Precipitation & Agglutination Reactions Flashcards
Define serology.
Study of fluid components in the blood, especially antibodies.
Define serum.
Liquid portions of the blood without coagulation factors.
Define antigen.
A foreign substance that initiates an immune response in the human body.
Define antibody.
A protein produced in response to antigens.
What are examples of antibodies?
Immunoglobulins.
What is a monoclonal antibody?
An antibody that recognizes one epitope.
What is a polyclonal antibody?
Recognizes multiple epitopes.
Define sensitivity.
To correctly identify the patients WITH the given disease you are looking for.
Define specificity.
To correctly identify the patients WITHOUT the given disease you are looking for.
What types of tests do highly sensitive tests work well?
Screening testing.
What types of tests do highly specific tests work well?
Confirmatory testing.
What is a qualitative result?
The presence or absence of a particular antibody.
What is a semi-quantitative test?
Provide an estimate of the quantity of a patient’s antibodies produced against infection.
What is a quantitative result?
A specific amount of analyte.
What is a precipitation assay?
Soluble antigen combines with soluble antibody which produces a visible, insoluble complex.
What is an agglutination assay?
An antigen-antibody complex where antigen is bound to a particle or cell.
What is a requirement for a precipitation reaction?
Antigen and antibody must have multiple binding sites for one another. Equal relative concentration of each.
Define affinity.
Amount of initial attraction between antibody Fab site and antigen epitope.
Define avidity.
Sum of all the attractive forces between antibody and antigen. The “strength” of the forces.
Define the Law of mass action equation.
K = [AgAb]/[Ab][Ag]
K, from the law of mass action, is dependent on what?
The strength of binding between the antibody and antigen.
As K increases, the amount of AgAb complexes form. This causes the reaction to be ___.
more visible.
Define prozone in the precipitation curve.
A large excess of antibody.
Define postzone in the precipitation curve.
A large excess of antigen.
How is the electric current effected with passive gel precipitation.
No electric current; slow movement of precipitants.
How is the electric current effected with non-passive gel precipitation.
Electric current present; fast movement of precipitants.
Describe nephelometry.
Light scattered at an angle and is measured indicating the amount of antigen or antibody present.
In nephelometry, light scatter will ___ when the amount of immune complexes ___.
increase; increase.
Describe turbidimetry.
Measures cloudiness; the reduction of light transmission causes greater absorbance.
Describe radial immunodiffusion.
A single passive diffusion technique where gel is infused with antibody, and the patient antigen sample is added to wells. Measurement of radius determines antigen concentration.
Describe ouchterlony double diffusion.
A double diffusion technique where antibody AND antigen diffuse from wells. Lines on the precipitation indicate antigen relationship.
What are the three types of patterns with the ouchterlony double diffusion technique?
Identity, partial identity, and nonidentity.
What type of ouchterlony double pattern is shown?
Identity / serological identity.
What type of ouchterlony double pattern is shown?
Nonidentity.
What type of ouchterlony double pattern is shown?
Partial identity.
Describe immunofixation electrophoresis.
A double-diffusion (non-passive technique) that measures antibody isotope levels. Unknown antigens are electrophoresed then antibody (monoclonal) is applied to gel.
In immunofixation electrophoresis, when do precipitates form?
When antigen-antibody combination forms.
In immunofixation electrophoresis, the first lane detects what?
All serum proteins.
List and describe the two steps of an agglutination reaction.
(1) Sensitization: initial binding.
2) Lattice formation: formation of large aggregates (visible
What are the two enhancement stains used for AbAg reactions?
LISS (low ionic strength saline)
PEG (polyethylene glycol)
How does LISS effect reactions?
Neutralizes surface charge and cells can get closer together.
How does PEG effect reactions?
Enhances viscosity and decreases water around cells which brings them closer together.
Define AHG (antihuman globulin).
(Coombs reagent) Attaches to the Fc portion of IgG and helps to bridge the gap between RBCs so that a visible agglutination reaction can occur.
Describe direct antiglobulin (DAT), Coombs test.
Antibodies are attached to cells inside the body and AHG is added to a patient’s red cells to cause agglutination.
Describe the indirect antiglobulin test (IAT).
Patient’s antibodies are added to purchased RBCs. AHG is added to sensitize RBCs which causes agglutination.
What are three enhancement factors in AgAb reactions?
(1) Temperature.
(2) pH.
(3) Type of antibody.
Describe direct agglutination.
Antigen is naturally present on the cell or particle.
Describe indirect (passive) agglutination.
An antigen that is artificially adsorbed to carrier particle or cell.
Describe reverse passive agglutination.
ANTIBODY is attached to carrier particle or cell. This technique is used to detect if the antigen is present.
Determine if this is passive or reverse passive agglutination.
Passive.
Determine if this is passive or reverse passive agglutination.
Reverse passive agglutination.
Describe agglutination inhibition.
Competition between particulate (purchased antigen) and soluble antigens (patient) for limited antibody-combining sites.
In agglutination inhibition, when no agglutination occurs what is this indicative of?
A positive result.
