Immunological testing Flashcards
What is ELISA? What does it measure?
Enzyme linked immunosorbent assay
Measures Ag or Ab, infection or hypersensitivity
What are the advantages of ELISA tests?
Quantifiable
Quick
Cheap
Reproducible
ELISA can detect specific antibodies in animal plasma/ serum. How does this work?
Coat plate wells with antibody Add serum samples Add enzyme tagged antibodies Add substrate (Wash between each stage to remove unbound reagents) Colour change = more antibody
Immune complex formations can be large or small. What does this depend on?
Relative amounts of antigen/antibody
Number of binding sites on antigen
Class of antibody
What is the method to test for agglutination for antibodies?
RBCs coated with antigen
Mixed with test serum
Positive sera = agglutination of RBCs
How do immunoflouresence tests work?
Specific antibodies conjugated to fluorscent dyes
Antibodies bind directly (primary) or indirectly (secondary) to the antigens
Put under short wavelength high energy light - immune complexes glow fluorescently
What is the most common serology test in small animal practice?
Snap test
Fast, done in practice
How does double diffusion work?
Wells cut in agar plate - one filled with antigen, one filled with antibody
Line of precipitation forms if two reactants meet
Double diffusion causes precipitation if the immune complex is produced at equivalence. What does this mean?
Equal amount of antibody to antigen
What does the concentration of each reactant depend on in double diffusion?
Concentration
Molecular size
Ability to diffuse
What are the 3 basic precipitation factors of double diffusion?
Lines of identity - identical antigens
Lines of non-identity - two lines that cross, serum contains 2 antigens
Lines of partial identity - serum contains an antigen with 2 different epitopes
What is single radial diffusion?
Antiserum containing known antibodies incorporated into agar
Wells cut out and antigen placed in wells
Immune complexes form radial diffusion ring - larger diameter = higher antigen concentration
What is immunoelectrophoresis used to detect?
B lymphocyte lymphoma (type of leukaemia)
What change in serum levels of antibodies does B-lymphocyte lymphoma result in
Increased antibodies
Due to clonal expansion of B cells
What is the difference between electrophoresis and immunoelectrophoresis?
Electrophoresis - identification of any increased antibodies
Immune - identifies specific pathological changes
How does electrophoresis work?
Patient serum (contains antigens) put into well in agar gel
Electric current is applied to cause antigens to migrate to either positive/negative ends of agar gel
Antiserum (containing specific antibodies) added to trough below agar plates
If immune complexes form - precipitation
Normal serum compared to patient serum
What is the snap assay?
Type of ELISA
Enzyme labeled conjugate mixed with serum/blood and added to sample well
Sample-conjugate mix flows through the matrix
The device is then activated by ‘snapping’ which releases wash buffer
Coloured precipitation = positive result
What is affinity?
Strength of reaction between single antigen and binding site (sum of attractive and repulsive forces)
What is avidity?
Overal strength of binding between an antigen with many determinants and multivalent antibodies
Complex size depends on relative amount of Ag/Ab, affinity and class of antibody. How can class of antibody affect complex size?
Different number of binding sites
E.g. IgG has 2 antigen binding sites, IgM has 10
What are useful properties of immune complex formation that helps with immunological testing?
Large complexes form (at optimal ratio of Ag:Ab)
Multidimensional lattice means visible precipitate can form
What holds the light chain of an antibody to the heavy chain?
Disulfide bond
What is an epitope?
The part of an antigen that interacts with the antigen specific receptor/antibody
What is immunoelectrophoresis mainly used to identify?
Myelomas (mainly)
Deficiencies
Acute bacterial infections
