Using Immunodiagnostics to Assess Host Immune Status Flashcards
What are the primary tissues sampled for assessing patient immune status?
blood, lymph nodes, spleen, bone marrow
what has urine been used to diagnose?
immune-based cancers (i.e. multiple myeloma or B cell lymphoma)
what is the most common blood site for collection in humans? companion animals?
median cubital vein
cephalic or lateral saphenous veins
why does the blood clot once a needle is stuck into the vein?
activates the host’s coagulation cascade which is designed to create a biological band aid at the injection point
what are some anti-coagulants in tubes used to collect blood?
EDTA, heparin, sodium citrate
what is the fluid component of blood?
plasma
what does plasma consist of?
proteins (i.e. albumin, immunoglobulins), lipids, carbohydrates, electrolytes, metabolic waste products, hormones, possibly platelets and water
if the blood clots in a tube with no anticoagulant, then what can you collect from the blood after centrifuge?
serum
what does serum contain?
same components as plasma minus the coagulation protein factors and platelets
what does the blood consist of?
45% cells and 55% plasma
how much of the blood cells make up RBCs and how much WBCs?
98% RBC
1% WBC
what are the 5 types of leukocytes?
neutrophils, lymphocytes, monocytes,
basophils and eosinophils
what is the purpose of the 5-point leukocyte differential?
help assess general shifts in sub-populations of leukocytes
define serology
assays involving antigen-antibody interactions
what biologic activities of antibodies are serology tests based on?
agglutination, precipitation and complement activation.
what is serotyping?
antibodies used to screen for the presence of
different epitopes of a particular microorganism (i.e. Flu virus)
what does the association constant help define?
binding strength with which the antigen epitope binds to the paratope (antigen-binding site) of the antibody and is called affinity
Antibodies with _____ binding affinity bind more rapidly to antigen, tend to maintain
these bonds and have greater sensitivity in assays.
high
what does avidity measure?
total strength of the antibody-antigen complex, which is dependent on antibody affinity for the epitope, structural conformation of the antibody and antigen interaction, plus the antibody and antigen valence
what can antigen epitope valence and determinant number coupled with antibody composition (whole antibody vs fragments) impact?
cross linking
what is required for agglutination and precipitation?
cross-linking
what antigens cannot form cross-linking complexes and what is an example?
single epitope on its surface (univalent/unideterminant)
ex. hapten
what is the ideal scenario for cross-linking to occur?
antigen to have multiple epitopes (multideterminant) expressed multiple time
on the antigen surface (multivalent)
n agglutination reactions, it is possible to semi-quantify the quantity of antibody in a sample
(i.e. serum) using a fixed concentration of _________________ and increasing dilutions of the antibody.
particulate antigen
what is the prozone?
in samples where the antibody concentration exceeds antigen (antibody excess), cross linking can take place and thus agglutination cannot occur
what is the zone of equivalence?
once the antibody is further diluted to an optimal ratio of antigen to antibody, crosslinking occurs and manifests as agglutination
what is a titer?
highest dilution of the antibody sample that still yields a discernible agglutination
what is pro-zone?
Once the antibody sample diluted above the titer, the amount of antibody is to low and
the amount of antigen is in excess preventing cross-linking
why can pro-zone be applicable to both precipitation reactions and agglutination?
since the only difference is that the target is soluble antigen
what is zeta potential?
in some antigen-antibody reactions, the surface of some antigens can have an electrical charge which can impede binding, affecting some antibody isotypes like IgG but not IgM
what is the advantage of using coombs test for agglutination?
negate the zeta potential cause by the antigen as the commercial antibodies target the Fc portion of the host antibodies placing them further away from the antigen
what is coombs test commonly employed for?
Immune-mediated hemolytic anemia (IMHA) and for hemolytic disease of the newborn (HDN)
when does agglutination occur using the coombs test?
when the anti-Ig is added
what is the pattern of identity?
a continuous single precipitation line indicating that the antigen is present in both samples
what is the pattern of non identity?
ines cross each other and form 2 different precipitation lines
what is pattern of partial identity?
cross reactive antigen with a weaker affinity, precipitation line resembling a boot or spur, it indicates that Ag2 has an epitope similar in structure to Ag1 (cognate Ag)
in a simple gel immunodiffusion assay, what does the presence of 3 precipitation lines confirm?
3 different antigen epitopes and would require 3 different antibodies
what is a qualitative test?
chemical-based test that determines whether a particular substance is present: positive + or negative
what is an example of a qualitative test?
simple and double gel immunodiffusion assays
what is a semi-quantitative test?
chemical-based test that determines whether a particular substance falls within or between a range of a particular value
what is an example of a semi-quantitative test?
vaccine titer assays
what is a quantitative test?
chemical-based test that determines the specific amount of substance present
what are examples of a quantitative test?
ELISA and Radioimmunoassays
what is radial immunodiffusion assay/mancini method?
it’s a precipitation assay but the mechanics is that the Ab or Ag is well-mixed into the agar prior to it being poured into the petri dish
in radial immunodiffusion assay, The higher the concentration of the Ag the______ the diameter of the circle.
larger
what kind of test would radial immunodiffusion assay be an example of?
quantitative test
what is electrophoresis?
proteins due to their charge have the ability to migrate toward a pole when an electrical current is applied
what samples are collected to run electrophoresis?
serum and urine
what kind of test is electrophoresis an example of?
qualitative assay
what does the western blot (immunoblot) test measure for?
analytical assay to measure proteins from tissue homogenate/extract
what are the steps to the western blot?
- samples are evaluated and diluted based on total protein, protein is loaded into vertical mounted gel apparatus and electrical charge is applied for a set time
- separated proteins transferred to protein binding nitrocellulose sheets
- enzyme-labeled Ab targeting the desired Ag or Ab protein is added, incubated, then washed followed by the addition of a super substrate for detection and detected on a special membrane imaging system
what kind of test is the western blot an example of?
semi-quantitated
what kind of tests use sensitivity and specificity?
direct binding immunoassays
what does sensitivity imply?
the test will correctly identify individuals who are positive
what does specificity immply?
the test will correctly identify those individuals who are negative
what is the most common isotope used in radioimmunoassays (RIA)?
gamma emitter 125I
what is the 125I assay based on?
competitive binding principle
describe how an RIA works
- a radioactive tagged analog of the substance in question is incubated with the patient’s serum containing the substance (i.e. Thyroxine) and antibodies (i.e. anti-Thyroxine)
- The two substances compete for the antibodies.
- After a series of wash steps in which the unbound component is discarded, the
radioactivity is measured and values calculated against known standard concentrations
what are the concentrations that RIA assays are capable of detecting substances?
as low as one trillionth of a gram/mL of serum or blood
what replaced RIA assays?
ELISA
describe what happens during a direct binding assay
- Ag tagged with radioisotope
- patient’s sample is added that has Ab to the labeled Ag
- incubation phase, wash steps with Ag-Ab complex forming a pellet at the bottom of the tube
- radioactivity counted on a gamma counter
describes what happens during an RIA competitive binding
- patient’s sample is competing for a fixed concentration of Ab with labeled commercial Ag
what is the result of RIA competitive binding?
pelleted samples with very low radioactivity are actually samples with high amounts of Ag
true or false: in RIA competitive binding, the higher the concentration of Ag in the patient’s sample, it outcompetes with the commercial labeled Ag
true
what is Enzyme-linked immunosorbent assay (ELISA)?
plate-based assay designed for detecting and enumerating peptides, proteins, antibodies and hormones.
in a solid phase immunoassay, what generates a color change?
incorporates an enzyme-bound (i.e alkaline phosphatase or peroxidase) antibody to a protein that reacts with a substrate
describe the 3 types of ELISA assays
- Direct ELISA- fast assay, few steps, less prone to error
- Indirect ELISA- high sensitivity, greater flexibility
- Sandwich ELISA- highest sensitivity, high specificity
describe the process of direct ELISA assay
- patient’s sample has the Ag. It is affixed to a reaction well via a coating procedure.
- A blocking agent is added to prevent “Non-specific binding”.
- The commercial antibody with the enzyme conjugated to it is added and incubated.
- this is followed by the substrate that is acted on by the enzyme.
- Following a specified incubation period, a weak acid is added to stop the reaction and the plate or tube is enumerated on a special spectrophotometer
describe what happens during the indirect ELISA test
- commercial Ag is absorbed to the plastic surface
- then patient sample containing Ab to the Ag are added
- commercial anti-species-specific antibodies conjugated with enzyme substrate
- then an acid solution to stop the reaction.
describe what happens during sandwich ELISA assay
involves the use of matched Ab pairs (i.e capture and detection Abs), which recognize specific non-overlapping epitopes on the same Ag (i.e. parvovirus, rabies). The captured antibody is bound to the plastic surface, then patient serum sample is added, then the detection enzyme-bound antibody and then the substrate
what can cell counting and cytologic assessment be evaluated based on?
whole blood or enriched leukocyte fraction (buffy coat)- CBC, blood smears.
how are cell phenotypic analysis primarily performed on?
enriched leukocytes stained with antibodies tagged to fluorochromes (anti-CD8-FITC, anti-CD4-PE) and analyzed by flow cytometry
what does cell signaling require and analyzed by?
requires cells to be enriched, lysed and the intracellular proteins (cytoplasmic or nuclear) analyzed by immunoblot (Western Blot)
what are cytokine assays?
single or multiple cytokines- these are traditionally ELISA or Fluorescent-based assays that can measure the cytokine levels from cellular secretions such as serum, urine, peritoneal fluid or lacrimal fluid.
what does cell gene expression require and what does it measure?
requires cells to be enriched, lysed and the mRNA analyzed (PCR and/or gene arrays).
This platform measures the expression (i.e. upregulated or downregulated) of a large number of genes, simultaneously. It requires sufficient sample to isolate total mRNA.
what is flow cytometry?
a laser(s) and fluid-based system that guides an individual cell through an aperture or window in which a laser beam is directed.
describe what happens during flow cytometry
As the cell passes through the aperture, it deflects the laser beam in multiple directions based on its size and granularity (i.e. smooth or rough surface). These deflected laser beans are caught by special detectors called Photomultiplier tubes (PMTs), which digitally enumerate the signals/cell and display them on a graph called a histogram. The power of this platform is that you can additionally tag these cells with commercial Fluorescent-tagged antibodies or probes that will identify other features of the cells (i.e. phenotype- anti-CD4, cell cycle, cell death)
