Antigen/ Antibody Interactions Flashcards
What causes precipitation of immune complexes? What point of the precipitin curve has optimal lattice formation?
Lattice formation into an insoluble complex; equivalence zone
What is the titer in agglutination?
The reciprocal of the most dilute solution at which cross-linking of a particulate antigen is seen.
What is the prozone of an agglutination curve?
The part of the precipitation curve in which too much antibody inhibits cross-linking of particles
What is the difference in precipitation and agglutination?
Precipitation occurs typically with smaller, initially soluble antigens, while agglutination occurs with larger antigens
How is slide agglutination used for diagnosis?
A slide can be coated with an antibody to or antigen of a particular pathogen and then patient sample is added. If agglutination occurs then the patient is positive for the pathogen
What is hemagglutination? What is its utility?
Agglutination of RBCs, which can be used to type blood based on the ABO/ Rh blood type
What is an isohemagglutinin?
An antibody (mostly IgM, some IgG) that agglutinates red blood cells from a member of the same species
How does an hemagglutination inhibition assay work? What is it used for?
Method used to detect infection with influenza virus. The virus has hemagglutinin protein which binds to RBCs- virus can agglutinate RBCs. Patient serum is incubate with the virus- if the patient has made antibody to virus it will bind to the virus and inhibit the binding of the virus to the RBCs.
How is the titer of a hemagglutination inhibition assay dtermined?
The titer is the reciprocal of the highest dilution in which inhibition (no agg.) is seen
How can hemagglutination inhibition assays be used to test vaccine efficacy?
Antibodies made against previous season’s virus are tested against the current year’s virus. A new vaccine must be developed if HI titers differ by more than 2
What is the difference in between a direct and indirect immunoassay?
Direct assays involve a detection antibody binding directly to antigen. Indirect assays involve a detection antibody binding to another antibody bound to antigen
What are the benefits of an indirect immune assay?
An indirect assay amplifies the response when a direct assay is not sufficient to visualize results, it is more cost-effective too
What is a Coombs Test?
Measures for the presence of antibodies to rhesus antigen (D antigen)
How is a direct Coombs tests performed?
RBCs are taken from the fetus and determine if they agglutinate with rabbit anti-human Ig
How is an Indirect Coombs Test performed?
Serum is taken from mother and incubated with Rh+ RBC, add rabbit anti-human Ig- if positive- agglutination
How is a diffusion assay performed?
Soluble antigens and/or Ab are added to wells in agar and allowed to diffuse towards each other to form an equivalence zone
How is radial immunodiffusion performed? For what is it used?
Ag s placed into an Ab-agar, Ag diffuses radially and precipitates at the equivalence zone such that the ring diamter is proportional to Ag concentration; Used to quantitate serum proteins
What is ouchterlony?
A double diffusion technique where Ag and Ab are placed in separate wells in a gel and can be used to identify cross reacting antibodies or antigens that share epitopes
How is immunoelectrophoresis performed?
Antigens are loaded into agar, subjected to electrical current, and migrate based on size and charge. Antiserum is added to the trough underneath and precipitation curves indicate interaction
How is a radioimmunoassay performed?
Radio- or fluoro- labeled Ag are used to measure the amount of antigen in a sample
What is ELISA? Is direct or indirect more common?
Enzyme-linked immunosorbent assay
What is a sandwich ELISA?
Antibody is coated at the bottom of well, antigen is added, and another antibody is added that recognizes the antigen, followed by a labeled anti-Ig antibody
Which method of ELISA is most sensitive?
Sandwich ELISA
How does flow cytometry work?
Cells are stained with fluorochrome label antibodies and drip one at a time through a laser that produces differing light refraction patterns depending on the cell type
For what do complement fixation assays test?
Tests for specific antigens or antibodies in a patient’s sample
How does the complement fixation assay work?
Patient’s serum is mixed with an antigen and a standardized amount of commercial complement. If a patient has proper antibody then it will form immune complexes and fix the complement. Serum is then added to sheep RBCs– if lysis occurs that means there was still complement in the serum, meaning there was no antibody. If there is no lysis that means all of the complement was fixed by immune complexes
