What are the target cells in chicken infectious anemia?
precursor T cells in the thymus
hemocytoblasts in bone marrow
In what age range would we see CIA and how is it transmitted?
chicks less than three weeks of age
fecal-oral or vertically via antibody negative breeder hens
vaccine exists for these hens, given at the start of their production (modified live); can be injected or added to water
What virus causes chicken infectious anemia?
What are common clinical signs in CIA?
anorexia, pale, decreased wt gain
mortality 10-50%; survivors recover slow
study of antigen antibody interactions in vivo. Principle of all antigen antibody reactions lies in the specific combination of epitopes on the antigen with the hypervariable regions or complementary determining regions of the antibody molecule
Serological assays differ
in their sensitivity, specificity, and speed. some are strictly qualitative, others may be qualitative or quantitative
Detection of antibody titer
4 fold increase between acute and covalescent serum samples collected 2-4 weeks apart against an etoliogic agent
Acute samples should be
frozen and should be submitted at the same time as convalescent sample
Disease can be diagnosed by
Detection of antibody titer rise, and detection of circulating antigen in certain infections, monitoring the level of humoral antibody immune response, and seroepidemiology
population survey using a serological test to screen for exposure and immunity to an infectious disease
What do all serological tests require?
Serum or plasma as the sample
Red top tube
lavendar top tube
plasma is required
fluid portion of blood remaining after the blood cells and materials responsible for clotting are removed.
devoid of antibodies
serum containing high concentration of antibodies
non cellular portion of blood obtained by collecting blood in an anticoagulant followed by centrifiguation
antibody that aggregates or clumps particular antigen (igM)
antispecie antibody. Antibody made against an immunoglobin usually by injecting immunoglobin into an animal of another species
antibody that forms precipitate with soluble antigen
appearance of antibody in the blood in response to infection, disease, or immunization.
Can be seronegative or seropositive
This should take note and include it in differential diagonsis.
Seropositive vs. seronegative
animal having detectable antigen-specific antibody. Animal with no detectable antigen specific antibody.
Seropositive is based off of
having titer being above a certain threshold level (cut off point). For example an animal with titer >1:4 may be classified as positive, whereas an animal with titer < or = to 1:4 may be negative
the type of microorganism as determined by its distictive antigenic properties.
(example ecoli with K antigen, O antigen, and H antigen
Variation within a serotype
a group of bacteria containing a common antigen or a group of viral species that are closly related antigenically.
the minimum concentration of a substance that can be reliably measured by a serological test. IT can measure a FASLE - NEGATIVE.
Relative Sensitivity of Tests measurement
need to use correct assay to detect protein bc there are limitations to every test .
the specificity of an antibody refers to its capacity to discriminate between ligands of similar structure by combining with them at different extents. The greater the differences in affinity for two cloesly related structures the more specific to the anibody FALSE POSTIVE RESPONSE
although Ag- Ab reactions are highly specific, in some cases antibody elicted by one antigen can cross react with an unrelated antigen. Cross reactivity occurs if two different antigens share an indentical epitope or if antibodies specific for one epitope also bind to an unrelated epotope pocessing similar chemical properties
The reciprocal of the highest dilution (lowest concentration) of serum antibody that produces a test reaction
Example of titer
highest dilution that produces a test reaction is 1 in 16, then the titer is 1/16. this means that the undiluted serum contains 16x the antibody for the reaction
Two fold serial dilution of serum
titration is accomplished by serially diluting the serum in an series of decreasing concentration in a test tube.
Tests involving enzyme labeling
enzyme covalently conjugated to an antibody reacts with a colorless substrate (chromogen) to generate a colored reaction product.
are both high specificity and high sensitivity
can detect or quantitate an antigen
Direct elisa after
How does direct elisa work
Monoclonal antibodies are bound to the walls of the well in a microtiter plate to a membrane or to a plastic wand. Then a secondary labeled antibody containing conjugated enzyme (horserashish peroxidase) is added (secondary ab is specific for antigen)
What happens after 2nd antibody is added?
secondary labeled antibody containing horseradish peroxidase is added which is specific for the antigen s it will bind to any remaining antigen
Color formed is directly proportional to
the amount of antigen present orignally
Indirect Elisa is used to detect
or qualitate ANTIBODY
Indirect elisa mechanism
primary antibody is added to an antigen coated microtiter
and detection of antibody is detected by adding an enzyme conjugated secondary antiisotype antobdy (antispecies) which binds to the first one
and then WASHED
Indirect elisa color detection
intensitiy of the color is proportional to the amount of enzyme linked to secondary antibody which is then inturn DETECTING PRIMARY antibody
What are flurochromes
chemical substances that are capable of absorbing a short wavelength of light and emitting a longer wavelength
Most common use of fluorescent dyes
FITC and rhodamine
to conjugate with the fc region of an antibody molecule without affecting the specificity of the antibody
FITC absorbs in the ___ and emits in ____
blue light, and intense yellow green
Rhodamine absobes in _____ andemits ____
yellow green and emits a deep red
Direct Fluorescent Antibody Test
prescence of antigen, in FRESH or FIXED TISSUE
Fixation of Tissue with FAT
how can FAT detect antigen
Can see FITC labeled antibody on fluroscent microscope
detection and quantitation of antibodies for demonstration of IDENTIFICATION OF ANTIGENS in tussue or cell cultures
What is the difference between FAT and IFAT
need three reactants : Antigen A , unlabeled antibody, flurochrome labeled antisotype 2nd antibody
Advantange over direct
primary antibody does noe need to be conjugated (do not need purification)
sensitivity of staining is increased bc of mutltiple fluorochrome antiisotyope antibodies can bind to each primary antibody
interaction between a ab and souble antigen in aq solution forms a lattice that eventually develops into a visible precipiate
formation of ag ab lattice depends on valency of both antibody and antigen
antibody must be bivalent
antigen must be multivalent (polyvalent) have different epiopes that react with different antibodies
each one ag molecule is linked to more than one
as neighboring ab molecules within lattice form ionic bonds with each other causing it it loose charge and become insonule
Qualitative evidence of Ag- ab reaction
using a test tube to see the presence of ab against an antigen in solution is to layer a small volume of one over the other- at the interface precipitation will occur forming a ring
placing a constant amount of ab in a series of tubes adn adding increasing amount of antigen to the tubes
plotting the amount of precipitate against increasing antigen concentration
excess of either antibody or antigen
interferes with maximal precipitation
uncombined antibody is present in supernattent and each antigen is completely covered with antibody preventing cross linking causing precip
both antigen and antibody are completely precipitated and no uncobmined ag or ab is present
ratio of ab to ag is optimal such that there is extensive cross linking and lattice formation
Lattice binding is
zone of antigen excess
Postzone. uncombined ag is present in supernatant and no precipitation since ab is bound to both two ag cuasing no complexes
agar gel immunodiffusiion reactions
will leave a line of precipitation if ab and ag diffuse towards one another. occurs in regions of equivalence
based on idea that ag and ab diffuse through a semisolif medium and from stable immune complexes and will diffuse towards one another forming preciptiation lines
application of outercolony
identification of soluble ag or ab in body fluids, like Coggins test which detects the presence of ab against equine infectious anemia virus in horses
can also compare antigens to see if they share epitope
latex agglutination tests are available to detect
rhematoid arthirtis in canine serum
k99+ in ecoli in stools of diarrhetic calces
S. aglalactiae in bovine mastatis
HA/ HI tests
number of viruses are capable of binding and agglutating RBC. antibodies direted gainst this may inhibit agglutination by blocking their viral attachment
inhibition of hemaglutiination by a specific antibody may be used to
identify a specific virus or to measure antibody levels in serum
preperation of 10% RBC suspenstion what do you use as anticoagulent
For HA /HI tests what RBC suspension is used?
0.5 % (1:200)
ability of ab to neutralize the biological activity of viable viruses in vitro