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Microbiology Lab > lab 9 > Flashcards

lab 9 Flashcards

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1
Q

Immunologic methods- applications

What do serological tests determine?

A

The presence of Ag or Ab in serum
The concentration of Ab in serum (i.e. Antibody titre)
-used to determine if exposure to Ag is current or was in the past
-used to determine the strength of immunity to a particular Ag

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2
Q

Immunologic methods- applications

In a lab setting, Ab and Ag are often used in a variety of immunological tests such as:

A

Serological test
Detection of an unknown microbe from a lab culture
Detection of a broad range of non clinical substances- used extensively in research

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3
Q

Define Antigen

A

a molecular structure that induces an immune response… said to be “antigenic” or “immunogenic”
-antigens can be materials such as proteins, carbohydrates, whole cells or viruses, or parts of cells or viruses etc

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4
Q

Define soluble antigen

A

small chemicals that are dissolved in a solution

-Ab’s will precipitate soluble Ags out of solution

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5
Q

Define Particulate antigen

A

whole/partial microbial cells or large particles that do not dissolve in a solution, and so are visible to the eye.
-Ab’s will agglutinate particulate Ag’s

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6
Q

Define Antibody

A

an immunoglobulin protein produced by B lymphocyte cells which attaches to Ag’s with high specificity leading to immune responses.

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7
Q

name five classes of Ab’s consisting of one or more monomers

A

IgG, IgA, IgM, IgD, IgE

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8
Q

Diagram, label and explain an Ab monomer

A 
2 identical longer heavy chains
2 identical shorter light chains
disulfide bonds
constant region
variable region
fragments- Fab and Fc
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9
Q

Define immunology

A

the study of the structure and function of the immune system

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10
Q

Define serology

A

a branch of immunology that studies blood products to detect the presence of antigens, antibodies or other immune substances in serum

  • directly identify microbe causing disease
  • identify antibodies in serum that indirectly indicates exposure to specific microbe
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11
Q

Name 5 immunologic tests

A 
Fluid precipitation
Slide agglutination of unknown microbe
Commercial Kit - monospot
ELISA
Gel precipitation tests
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12
Q

What does the Gel precipitation tests detect/identify

A

Detection of Ag’s in serum using known Ab
Determination of Ab titre
Identification of source of ground meat
Use of Agarose Plate - no band=negative. band=postivie

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13
Q

What is The Zone of Equivalence

A

The zone at which Ab and Ag are at optimal proportions and form a large “insoluble lattice”

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14
Q

What are the 3 methods used to identify unknown microbes to the level of genus and species

A

Phenotypic - using morphology, physiology and biochemistry
Genetic - using nucleotide sequences within nucleic acids
Immunological - using specific antibodies that react to specific microbes

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15
Q

What is Slide Agglutination test

A

Simple qualitative assay to detect the presences of Ab or an Ag on a glass or cardboard slide

  • if agglutination is visible, Ag or Ab is identified
  • can be used to identify specific microbe, other antigens or presence of Ab’s
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16
Q

What does a Monospot Commercial Kit do

A

Diagnosis of infectious mononucleosis caused by Epstein-Bar virus (EBV)

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17
Q

What does a Monospot Commercial Kit detect

A

heterophile Ab in blood that react with EBV

  • heterophile Ab’s are a type that reacts with the target Ag (e.g. EBV) but also cross reacts with other Ag’s
  • limitation exists
  • if needed, other tests are used to detect actual EBV
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18
Q

What does ELISA state for

A

Enzyme Linked ImmunoSorbent Assay

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19
Q

What is ELISA do and used for

A

A quantitative assay used to detect the presence and concentration of an Ab or an Ag in a sample
Use for various Ag’s
-microbes, viruses, toxins, enzymes, allergens, misc biochemical
Medicine, Agriculture, Food industry, Industry, etc
-ELISA used for screening for HIV, followed by Western Blot for confirmation

20
Q

4 Types of ELISA tests

A

Direct
Indirect
Sandwich
Competitive

21
Q

What does Indirect ELISA do

A

Antigen is added to well - sticks to polystyrene
Primary Ab is added - has specificity of Ag and binds to Ag
Secondary Ab/enzyme complex is added - has specificity to Fc of primary Ab. Enzyme has specificity to substrate
Colorless Substrate is added → if enzyme is present, it acts on substrate → color change occurs. Color change = antigen present

22
Q

What is Antibody Titre

A

quantitative assay to determine amount of Ab present in serum to a particular Ag

23
Q

Why perform an Antibody Titre test?

A

Determine immune status
-no exposure if titre = 0
-past or current infection if titre is >0
-determine if current infection - if first titre = 160 and 2 weeks later titre= 640
Determine strength of immune response
Determine if seroconversion occurred following vaccination

24
Q

What is seroconversion

A

is the development of detectable specific antibodies to microorganisms in the blood serum as a result of infection or immunization

25
Q

What is Fluid Precipitation Test

A

Or ring test takes advantage of a phenomenon where small soluble antigens and matching antibodies form complexes which cross link with each other and form a lattice

26
Q

Interpret titres of 3 students suspected of having mononucleosis
Pt A= day 1, 160. day 7, 160. day 14, 160
Pt B= day 1, 0. day 7, 0. day 14, 0
Pt C= day 1, 80. day 7, 160. day 14, 1280

A

Pt A - had mononucleosis or at least was sensitized to Epstein-Bar virus
Pt B - has never had an immune response to the virus
Pt C - currently has the disease mononucleosis

27
Q

Define antiserum

A

a blood serum containing antibodies against specific antigens, injected to treat or protect against specific diseases.

28
Q

Define Agglutination

A

Ag or Ab is identified by “clumping” on plate, clear plate is visible

29
Q

Slide Agglutination test info

A

Agglutination looks like grains of sand and it must be uniform. No agglutination will just look turbid

30
Q

What is Burkitt’s lymphoma? Who is at risk? Where more prevalent?

A

Aggressive form of non-Hodgkin’s lymphoma (B cells).

Most common in children living in sub-Saharan Africa. Immunocomprimised

31
Q

How is Infectious Mononucleosis spread?

A

person to person contact. by saliva (kissing) or on hands and toys. Sharing food/drinks/silverware with infected person. If infected person sneezes/coughs near you

32
Q

ELISA test result colors: positive? negative?

A

Blue color indicates presence of HIV. Clear color indicates lack of HIV

33
Q

What materials are used for ELISA test?

A

Multi-channel pipetter, automatic plate washer, automatic plate reader - reads absorbance in liquid in each well - quantitative measurements

34
Q

Characteristics of an Agarose plate

A

Not for growth
No proteins or nutrients
Diffusion medium

35
Q

Characteristics of an Agarose plate

A

Not for growth
No proteins or nutrients
Diffusion medium

36
Q

What happens to antigens when an antibody attaches?

A

When antibodies attach to an antigen, that antigen is coated, precipitated or agglutinated. = inactivating the antigen

37
Q

List 2 reasons why it is important to use controls when testing?

A

Controls are used to monitor the validity of the results. If the reaction of the controls are not as expected the test results are invalid

38
Q

What does Antitoxin contain

A

Antibodies that will neutralize exotoxins

39
Q

What is Adjuvant

A

is a chemical included with vaccines to enhance an immune response

40
Q

What is monoclonal antibody?

A

They are produced by a hybridoma cell which is a plasma cell fused with an myeloma cell

41
Q 
What is the role of the following in the ELISA test?
Washing walls after each step?
Use of a primary antibody?
Use of a secondary antibody?
Running test in triplet?
A
  • To avoid reaction, to rinse whatever Ab or substrate used prior to it doesn’t react with the next one used
  • Bind to the agent - has specificity to Ag and binds to Ag
  • Bind to the Fc portion of primary antibodies - specificity to provides enzyme
  • Results are more accurate using 3 tests
42
Q

What are the limitation to the Monospot test?

A

Lower sensitivity and negative test is not conclusive
Some people do not produce detectable heterophiles antibodies
Detectable levels of heterophiles antibodies may persist for years in some people
Other tests are available to detect actual antigens of EBV (Monospot test is for detecting heterophile antibodies which are cross reactive antibodies to multiple antigens similar to EBV

43
Q

What is added to Mac plate to inhibit gram positive and fastidious gram negative bacteria

A

Bile salts and CV

44
Q

particular antigens are used in which test

A

Agglutination

45
Q

One the antibody V (variable) structure, what is the function

A

This is the variable region that is specific for the epitope of the antigen. This region is used to bind the epitope of the specific antigen

46
Q

Which of following cell structures are examples of where antigens may be found or the molecule may act as an antigen?

a. microbial cells and viruses
b. proteins and polysaccarides
c. cell membranes and bacterial capsules
d. all of the above

A

all of the above

Microbiology Lab (12 decks)

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