Labeled Immunoassay

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Labeled Immunoassay
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Immunoassay

• An immunoassay is a test that uses antibody and
  antigen complexes as a means of generating a
  measurable result.
• An antibodyantigen complex is also known as an
  immuno-complex. Immuno refers to an immune
  response that causes the body to generate
  antibodies, and assay refers to a test.
• The assay takes advantage of the specific binding
  of an antibody to its antigen.
• The antibodies used must have a high affinity for
  the antigen.

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Immunoassay

• Both the presence of antigen or antibodies can be
  measured.
• Example, when detecting infection the presence of
  antibody against the pathogen is measured.
• For measuring hormones such as insulin, the
  insulin acts as the antigen.
• For numerical results, the response of the fluid
  being measured must be compared to standards of a
  known concentration.
• This is usually done through the plotting of a
  standard curve on a graph paper, and then the
  quantity of the unknown is found from the curve.

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History and Background

• In the year 1959, Drs. Rosalyn Yalow Soloman
  Berson invented the radioimmunoassay, which
  applied the use of radioisotopes in the
• measurement of insulin.
• The RIA is the predecessor of modern immunoassays.

Dr. Rosalyn Yalow became the first female to win
a Nobel Prize with her work on the
radioimmunoassay.
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Immunoassay

• The first immunoassays were described for the
  measurement of insulin and thyroxine,
  respectively.
• There are now hundreds of immunoassays for scores
  of analytes including
• hormones,
• tumor markers,
• drugs, antibodies,
• and cardiac markers
• covering the fields of
• endocrinology,
• oncology,
• hematology,
• toxicology,
• serology,
• infectious diseases
• Developments in antibodies, labels, and
  automation have resulted in highly specific and
  sensitive assays.

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Labeled immunoassays

• Labeled immunoassays are designed for antigens
  and antibodies that may be
• small in size
• or present in very low concentrations.
• The presence of such antigens or antibody is
  determined indirectly by using a labeled reactant
  to detect whether or not specific binding has
  taken place.

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Constituents of Labeled assay

• For detection of an analyte, the following are
  usually a part of the assay
• Labeled and nonlabeled analytes
• Specific antibody
• Standards or calibrators
• A method to separate the bound from free
  components
• A method for detection of the label.

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1- Labeled analyte

• A labeled reactant is used to detect whether or
  not specific binding has taken place.
• The label used in immunoassay
• must not alter the reactivity of the molecule,
• and it should remain stable for the shelf life of
  the reagent.
• Labels attached to analytes and antibodies may
  be
• radioactive, usually iodine-125 (radioimmunoassay
  and immunoradiometric assays),
• enzymes such as alkaline phosphatase and
  horseradish peroxidase, (enzyme immunoassay or
  immunometric assay, or enzyme-linked
  immunosorbent assay ELISA),
• chemiluminescent (e.g., acridinium ester),
• or fluorescent (e.g., fluorscein).

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Methods of coupling indicator labels to antigen
or antibody

• Different methods by which the indicator label
  may be coupled to antigen or antibody.
• For example, the radioactive isotope iodine is
  covalently linked to tyrosine residues present on
  antibodies and most antigens.

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Methods of coupling indicator labels to antigen
or antibody

• Fluorochromes or enzymes may be coupled to
  antigens or antibodies using glutaraldhyde, a
  bifunctional reagent that covalently cross links
  two aminoacids together.
• Alternatively enzymes can be linked to
  streptavidin for use in systems where biotin has
  been attached to either antigen or antibody.

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The biotin-Streptavidin/ Avidin indicator label
system

• Biotin is a vitamin that can bind tightly to
  either avidin or streptavidin.
• Avidin streptavidin are proteins.
• The natural attraction of these two proteins for
  one another is a property that has been exploited
  to facilitate coupling of indicator molecules to
  antigens or antibodies.

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The biotin-Streptavidin indicator label system
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Production of Antibodies

• The production of antibodies is an important
  process in the use of immunoassays because it is
  the antibody-antigen complexes form the basic.
• Antibodies can be called monoclonal or
  polyclonal, depending upon the technique used to
  produce them.

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Polyclonal antibodies

• Polyclonal antibodies may be produced in mammals
  such as rabbits or sheep.
• When a foreign substance enters the body, it
  stimulates the immune system to produce
  antibodies to the substance.
• Using this natural reaction, an analyte in as
  pure form as possible is injected into the animal
  stimulating the production of antibodies.
• Antiserum usually contains a mixture of
  antibodies that recognize and bind to the same
  antigen, but they may attach to different
  epitopes.

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Monoclonal antibodies

• Monoclonal antibodies production result in very
  specific antibodies that bind only to one antigen
  epitope, which in turn reduces the occurrence of
  false positives in the immunoassay

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Monoclonal Antibodies
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3- Standards or calibrators

• Calibrators are solutions with known values that
  establish the relationship between the amount of
  signal produced in the assay and analyte
  concentration.

• By running a set of calibrators, a calibration
  curve is set up in the instruments software and
  correlates certain values of signal to known
  analyte concentrations.
• By comparing levels of signal produced by patient
  samples to this calibration curve, a patient
  analyte concentration value, or result, can be
  determined.

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4- Separation Methods

• In most assays, once the reaction between antigen
  and antibody has taken place, there must a way of
  separating reacted from unreacted analyte.
• This can be accomplished by several different
  means. Unreacted analyte can be removed by
• Adsorption on particles such as dextran-coated
  charcoal,
• These adsorb out the smaller unbound molecules,
  which are then separated from bound molecules by
  centrifugation or filtration.
• The amount of label remaining in the supernatant
  provides an indirect measure of analyte present
  in the patient's sample.

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4- Separation Methods

• Another means of separation involves
  precipitation of antigen-antibody complexes.
• Complexes can be precipitated by adding
  concentrated solutions of ammonium sulfate, or
  ethanol
• Antigen-antibody complexes can also be removed
  from solution by the use of a second
  precipitating antibody (anti-antibody).

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4- Separation Methods

• Currently, most immunoassays use a solid-phase
  stage for separation.
• Numerous substances, such as polystyrene test
  tubes, microtiter plates are used for this
  purpose.
• Antigen or antibody is attached by physical
  adsorption , and when specific binding takes
  place, complexes remain attached to the solid
  phase.
• This provides a simple way to separate bound and
  free reactants.

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5- Methods for detection of label

• The last step common for all immunoassays is
  detection of the labeled analyte.
• The method depends on the label e.g. 125I is
  easily detected in a ?-counter
• Enzymes are generally used to produce coloured
  products from colourless substrates that can be
  determined easily in a spectrophotometer or
  colorimeter.
• Automated plate readers are commercially
  available which make reading large numbers of
  samples relatively easy.

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Quality Control

• It is essential that quality control procedures
  be established.
• This is done to limit random errors, such as
• temperature fluctuations,
• minor changes in the concentration of reagents,
• and changes in detector efficiency.
• A negative control and a positive control should
  be run.
• This serves as a check on the quality of the
  reagents to make sure that the label is readily
  detectable under current testing conditions.

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Types of Labeled Immunoassays

• 1- Competitive and Noncompetitive Immunoassays
• 2- Homogeneous and Heterogeneous Immunoassay
  Methods

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Competitive Immunoassays
In competitive formats, unlabelled analyte
(usually antigen) in the test sample is measured
by its ability to compete with labeled antigen in
the immunoassay.
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Competitive Immunoassays
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Noncompetitive Immunoassays

• Noncompetitive (sandwich) immunoassays generally
  provide the highest level of assay sensitivity
  and specificity.
• The reaction mixture typically includes an excess
  of labeled antibody, so that all metabolite is
  bound.
• The amount of antibody-antigen complex is then
  measured to determine the amount of analyte
  present in the sample.
• The labeled antibody, is directly proportional to
  the amount of antigen present in the sample.

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Noncompetitive Immunoassays
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Homogeneous VS Heterogeneous Methods

• Immunoassay methods that require separation of
  bound Ab-Ag complex are referred to as
  heterogeneous immunoassays.
• Those that do not require separation are referred
  to as homogeneous immunoassays.
• Homogeneous methods have been generally applied
  to the measurement of small analytes such as
  abused and therapeutic drugs.
• Since homogeneous methods do not require the
  separation of the bound Ab-Ag from the free Ag,
  they are generally much easier and faster to
  perform.

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Homogeneous VS Heterogeneous Methods
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Homogeneous immunoassays
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Types of Immunoassays

• Within the categories of competitive,
  noncompetitive, homogenous, and heterogeneous,
  there are specific types, which include

• Radioimmunoassays (RIAs) utilize a radioactive
  label (usually 125I, 3H or 14C), which emits
  radiation that can be measured with a beta or
  gamma counter.

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Types of Immunoassays Contd

• In the Enzyme Multiplied Immunoassay (EMIT), the
  drug in the sample and the drug labeled with G6PD
  compete for antibody binding sites.
• Binding inhibits enzyme activity, while free
  enzyme remains active to interact with.
• Enzyme activity/absorbance is directly
  proportional to drug concentration.

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Types of Immunoassays Contd

• Enzyme linked immunosorbant assay (ELISA)
  competitive, heterogeneous EIA
• Reaction components are absorbed or bound to the
  surface of a solid phase, commonly a well of a
  microtiter plate
• Absorbance is measured using a micro-plate reader
• Sample absorbance is inversely proportional to
  drug concentration

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Types of Immunoassays Contd

• In the Fluorescent Polarized Immunoassay, the
  drug in the sample competes with
  fluorescein-labeled drug for antibody binding
  sites.
• Reaction mixture is excited by planepolarized
  light.
• As the tracer returns to a lower energy state, it
  emits light polarization is measured.

• The polarization value of the sample is
  inversely proportional to analyte concentration.

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Immunoassay Results

• Qualitative
• Single point calibration at a specific cutoff
• Results are either positive or negative
  (i.e. above or below the cutoff)
• Possible false positives monoclonal antibodies
  restrict this slightly.
• Quantitative
• Provides numeric results that are an estimate of
  drug/compound concentration based on the
  measurement of labeled analyte in the solution,
  and taking into consideration the
  competitive/noncompetitive nature of the device.
• In terms of use on drugs, this is sometimes
  complicated by possible cross-reactivities.