunoassay?

A 
Homogeneous = no wash phase in testing procedure
Heterogenous = wash phase in testing procedure
63
Q

Explain the principle of flow cytometry and its clinical applications:

A

the passage of cells in single file in front of a laser so they can be detected, counted and sorted. Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths

64
Q

Using flow cytometry, if a population of cells falls between normal myeloid cells and normal lymphoid cells, what would be the most likely explanation?

A

There is an abnormal cell population

65
Q

In flow cytometry, what is measure using forward-angle light scatter and using side scatter?

A

Forward light scatter—indicator of cell size

Side light scatter—indicator of granularity and intracellular complexity

66
Q

Concentration from dilution factor.

A

Concentration = (solute/solvent)

Immunology (4 decks)

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