lab 9 Flashcards
Immunologic methods- applications
What do serological tests determine?
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
Immunologic methods- applications
In a lab setting, Ab and Ag are often used in a variety of immunological tests such as:
Serological test
Detection of an unknown microbe from a lab culture
Detection of a broad range of non clinical substances- used extensively in research
Define Antigen
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
Define soluble antigen
small chemicals that are dissolved in a solution
-Ab’s will precipitate soluble Ags out of solution
Define Particulate antigen
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
Define Antibody
an immunoglobulin protein produced by B lymphocyte cells which attaches to Ag’s with high specificity leading to immune responses.
name five classes of Ab’s consisting of one or more monomers
IgG, IgA, IgM, IgD, IgE
Diagram, label and explain an Ab monomer
2 identical longer heavy chains 2 identical shorter light chains disulfide bonds constant region variable region fragments- Fab and Fc
Define immunology
the study of the structure and function of the immune system
Define serology
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
Name 5 immunologic tests
Fluid precipitation Slide agglutination of unknown microbe Commercial Kit - monospot ELISA Gel precipitation tests
What does the Gel precipitation tests detect/identify
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
What is The Zone of Equivalence
The zone at which Ab and Ag are at optimal proportions and form a large “insoluble lattice”
What are the 3 methods used to identify unknown microbes to the level of genus and species
Phenotypic - using morphology, physiology and biochemistry
Genetic - using nucleotide sequences within nucleic acids
Immunological - using specific antibodies that react to specific microbes
What is Slide Agglutination test
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
What does a Monospot Commercial Kit do
Diagnosis of infectious mononucleosis caused by Epstein-Bar virus (EBV)
What does a Monospot Commercial Kit detect
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
What does ELISA state for
Enzyme Linked ImmunoSorbent Assay
What is ELISA do and used for
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
4 Types of ELISA tests
Direct
Indirect
Sandwich
Competitive
What does Indirect ELISA do
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
What is Antibody Titre
quantitative assay to determine amount of Ab present in serum to a particular Ag
Why perform an Antibody Titre test?
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
What is seroconversion
is the development of detectable specific antibodies to microorganisms in the blood serum as a result of infection or immunization
What is Fluid Precipitation Test
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
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
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
Define antiserum
a blood serum containing antibodies against specific antigens, injected to treat or protect against specific diseases.
Define Agglutination
Ag or Ab is identified by “clumping” on plate, clear plate is visible
Slide Agglutination test info
Agglutination looks like grains of sand and it must be uniform. No agglutination will just look turbid
What is Burkitt’s lymphoma? Who is at risk? Where more prevalent?
Aggressive form of non-Hodgkin’s lymphoma (B cells).
Most common in children living in sub-Saharan Africa. Immunocomprimised
How is Infectious Mononucleosis spread?
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
ELISA test result colors: positive? negative?
Blue color indicates presence of HIV. Clear color indicates lack of HIV
What materials are used for ELISA test?
Multi-channel pipetter, automatic plate washer, automatic plate reader - reads absorbance in liquid in each well - quantitative measurements
Characteristics of an Agarose plate
Not for growth
No proteins or nutrients
Diffusion medium
Characteristics of an Agarose plate
Not for growth
No proteins or nutrients
Diffusion medium
What happens to antigens when an antibody attaches?
When antibodies attach to an antigen, that antigen is coated, precipitated or agglutinated. = inactivating the antigen
List 2 reasons why it is important to use controls when testing?
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
What does Antitoxin contain
Antibodies that will neutralize exotoxins
What is Adjuvant
is a chemical included with vaccines to enhance an immune response
What is monoclonal antibody?
They are produced by a hybridoma cell which is a plasma cell fused with an myeloma cell
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?
- 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
What are the limitation to the Monospot test?
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
What is added to Mac plate to inhibit gram positive and fastidious gram negative bacteria
Bile salts and CV
particular antigens are used in which test
Agglutination
One the antibody V (variable) structure, what is the function
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
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
all of the above
