lecture 8 Flashcards
detect antibodies through
infectious agents
vaccine for antigens (measure primary or memory responses)
autoantibodies: anti-nuclear antibodies
maternal antibodies
reagent antibodies
allow us to detect antigens of pathogens in tissues
cell surface antigens detected by antibodies tell us
blood types
lymphocyte subsets: lymphoma, leukemia, immunodeficiency
tumors and tumor markers in tissues and blood
the specificity of antibody mediated immune responses can be exploited for
diagnostic purposes
serology confirms
results of other methods (virus isolation, PCR etc)
surveillance of infections in herds (detect antibody or monitor antibody levels)
serology
test of choice for many agents for which no test is available
test results from any serological test means
there has been an exposure to pathogens and/or some protein (components of pathogens as vaccine - antibodies present bc there was vaccine)
in young animal it also means they could receive antibodies from mother
serum test sample
top fraction after blood sample clots
contains serum proteins such as antibodies and many other proteins
antibodies will tell what antigens present in body
body cavity fluids test sample
peritoneal fluid: feline infectious peritonitis (FIP) virus
cerebral spinal fluid: west nile virus (WNV - TLR3 issue)
whole blood in anticoagulant
contains RBC, WBC and platelets
ex: coombs’ test, flow cytometric immunophenotyping
tissue sections
necropsy or surgical biopsy specimens
ex: immunohistochemistry or immunofluorescence for bovine viral diarrhea virus (BVDV) FIP
polyclonal antibodies
type of serologic reagent used in immunodiagnositics
multiple B cell clones make antibodies to different epitopes
antibodies generated against a certain pathogen or molecule
antiglobulin or antiserum consists of polyclonal antibodies made against immunoglobulins of different species
isotypes and half lifes are important in
antibody test result interpretation
monoclonal antibodies
type of serologic reagents used in immunodiagnostics
single B cell clone makes antibody to one epitope
made w hybridoma technology
hybridoma
hybridoma
hybrid cell line generated by fusion of a B cell (ANTIBODY PRODUCER) w an immortal neoplastic B cell (myeloma) - bad
monoclonal antibody production
host animal immunized w antigen of interest -> spleen lymphocytes harvested and fused w immortalized myeloma cells -> transfer to media w selection agent for immortalized hybridoma cells -> individual hybridoma cells are screened for antibody production -> cells that make antibody of desired specificity are expanded from a single antibody-producing clone
all descendent cells make the identical antibody
major types of immunodiagnostic tests
immunofluorescene assay (IFA)
enzyme assay
precipitation assay
agglutination assay
immunofluorescence assays (IFA)
antigen detection
antibody detection
serum or virus neutralization assay
can be direct or indirect
enzyme assays
enzyme linked immunoabsorbant assay (ELISA) - indirect
immunohistochemistry
western blot
need to wait how many days before can detect antibody in serum?
14 days
direct immunofluorescence
uses a primary antibody to detect the target protein
indirect immunofluorescence
uses antibodies in patients serum (PRIMARY ANITBODY) to detect an antigen followed by a secondary antibody to detect the primary antibody
ELISA
enzyme linked immunosorbent assay
enzyme assay - indirect
absorbance is proportional to the amount of antibody attached to the antigen and proportional to the amount of antigen attached to well
SN
serum neutralization
HI
hemagglutination inhibition assay
IFA
indirect fluorescent asay
CF
complement fixation
WB
western blot
enzyme assay
AGID
agar gel immunodiffusion assay
IFA indirect test results
positive (antibody is present in serum) is fluorescent
fluorophore binds to antibody (Ig) in patient serum and will cause stain
antiPCV antibody test
can use other animal to produce antibody to virus that would bind to PRRS virus in mouse? detects antibodies
antiPCV antibody test (ANA)
used in the diagnosis of immune response to PCV2 or any other viruses
IFA
serum neutralization test
tests function of the patient antibody, whether or not its antibodies block the virus infection or not
antibody present in serum + virus (incubation) -> target cell not infected - no fluorescence (FAVN)
no antibody present in serum + virus -> specific monoclonal antibody binds to flurophore and shows flourescence
FAVN
fluorescent antibody virus neutralization test
serum neutralization test
ELISA indirect test
well is precoated w antigen -> test serum is added w chosen antibodies to test for -> labeled antiglobulin is added -> enzyme substrate added and will wash away any nonbound antibody -> color changes when bound
ELISA competitive test
no antibodies = color = negative
antibodies present = no color = positive
labeled and unlabeled (patient serum) antibodies compete for binding to antigen -> addition of enzyme substrate leads to a color change that is inversely proportional to amount of patient serum (monoclonal antibodies!) antibody bound
more accurate
ELISA sandwich test
well is precoated w capture antibody -> test antigen is added -> detection antibody is added -> labeled antiglobulin is added -> color changes after enzyme substrate is added
antigen is bound to plate by means of an antibody (captured antibody!)
presence of bound antigen is detected by sequential addition of a secondary antibody (detection antibody) and an enzyme labeled antiglobulin
addition of enzyme substrate leads to color change proportional to amount of bound antigen
color = positive!!
