Exam 1: Immunological Techniques Flashcards
Serum
Clear yellow fluid that is released when blood can clot and clot contracts.
Most clotting factors removed in process.
Plasma
Claer yellow fluid that forms the liquid phase of blood.
Can be obtained from blood using anticoagulants.
(Clotting factors are still present)
Titer
Highest dilution of a reactant able to give a reaction.
(Ab or Ag)
Serology
Subdiscipline of immunology that studies the reaction of Ab with Ag.
Precipitation
Rxn between soluble Ag with Ab (precipitin) resulting in the loss of solubility.
Agglutination
Clumping of an insoluble Ag by Ab (agglutinin)
Antiserum
Serum that contains Ag specific Ab
Prozone Effect
Zone of Ab excess
- No free Ag in reaction mixture
- Free Ab in supernatant
- May need to dilute serum to see reaction
Direct ELISA
- Unlabeled Ag attached to inert substance
- Bound Ag detected by an Ab that has been labeled with an enzyme.
- Substrate added
- Colometric or fluorescent change detected
Indirect ELISA
- Unlabeled Ag bound to plate.
- Serum added to plate & Ab/Ag interaction allowed to occur.
- Enzyme conjugated with anti-Ig added which binds to previous Ab.
- Excess or unbound enzyme-anti-Ig washed away.
- Enzyme substrate added.
- Color change quantitated.
Sandwich ELISA
Used for quantitation of antigen.
- Ab conjugated to plate.
- Ag added.
- Enzyme-labeled Ab added.
- Substrate added.
- Color change detected.
Sample Titer
The reciprocal of the highest dilution of serum that gives a positive reaction and beyond which there are only negative reactions.
Serological Diagnosis
A definitive serological diagnosis for an acute infection shows a four-fold increase in antibody titer between two serum samples drawn at least 10-14 days apart.
Referred to as acute and convalescent serum samples.
Identification Techniques
Table
Immunohistochemistry is used to…
detect and localize antigens in tissues and cells
Direct
Immunohistochemistry
Uses a labeled Ab to a specific antigenic component.
- Sample immobilized on a slide and treated with a tagged primary Ab.
- Sample washed to remove unbound Ab.
- Sample viewed by LM or fluorescent microscopy.
Indirect
Immunohistochemistry
Secondary antibody has been labeled which allows for detection of lower Ag concentrations.
- Sample immobilized on a slide and treated with primary Ab or serum.
- Sample washed to remove primary Ab.
- Secondary Ab added which reacts with primary Ab (eg anti-Ig Ab)
- Sample washed to remove unbound secondary Ab.
- Sample viewed by LM or fluorescent microscopy.
Flow cytometry
&
Fluorescence-activated cell sorting (FACS)
Useful to identify status of a single cell in a mixed population.
Utilizes tagged Ab to identify different molecules on the cell surface or expressed in the cytoplasm allowing visually identical cells to be differentiated.
- Cells stained with fluorescently labeled Ab specific for the cell type analyzed.
- Suspension forced through nozzle which seperates it into droplets each containing a single cell.
- Droplets passed through a laser beam.
- As each cell scatters the laser, it is detected.
- If cell is bound by fluorescent Ab, then signal emitted and recorded.
Western Immunoblotting
Gel electrophoresis + Ab-Ag Interactions
Useful in analysis of intracellular or serum proteins.
Allows ID of Ab reactivity to specific components.
Commonly used to confirm reactive serology for HIV diagnosis:
- Viral antigens denatured and separated by SDS-PAGE
- Transfer from gel onto membrane
- Serum added and bind to viral Ag on membrane
- Membrane washed and incubated with enzyme-conjugated anti-human IgG
- Enzyme substrate added to visualize Ab/Ag complexes
- Color/band that forms identifies MW of protein
Precipitin Curve
Increasing [Ag] added to constant [Ab]
-
Zone of Antibody Excess
- [Ag] insufficient to react with and precipitate all Ab present
- Free Ab in supernatant
-
Zone of Equivalence
- [Ag] sufficient to combine with and precipitate all Ab present
- No free Ab or Ag in supernatant
-
Zone of Antigen Excess
- [Ag] exceeds that required to bind all Ab
- Reduction of Ab-Ag precipitation
- Due to solubilization of Ag-Ab complexes by excess Ag
Direct Agglutination
- Particulate Ag reacted with specific Ab
- Ag ⇒ RBC, bacteria, latex bead
- Ab ⇒ two or more receptors
- Ab-Ag complexes can be cross-linked
- Results in visible clumping
Indirect Agglutination
“Passive agglutination”
- Soluble Ag absorbed or covalently linked to a carrier particle
- Reacted with bivalent + Ab similar to direct agglutination
Polyclonal Antibodies
Ig that react against an antigen and originate from many different plasma cells.
Derived from the total pool of activated B-cells within an organism.
(Multiple specificities and isotypes)
Monoclonal Antibody
Ig reacting against an Ag produced by a colony originating from a single plasma cell.
Usually prepared by somatic cell hybridization or hybridoma technology.
(Single specificity and single isotype)
Hybridoma Formation
- Animals immunized with Ag ⇒ Ab made
- Spleen removed and a single-cell suspension made
-
Cells fused with B cell myeloma cell line
- By incubating cells with polyethylene glycol (PEG)
-
Fusion treated with HAT media
- Hypoxanthine aminopterin thymidine (HAT)
-
Screen HAT-resistant clones by ELISA or RIA
- Myeloma cell provides immortality
- Spleen cells provides HGPRT for purine nucleotide synthesis
