Exam 5 Immunochemistry

What is Immunochemistry based on?
Binding of ABY to AGN for detection on an analyte
Define AGN
substance capable of inducing the formation of an antibody
Define ABY
protein molecule that combines with a specific antigen
Define Analyte
substance of interest in the patient’s serum, plasma or urine
Define Binding Reagent
Ab that binds to the patient Ag or Ab
Define Label
allows for measurement of the end-product of the patient-reagent reaction
Define Substrate or Developer
added to produce an end-product
(ex. color)
What is the Label or “Tag” for the following:
RIA
125 I
What is the Label or “Tag” for the following:
EIA
Horseradish peroxidase,
Alkaline phosphatase,
Other enzymes
What is the Label or “Tag” for the following:
Chemiluminescence
Acridinium ester
What is the Label or “Tag” for the following:
Fluorescence Immunoassay
Various fluoresceins
What is the Detection method for the following:
RIA
Gamma counter
What is the Detection method for the following:
EIA
Photometer,
fluorometer,
luminometer
What is the Detection method for the following:
Chemiluminescence
Luminometer
What is the Detection method for the following:
Fluorescence Immunoassay
Fluorometer
What is the Principle/Advantage of/for the following:
EIA
Enzymes amplify the amount of product generated; product may be color/UV, fluorescence or chemiluminescence.
Long shelf-life; stable, easy to
detect
What is the Principle/Advantage of/for the following:
Chemiluminescence
Products release light energy;
high sensitivity
What is the Principle/Advantage of/for the following:
Fluorescence Immunoassay
Molecules absorb E at one wavelength and emit E at another; high specificity
Competitive Binding Reactions
Patient analyte (AGN) and labeled AGN compete for a limited number of binding sites on a reagent ABY
Noncompetitive-Binding Reactions – “Sandwich” Technique
-Patient antigen is allowed to bind to an excess amount of immobilized reagent antibody
-A second labeled antibody
then binds to Ag-Ab complexes
Define the following separation technique:
Homogeneous
No separation of free or bound labeled reagents is required
Define the following separation technique:
Heterogeneous
Separate step “wash” is required to physically separate the bound labeled reagent from the free labeled AGN before measurement can be performed
How can Heterogeneous Separation be achieved?
Adsoprtion
Precipitation
Buffer Wash
etc.
Indirect ELISA is used for detecting unknown _____?
ABY
True or False:
Direct ELISA – enzyme-tagged AGN & patient AGN compete for sites on an ABY
True
Do you perform a washing step in Direct ELISA?
Yes
The amount of product in Direct ELISA is ______ _______ to the amount of patient AGN preset
Inversely Proportional
What is Direct ELISA used to measure?
Drugs, hormones, proteins, markers, etc.
Is Direct ELISA Homogeneous or Heterogeneous?
Heterogeneous
Describe Chemiluminescent Assays
Chemical reaction in which an unstable product is formed; when it reverts to ground energy state, it releases energy in the form of visible photos on light
Name 2 labels that are commonly used for Chemiluminescent Assays
Lluminol
Acridnium ester
Chemiluminescent Assay – The # of photons is directly proportional to what?
The level of patient analyte
What does EMIT stand for?
Enzyme-Multiplied Immunoassay Technique
What does FPIA stand for?
Fluorescence Polarization Immunoassay
Is EMIT homogeneous or heterogeneous?
Homogeneous
Is EMIT competitive or non-competitive?
Competitive
Is FPIA homogeneous or heterogeneous?
Homogeneous
Is FPIA competitive or non-competitive?
Competitive
Is ELISA competitive or non-competitive?
Competitive
Is ELISA homogeneous or heterogeneous?
Heterogeneous
With EMIT what is competing for sites on the reagent ABY?
Patient AGN
Labeled AGN
EMIT – The Binding of labeled AGN with ABY hinders what?
Enzyme’s activity
True or False:
EMIT – Only free labeled AGN will react with substrate to cause product development
True
EMIT – The amount of product is ______ _____ to the amount of patient AGN present in the test sample
Directly Proportional
FPIA – what is used to excite a fluorescent-tagged AGN?
Polarized Light
What does FPIA measure?
Change in the degree of polarized light emitted by the label
What happens to polarization with smaller Free Labeled AGNs?
e- do not stay in line with the polarized light source
Polarization is Lost
What happens to polarization with larger Bound labeled AGNs?
Able to rotate
Increased polarized fluorescence is emitted
FPIA – The amount of polarized fluorescent light is ____ _____ to the concentration of patient AGN
Inversely proportional
FPIA – Polarization Increases as Patient AGN _______
Decreases