Serology Flashcards
Serology
study of serum
Immunodiagnostics are used for…
- Detection of Ags
- Detection of Abs
- Diagnosis of immunologic diseases
- Measurement of blood concentrations of pharmacologic agents/hormones
General formula for testing for Ags
Sample + Abs => detectable Ag:Ab reaction (if present in sample)
General formula for testing for Abs
Sample + Ags => detectable Ag:Ab reaction (if present in sample)
Antiserum
serum from animal deliberately immunized for a particular Ag & that has Abs to react with that particular Ag
generally said to be polyclonal
monoclonal antibodies
Abs derived from a single clone of B-cells
Antiglobulins
Abs that react with immunoglobulins of a different species because those Igs make good Ags in the different species.
Antiglobulins use in diagnostic tests
used to detect Abs from a particular species
False Positive
test is positive but the animal isn’t infected with the agent
False Negative
Test is negative but the animal is infected with the agent
False Positive
testing for Ag
- recently vaccinated for the agent (& ags still present)
- infected with an agent that cross-reacts
False Negative
testing for ag
- low level of Ag present in specimen
- early in infection
- late in infection
False Positive
testing for ab
- cross-reactive abs are being detected
- previous vaccination or infection
False Negative
testing for ab
- low level of Ab present in specimen
- early in infection
- poor immune response to infection
Secondary Binding Assays
detect consequences of Ag:Ab binding
aka detect function!
Agglutination
USED TO DETECT Abs (& occasionally Ag)
- Ag is on a particle
- specific Abs cross-link the particles => clumping of particles
- Ag:Ab reaction detected by clumping
can be done on a slide or in wells (can do titers)
Agglutination to test Ags done by..
latex particles coated with Abs
Immunoprecipitation
NOT an immunodiagnostic test. (basis for other tests)
- Ag in solution
- specific Abs cross-link soluble Ags for large precipitating complexes
- Precipitate forms when Ag & Ab at ~equivalent [molar]
Agar Gel Immunodiffusion (AGID) Test
Detect Abs
Agar Gel Immunodiffusion (AGID) test
performed by…
- Ags & Abs placed in wells cut into an agar gel matrix
- Ags & Abs diffuse in agar & form precipitate when they meet at optimal proportions
- Ag:Ab reaction detected by formation of line of precipitate (precipitin line)
Complement Fixation
detects Abs
reported as titers
Complement Fixation
performed by..
- Ags & Abs mixed & source of complement is added
- Ag:Ab reaction detected by indicator system (sheep RBCs [SRBC] & Ab to SRBCs)
- no reaction in 1st step, complement can lyse SRBCs coated w/ Abs.
- reaction in 1st step, complement is consumed & no SRBCs lysed.
Complement Fixation results
positive test: Absence of SRBC lysis (complement consumed)
negative test: Presence of SRBC lysis (complement available)
Hemagglutination Inhibition
clinically, detect Abs
in lab, can ID virus isolated from clinical specimens using Abs w/ known specificity
done in a plate & reported as a TITER
Hemagglutination Inhibition mechanism
- some viruses cause agglutination of RBCs of certain species
- Ag:Ab reaction detected by inhibition of hemagglutination by virus
Serum Neutralization (SN) Test
Used to detect neutralizing Abs
Results reported as Titers
Serum Neutralization (SN) Test mechanism for viruses
- Virus grown in tissue culture cells.
- Growth detected by change in (or death) of cells
Serum Neutralization (SN) Test mechansim for toxins
-Toxin added to cells affected by the toxin
Serum Neutralization (SN) Test mechanism general
- 1st, virus or toxin incubated with Abs prior to adding it to cell indicator system
- neutralizing Abs detected by ability to inhibit damaging effects of the virus/toxin.
Primary Binding Assays
detect Ag:Ab binding DIRECTLY
aka detect BINDING
Types of Primary Binding Assays
Fluorescent Antibody test, ELISA (Ag & Ab), Western Blot, Immunohistochemistry, Immunochromatography
Types of Secondary Binding Assays
Agglutination, Immunoprecipitation, Agar Gel Immunodiffusion Test, Complement Fixation, Hemagglutination Inhibition, Serum Neutralization test
Fluorescent Antibody (FA) Test
Direct: detects Ag
Indirect: detects Ab
results reported as titers or qualitative results
Fluorescent Antibody Test mechanism
fluorescent molecule (fluorescein) covalently bonded to Ab. Ag:Ab reaction detected by presence of fluorescence on slide
Direct Fluorescent Antibody Test
tests for presence of Ag on a slide using a labeled Ab to the Ag
Indirect Fluorescent Antibody Test
tests for presence of Abs to an Ag on a slide using labeled antiglobulin reagent
Enzyme-Linked ImmunoSorbent Assay (ELISA)
for Ags
- Ab to specific infectious agent attached to surface
- Sample being tested for Ag added & unbound substances washed away.
- Enzyme covalently attached to 2nd Ab specific for infectious agent
- Enzyme converts colorless substrate (chromagen) to a colored product
- Ag:Ab reaction detected by presence of color reaction when chromagen added
Enzyme-Linked ImmunoSorbent Assay (ELISA)
for Ab
- Ag of infectious agent attached to surface
- Sample being tested for presence of Ab to the infectious agent added
- Unbound substances washed away
- Enzyme is covalently attached to: (1) Ag of infectious agent (2) antiglobulin
- Chromagen converted to colored product in enzyme’s presence
- Ag:Ab reaction detected by presence of colored reaction
Immunoblot, Western Blot
detect Abs
clinically used to demonstrate specificity of Ab for particular Ag in mix of Ags
Immunoblot, Western Blot mechanism
- Ags separated according to MW by agar gel electrophoresis then transferred to a membrane
- Ag:Ab reaction detected by addition of enzyme-labeled Ab, followed by chromogen
Immunohistochemisty
tests for Ag presence in particular location in a tissue specimen
(& occasionally Ab)
Immunohistochemistry mechanism
- Ag is present in a tissue section on a slide (biopsy)
- Ag:Ab reaction detected by addition of enzyme-labeled Ab, followed by chromogen.
Immunochromatography
detects Ags in blood sample
Immunochromatography mechanism
- Filter strip prepped w/ gold-labeled Abs @ one site & another site has unlabeled Abs (both recognize Ag being tested)
- Sample added to Gold-Ab site & Ag binds to gold-Ab if present.
- Buffer is added to cause gold-Ab to migrate toward unlabeled Ab @ 2nd site
- If Ag present, binds to Ab @ 2nd site, concentrating gold-Ab & making line.
Immunochromatography results in absence of Ag
Gold-Ab migrates to 2nd site when buffer is added, but NO line forms.
Complement Fixation detection method
activation of complement
Hemagglutination Inhibition detection method
inhibition of virus’s ability to agglutinate RBCs
Enzyme-Linked ImmunoSorbent Assay (ELISA)
detects Ag & Ab
Serum Neutralization detection method
neutralization of virus infectivity or toxin
Agar Gel Immunodiffusion (AGID) detection method
precipitation of soluble antigens
Immunoblot, Western Blot detection method
Ag separated according to MW by electrophoresis.
Ag or Ab labeled w/ enzyme. enzymatic reaction causes formation of colored product.
Immunohistochemistry (IHC) detection method
Ags are on glass slide (tissue section or cytology).
Ab or Ag labeled with enzyme. Enzymatic reaction causes formation of colored product.
Immunochromatography detection method
Ab is labeled w/ gold particles that migrate across a filter, capture of gold-labeled Ag:Ab complex leaves a visible line on the filter.
