MICRO EXAM 2 CH10 Flashcards
What occurs in an immunoassay between Ab-Ag?
An antibody molecule recognizes and binds to an antigen.
What is binding of antibody and antigen related to?
Conc of each reactant.
Specificity of Ab to Ag.
Affinity and avidity.
Environmental conditions (temp, pH and time).
Serology?
The study of serum.
What is an antibody?
Proteins in the serum that bind to antigen.
What is an antigen?
Anything that causes an immune response.
Usually a high molecular weight protein or polysaccharide.
What is an immune complex?
Ab attached to an Ag.
What is an immunogen?
The Ab-eliciting property of the molecule. (Is what triggers an immune response).
What is an epitope?
A small part of a molecule that is recognized by the immune system.
What is affinity?
The attraction between the Ab and Ag.
What is high affinity binding?
Strong binding interaction between Ab and Ag.
What is low affinity binding?
Weak binding interaction between Ab and Ag.
What is avidity?
Overall strength of Ab-Ag bond that has been formed.
Avidity and cross-reactivity relationship?
High avidity = low cross-reactivity and vice versa.
What is the law of mass action?
Governs the reversibility of the Ab-Ag rxn.
At equilibrium, ratio between Ab and Ag remain constant.
What are polyclonal Abs?
Mixture of multiple Ab that has been derived from different cells against multiple epitopes found on Ag(s).
What are monoclonal Abs?
Only one cell that has been exposed to one epitope and specific to only one epitope.
High specificty and avidity.
1st step of polyclonal Ab production?
Inject Ag into rabbit.
2nd step of polyclonal Ab production?
Ag activates B cells.
3rd step of polyclonal Ab production?
Plasma B cells produce polyclonal Abs.
4th step of polyclonal Ab production?
Obtain antiserum from rabbit containing polyclonal Abs.
1st step of monoclonal Ab production?
(Innocculation) Inject rabbit with antigen.
2nd step of monoclonal Ab production?
(Innocculation) Obtain whole blood from rabbit, centrifuge for plasma.
3rd step of monoclonal Ab production?
(Production) Mixing antiserum and myeloma cells to develop hybridoma cells.
4th step of monoclonal Ab production?
Hybridomas are cloned for individual cells.
5th step of monoclonal Ab production?
Test for monoclonal Abs.
What is cross-reactivity?
When an antibody binds to an antigen that is not the exact target, but share similar epitopes.
What immunoglobulin creates a primary response?
IgM
What immunoglobulin creates a secondary response?
IgG
known as anamnestic response
What is a titer?
Measurement of the amount of Abs for specific Ags.
Highest or last dilution is the result
How is a titer reported?
1:16 dilution = 16
How does an agglutination assay work?
Agglutinating Abs reacts with Ags on the surface of microscopic particles to form visible clumps.
What are agglutination tests performed on?
Glass, plastic or cardboard slides.
Tubes as well
What is a direct agglutination?
Natural carrier particles of patient Ab and Ag.
Example of direct agglutination?
RBCs, blood typing
Example of indirect agglutination?
Latex beads coated with Ag.
What is indirect agglutination?
Artificial carrier particles
What is the prozone phenomenon in latex agglutination?
Occurs when the concentration of Abs exceeds the concentration of Ags.
Also known as the zone of Ab excess.
What is the postzone in latex agglutination?
Concentration of Ags exceed the Ab concentration.
What kind of results can a postzone latex agglutination give?
False negs
What is the zone of equivalence?
Optimal ratio of concentration between Ab and Ag.
What is a labeled immunoassay?
Ag or Ab is labeled (tagged) with a substance that can be detected later on and allows for the detection of an antibody.
Types of tags?
Radioactive isotopes (RIA)
Enzymes (EMIT, ELISA)
Fluorescent molecules
Luminescent labels
Types of immunoassays?
Heterogenous and homogenous
What is heteogenous assays?
Separation assay that requires multiple steps.
What is homogenous assays?
Does not require a separation step.
What is a fluorescence polarization immunoassay?
It is a competitive and homogeneous assay.
How does fluorescence polarization immunoassay work?
Ag (reagent) that is tagged and an untagged Ag (patient) compete for specific Ab in a cuvette.
Results for fluorescence polarization immunosassay?
Larger molecules (tagged Ag) emit polarized light.
What is luminescence/chemiluminescence?
Process of exciting molecules by chemical means and measuring the light emitted as the molecules turn back to their unexcited state.
Competitive and heterogeneous
What is direct fluorescent Ab test (DFA)?
A conjugated Ab to fluorescent tag binds to an Ag. Looks for Ag.
Example of IFA?
FTA-ABS for syphillis
What is indirect fluorescent Ab test (IFA)?
Ag fixed onto slide with primary Ab of interest (not tagged). Secondary Ab conjugated to fluorescent tag binds to primary Ab.
What is immunofluorescent assays?
Rapid Ag or Ab detection.
Mono or polyclonal Abs are conjugated with fluorochromes.
What is competitive immunoassays?
Labeled known and patient unknown are added to a rxn to compete for the target.
Examples of fluorochromes?
Fluorescien
Rhadomine
Is the concentration inversely proportional in a competitive immunoassay?
Yes
What is a membrane-bound immunoassay?
An immunoassay that uses a membrane that has bound Abs.
What is the material used for a membrane-bound immunoassay?
Nitrocelulose, nylon, etc
What is an immunochromatographic test?
Detects Ag in lateral flow method. Abs bound to membrane.
1st step of immunochromatographic test?
Specimen placed in buffer
2nd step of immunochromatographic test?
Process sample is placed onto the membrane.
3rd step of immunochromatographic test?
Buffer is carried laterally along the membrane by capillary action.
4th step of immunochromatographic test?
Labeled rabbit Ab suspended in buffer.
5th step of immunochromatographic test?
If Ag is present, Ag labeled Ab latex is formed = pos line
