MLS117 Antigen Antibody Reaction (Part 3) IMMUNOLOGY & SEROLOGY

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MARK MAGBUHOS, RMT | March 7, 2021 LE 2 TRANS 08

OUTLINE
I. Labeled Immunoassays IX. Capture Assays
II. Competitive versus A. Rapid Immunoassays -
Noncompetitive Assays Membrane-Based Cassette
III. Antibodies Assays
IV. Standards or Calibrators X. Homogeneous Enzyme
V. Detection of the Label Immunoassay
VI. Quality Control XI. Advantages and Disadvantages
VII. RADIOIMMUNOASSAY - of Enzyme Immunoassay
Competitive Binding XII. FLUORESCENT
Assays IMMUNOASSAY
A. Advantages and A. Direct versus Indirect II. Competitive versus Noncompetitive Assays
Disadvantages of Immunofluorescent Assays •There are two major formats for all labeled assays:
Radioimmunoassay B. Indirect Immunofluorescent  Competitive and noncompetitive.
VIII. ENZYME Assays •In a competitive immunoassay
IMMUNOASSAY XIII. Direct versus Indirect  All the reactants are mixed together simultaneously, and
A. Heterogeneous Immunofluorescent Assays labeled antigen competes with unlabeled patient antigen for a
Enzyme Immunoassay XIV. Other Fluorescent limited number of antibody-binding sites.
-Competitive EIA Immunoassays •The amount of bound label is inversely proportional to the
XV. Advantages and concentration of the labeled antigen.
B. Heterogeneous
Disadvantages of Fluorescent •This means that the more label detected, the less there is of patient
Enzyme Immunoassay
Immunoassay antigen.
-Noncompetitive EIA
•In a typical noncompetitive immunoassay
XVI. CHEMILUMINESCENT
 Antibody, often called capture antibody, is first passively
IMMUNOASSAYS
absorbed to a solid phase.
A. Advantages and
•Unknown patient antigen is then allowed to react with and be captured
Disadvantages of
by the antibody.
Chemiluminescent Assays
•After washing to remove unbound antigen, a second antibody with a
label is added to the reaction.
LEGEND
•In this case, the amount of label measured is directly proportional to
Remember Lecturer Book Previous Presentation the amount of patient antigen.
Trans •In both types of assays, the label must not alter the reactivity of the
molecule, and it should remain stable for the shelf life of the reagent.
•Radioactivity, enzymes, fluorescent compounds, and
OBJECTIVES: chemiluminescent substances have all been used as labels.
At the end of the lecture, the student should be able to: III. Antibodies
•Describe the difference between competitive and noncompetitive •In any assay, it is essential for the antibody used to have a high affinity
immunoassays. for the antigen in question.
•Identify characteristics that an antibody must have to be used for •In competitive binding assays, there is random interaction between
immunoassay. individual antigen and antibody molecules.
•Explain how the principle of competitive binding is used in •Therefore, the higher the affinity of antibody for antigen, the larger the
radioimmunoassays. amount of antigen bound to antibody and the more accurately specific
•Distinguish between heterogeneous and homogeneous enzyme binding can be measured.
immunoassay. IV. Standards or Calibrators
•Explain the principle of sandwich or capture immunoassays. •Standards
•Describe uses for rapid immunoassays.  also known as calibrators, are unlabeled analytes that are
•Describe applications for homogeneous enzyme immunoassay. made up in known concentrations of the substance to be
•Compare and contrast enzyme immunoassay and radioimmunoassay measured.
regarding ease of performance, shelf life, sensitivity, and clinical •They are used to establish a relationship between the labeled analyte
application. measured and any unlabeled analyte that might be present in patient
•Describe the difference between direct and indirect specimens.
immunofluorescence techniques. •Differing amounts of standards are added to antibody–antigen mixtures
•Relate the principle of fluorescence polarization immunoassay. to ascertain their effect on binding of the labeled reagent.
•Discuss advantages and disadvantages of each type of immunoassay. •Most instruments then extrapolate this information and do a best-fit
•Choose an appropriate immunoassay for a particular analyte. curve (one that is not absolutely linear) to determine the concentration
I. Labeled Immunoassays of the unknown analyte.
•Labeled immunoassays V. Detection of the Label
 Are designed for antigens and antibodies that may be small in •The last step common to all immunoassays is detection of the labeled
size or present in very low concentrations. analyte.
•The presence of such antigens or antibodies is determined indirectly •For radioimmunoassays
by using a labeled reactant to detect whether or not specific binding has  this involves a system for counting radioactivity
taken place. •While for other labels such as enzymes, fluorescence, or
 The substance to be measured is known as the analyte. chemiluminescence
 Examples include bacterial antigens, hormones, drugs, tumor  Typically a change in absorbance in a substrate is measured
markers, specific immunoglobulins, and many other by spectrophotometry.
substances. •All systems must use stringent quality controls.
•Analytes are bound by molecules that react specifically with them. VI. Quality Control
 Typically, this is antibody. •When measuring analytes that are present in very limited quantities, it
•One reactant, either the antigen or the antibody, is labeled with a is essential to establish quality-control procedures.
marker so that the amount of binding can be monitored. •Because the goal of testing is to

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X.0Y Antigen Antibody Reaction (Part 3)
 determine whether patient levels are increased or decreased
over normal values,
 test performance must be monitored to limit random errors.
•One means of doing this is to run a blank tube, usually phosphate-
buffered saline, with every test. This is not expected to have any
detectable label but serves as a check for nonspecific adsorption and
for inadequate washing between steps.
•Any readings indicative of label in the blank are known as
background.
 If the background is too high, wash steps need to be made
more efficient.
•A negative control and a high and a low positive control should be run
in addition.
 This serves as a check on the quality of the reagents to make
sure the label is readily detectable under current testing
conditions.
•All controls and the patient sample are usually run induplicate.
 If any controls are out of range, test values should not be
reported.
VII. RADIOIMMUNOASSAY -Competitive Binding
Assays
•The first type of immunoassay developed was radioimmunoassay
(RIA), pioneered by Yalow and Bersonin the late 1950s.
•A radioactive substance is used as a label.
 Radioactive elements have nuclei that decay spontaneously,
emitting matter and energy.
•Several radioactive labels
 including 131I; 125I; and tritiated hydrogen, or 3 H, have
been used, but 125I is the most popular.
 It has a half-life of 60 days, and because it has a higher
counting rate than that of 3 H, the total counting time is less.
•It is easily
1. incorporated into protein molecules, and
2. It emits gamma radiation, which is detected by a gamma
counter.
3. Very low quantities of radioactivity can be easily measured.
•RIA
 was originally based on the principle of competitive binding.
Thus, the analyte being detected competes with a
radiolabeled analyte for a limited number of binding sites on a
high-affinity antibody.
•The concentration of the radioactive analyte is in excess, so all binding
sites on antibody will be occupied. If patient antigen is present, some of
the binding sites will be filled with unlabeled analyte, thus decreasing
the amount of bound radioactive label (Fig. 10–1).
•When bound and free radiolabeled antigens are separated and a
washing step has occurred, the amount of label in the bound phase is
indirectly proportional to the amount of patient antigen present.
 This can be illustrated by the following equation:
6Ag* + 2Ag + 4Ab -> 3Ag*Ab + 1AgAb + 3Ag* + 1Ag
•In this example, labeled and unlabeled antigens occur in a 3:1 ratio.
Binding to a limited number of antibody sites will take place in the same
ratio. Thus, on the right side of the equation, three of the four binding
IMMUNOLOGY & SEROLOGY
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sites are occupied by labeled antigen, while one site is filled by
unlabeled antigen.
•As the amount of patient antigen increases, fewer binding sites will be
occupied by labeled antigen, as demonstrated by the next equation:
 6Ag* + 18Ag + 4Ab -> 1Ag*Ab + 3AgAb + 5Ag* + 15Ag
•In this case, the ratio of labeled to unlabeled antigen is 1:3. Binding to
antibody sites takes place in the same ratio, and the amount of bound
label is greatly decreased in comparison to the first equation.
•In this type of RIA, use of a constant amount of radiolabeled antigen
with standards of known concentration will result in a standard curve
that can be used to extrapolate the concentration of the unknown
patient antigen.
•The detection limits of competitive assays are largely determined by
the affinity of the antibody.
•These limits have been calculated to be as low as 10 fmol/L, or
600,000 molecules in a sample volume of 100 uL.
A. Advantages and Disadvantages of Radioimmunoassay
•Examples of substances that are measured by RIA include thyroid-
stimulating hormone and total serum IgE.
•RIA
 Is an extremely sensitive and precise technique for
determining trace amounts of analytes that are small in size.
•However, chief among the disadvantages of all RIA techniques is the
health hazard involved in working with radioactive substances.
•Enzyme immunoassays have largely replaced RIA because of their
comparable sensitivity and the availability of automated instrumentation
that allows for processing of a large number of samples in less time.
VIII. ENZYME IMMUNOASSAY
•Enzymes
 Are naturally occurring molecules that catalyze certain
biochemical reactions.
 They react with suitable substrates to produce breakdown
products that may be chromogenic, fluorogenic, or
luminescent.
 Some type of spectroscopy can then be used to measure the
changes involved.
•Enzyme labels can either be used
 qualitatively to determine the presence of an antigen or
antibody or
 Quantitatively to determine the actual concentration of an
analyte in an unknown specimen.
•Enzymes used as labels for immunoassay are
 Typically chosen according to the number of substrate
molecules converted per molecule of enzyme, ease and
speed of detection, and stability.
•Typical enzymes that have been used as labels in colorimetric
reactions
 Include horseradish peroxidase, glucose-6-phosphate
dehydrogenase, alkaline phosphatase, and b-D-
galactosidase.
•Alkaline phosphatase and horseradish peroxidase
 Have the highest turnover (conversion of substrate) rates,
high sensitivity, and are easy to detect, so they are most
often used in such assays.
•Enzyme assays are classified as either heterogeneous or
homogeneous on the basis of whether a separation step is necessary.
•Heterogeneous enzyme immunoassays
 Require a step to physically separate free from bound
analyte.
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•In homogeneous immunoassays
 On the other hand, no separation step is necessary, because
enzyme activity diminishes when binding of antibody and
antigen occurs.
A. Heterogeneous Enzyme Immunoassay -Competitive
EIA
•The first enzyme immunoassays (EIAs) were competitive assays
based on the principles of RIA.
•Enzyme-labeled antigen competes with unlabeled patient antigen for a
limited number of binding sites on antibody molecules that are attached
to a solid phase.
•After carefully washing to remove any nonspecifically bound antigen,
enzyme activity is determined.
•Enzyme activity is inversely proportional to the concentration of the test
substance, meaning that the more patient antigen is bound, the less
enzyme-labeled antigen can attach.
B. Heterogeneous Enzyme Immunoassay -
Noncompetitive EIA
•Noncompetitive assays are often referred to as indirect enzyme-linked
immunosorbent assays (ELISA)
 Because the enzyme-labeled reagent does not participate in
the initial antigen–antibody binding reaction.
•This type of assay is one of the most frequently used immunoassays in
the clinical laboratory due to its sensitivity, specificity, simplicity,
and low cost.
•Either antigen or antibody may be bound to solid phase.
 A variety of solid-phase supports are used, including
microtiter plates, nitrocellulose membranes, and magnetic
latex beads.
•When antigen is bound to solid phase, patient serum with unknown
antibody is added and given time to react. After a wash step, an
enzyme-labeled antiglobulin is added.
•This second antibody reacts with any patient antibody that is bound to
solid phase. If no patient antibody is bound to the solid phase, the
second labeled antibody will not be bound. After a second wash step,
the enzyme substrate is added.
•The amount of enzyme label detected is directly proportional to the
amount of antibody in the specimen (Fig. 10–2).
•This type of assay has been used to measure antibody production to
infectious agents that are difficult to isolate in the laboratory and has
been used for autoantibody testing.
•This technique remains the preferred screening method for detecting
antibody to HIV, hepatitis A, and hepatitis C.
•ELISA-based tests
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 Are also used to identify Epstein-Barr-specific antibodies
produced in infectious mononucleosis.
IX. Capture Assays
•If antibody is bound to the solid phase, these assays are often called
sandwich immunoassays, or capture assays. Antigens captured in
these assays must have multiple epitopes.
•Excess antibody attached to solid phase is allowed to combine with the
test sample to capture any antigen present.
•After an appropriate incubation period, enzyme-labeled antibody is
added. This second antibody recognizes a different epitope than the
solid-phase antibody and completes the “sandwich.”
•Enzymatic activity is directly proportional to the amount of antigen in
the test sample (Fig. 10–3).
•Capture assays
 Are best suited to antigens that have multiple determinants,
such as antibodies, polypeptide hormones, proteins, tumor
markers, and microorganisms, especially viruses.
•When used with microorganisms, the epitope must be unique to the
organism being tested, and it must be present in all strains of that
organism.
•Use of monoclonal antibodies has made this a very sensitive test
system.
•Rotavirus
 In stool and respiratory syncytial virus in respiratory tract
secretions are two examples of capture assays.
•In addition, recently developed ELISAs have made it much easier to
detect parasites such as Giardia lamblia and Cryptosporidium in the
stool.
•Antigen-detection tests have also proved useful for identifying fungi
such as Aspergillus, Candida, and Cryptococcus.
•Another major use of capture assays is in the measurement of
immunoglobulins, especially those of certain classes.
•When capture assays are used to measure immunoglobulins, the
specific immunoglobulin class being detected is actually acting as
antigen, and the antibody is antihuman immunoglobulin.
•Indirect ELISA tests
 Are more sensitive than their direct counterparts, because all
patient antigen has a chance to participate in the reaction.
•However, there is more manipulation than in direct tests, because
there are two incubations and two wash steps.
•Heterogeneous enzyme assays,
 In general, achieve a sensitivity similar to that of RIA.
•In sandwich assays
 capture antibody on solid phase must have both a high
affinity and a high specificity for this test system to be
effective.
•However, there may be problems with nonspecific protein binding or
the presence of antibodies to various components of the testing system.
If this is suspected, serum can be pretreated to avoid this problem.
•Sandwich assays
 Are also subject to the hook effect, an unexpected fall in the
amount of measured analyte when an extremely high
concentration is present.
•This typically occurs in antigen excess, where the majority of binding
sites are filled, and the remainder of patient antigen has no place to
bind. If this condition is suspected, serum dilutions must be made and
then retested.
A. Rapid Immunoassays -Membrane-Based Cassette
Assays
•Membrane-based cassette assays
 Are a relatively new type of enzyme immunoassay.
 They are rapid, easy to perform, and give reproducible
results.
•Typically these are designed as single-use, disposable assays in a
plastic cartridge.
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•The membrane is usually nitrocellulose, which is able to easily
immobilize proteins and nucleic acids.
•The rapid flow through the membrane and its large surface area
enhance the speed and sensitivity of ELISA reactions.
•Either antigen or antibody can be coupled to the membrane, and the
reaction is read by looking for the presence of a colored reaction
product.
•Some test devices require the separate addition of patient sample,
wash reagent, labeled antigen or antibody, and the substrate.
•Another type of rapid assay, called immunochromatography.
•The analyte is applied at one end of the strip and migrates toward the
distal end, where there is an absorbent pad to maintain a constant
capillary flow rate. The labeling and detection zones are set between
the two ends.
•As the sample is loaded, it reconstitutes the labeled antigen or
antibody, and the two form a complex that migrates toward the
detection zone.
•An antigen or antibody immobilized in the detection zone captures the
immune complex and forms a colored line for a positive test (Fig. 10–4).
This may be in the form of a plus sign. Excess labeled
immunoreactantmigrates to the absorbent pad.
•This type of test device has been used to identify microorganisms such
as Streptococcus pyogenes and Streptococcus agalactiae and has
been used to test for pregnancy, for troponin in a heart attack, and for
hepatitis B surface antigen, to name just a few examples.
•Test results are most often qualitative rather than quantitative.
X. Homogeneous Enzyme Immunoassay
•A homogeneous enzyme immunoassay
 is any antigen-antibody system in which no separation step is
necessary.
•Homogeneous assays are generally less sensitive than heterogeneous
assays, but they are rapid, simple to perform, and adapt easily to
automation. No washing steps are necessary.
•Their chief use has been in the determination of low-molecular-weight
analytes such as hormones, therapeutic drugs, and drugs of abuse in
both serum and urine.
•Homogeneous assays
 Are based on the principle of change in enzyme activity as
specific antigen–antibody combination occurs.
 Reagent antigen is labeled with an enzyme tag.
•When antibody binds to specific determinant sites on the antigen, the
active site on the enzyme is blocked, resulting in a measurable loss of
activity.
•Free analyte (antigen) competes with enzyme-labeled analyte for a
limited number of antibody-binding sites, so this is a competitive assay.
•Enzyme activity is directly in proportion to the concentration of patient
antigen or hapten present in the test solution. A physical separation of
bound and free analyte is thus not necessary.
•The sensitivity of homogeneous assays is determined by the following:
1. Detectability of enzymatic activity
2. Change in that activity when antibody binds to antigen
3. Strength of the antibody’s binding
4. Susceptibility of the assay to interference from endogenous
enzyme activity, cross-reacting antigens, or enzyme inhibitors.
•Typically,
 a sensitivity of micrograms per milliliter is reached,
 Far less than that achievable by heterogeneous enzyme
assays
 Because the amplification properties of enzymes are not
utilized.
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•This technique is usually applied only to
 Detection of small molecules that could not be easily
measured by other means.
•Enzymatic activity may be altered by steric exclusion of the substrate,
or there may also be changes in the conformation structure of the
enzyme, especially in the region of the active site.
•Two enzymes that are frequently used in this type of assay are malate
dehydrogenase and glucose-6-phosphate dehydrogenase.
XI. Advantages and Disadvantages of Enzyme
Immunoassay
•Enzyme immunoassays
 Have achieved sensitivity similar to that of RIA without
creating a health hazard or causing disposal problems.
•The use of nonisotopic enzyme labels with high specific activity in
noncompetitive assays increases sensitivity and does so using shorter
incubation times than the original RIAs.
•There is no need for expensive instrumentation, because most assays
can be read by spectrophotometry or by simply noting the presence or
absence of color.
•Disadvantages
 Include the fact that some specimens may contain natural
inhibitors.
•Additionally, the size of the enzyme label may be a limiting factor in the
design of some assays.
•Nonspecific protein binding is another difficulty encountered with the
use of enzyme labels.
XII. FLUORESCENT IMMUNOASSAY
•In 1941, Albert Coons demonstrated that antibodies could be labeled
with molecules that fluoresce.
 These fluorescent compounds are called fluorophores or
fluorochromes.
•They can absorb energy from an incident light source and convert that
energy into light of a longer wavelength and lower energy as the excited
electrons return to the ground state.
•The time interval between absorption of energy and emission of
fluorescence is very short and can be measured in nanoseconds.
•Ideally, a fluorescent probe should exhibit high intensity, which can be
distinguished easily from background fluorescence. It should also be
stable.
•The two compounds most often used are fluorescein and rhodamine,
 Usually in the form of isothiocyanates, because these can be
readily coupled with antigen or antibody.
•Fluorescein absorbs maximally at 490 to 495 nm and emits a green
color at 517 to 520 nm.
 It has a high intensity, good photostability, and a high
quantum yield.
 Tetramethylrhodamineabsorbs at 550 nm and emits red
light at 580 to 585 nm.
•Because their absorbance and emission patterns differ, fluorescein
and rhodamine can be used together.
•Other compounds
 Used are phycoerythrin, europium (b-naphthyl
trifluoroacetone), and lucifer yellow VS.
•Fluorescent tags or labels were first used for histochemical
localization of antigen in tissues.
•This technique is called immunofluorescent assay (IFA)
 A term restricted to qualitative observations involving the use
of a fluorescence microscope.
•In this manner, many types of antigens can be detected either in fixed
tissue sections or in live cell suspensions with a high degree of
sensitivity and specificity.
•The presence of a specific antigen is determined by the appearance of
localized color against a dark background.
•This method is used for rapid identification of microorganisms in cell
culture or infected tissue, tumor-specific antigens on neoplastic tissue,
transplantation antigens, and CD antigens on T and B cells through the
use of cell flow cytometry.
A. Direct Immunofluorescent Assays
•Fluorescent staining can be categorized as direct or indirect,
depending on whether the original antibody has a fluorescent tag
attached.
•In a direct immunofluorescent assay
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 Antibody that is conjugated with a fluorescent tag is added
directly to unknown antigen that is fixed to a microscope
slide.
•After incubation and a wash step, the slide is read using a
fluorescence microscope.
•Antigens are typically visualized as bright apple green or orange-
yellow objects against a dark background.
•Direct immunofluorescent assay
 Is best suited to antigen detection in tissue or body fluids,
while indirect assays can be used for both antigen
andantibody identification.
•Examples of antigens detected by this method include Legionella
pneumophila, Pneumocystis carinii, Chlamydia trachomatis, and
respiratory syncytial virus (RSV) (Fig. 10–5).
B. Indirect Immunofluorescent Assays
•Indirect immunofluorescent assays
 Involve two steps, the first of which is incubation of patient
serum with a known antigen attached to a solid phase.
•The slide is washed, and then an antihuman immunoglobulin
containing a fluorescent tag is added. This combines with the first
antibody to form a sandwich, which localizes the fluorescence.
•In this manner, one antibody conjugate can be used for many different
types of reactions, eliminating the need for numerous purified, labeled
reagent antibodies.
•Indirect assays
 result in increased staining, because multiple molecules can
bind to each primary molecule, thus making this a more
sensitive technique.
•Such assays are especially useful in antibody identification and have
been used to detect
 treponema, anti-nuclear, chlamydial, and toxoplasma
antibodies, as well as antibodies to such viruses such as
herpes simplex, EpsteinBarr, and cytomegalovirus.
IMMUNOLOGY & SEROLOGY
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XIII. Direct versus Indirect Immunofluorescent Assays
•Both techniques allow for a visual assessment of the adequacy of the
specimen.
 This is especially helpful in testing for chlamydia and RSV
antigens.
•Immunofluorescent assays in general, however, face the issue of
subjectivity in the reading of slides.
 Only experienced clinical laboratorians should be responsible
for reporting out slide results.
XIV. Other Fluorescent Immunoassays
•Quantitative fluorescent immunoassays (FIAs)
 Can be classified as heterogeneous or homogeneous,
corresponding to similar types of enzyme immunoassays.
 In this case, the label is fluorescent, and such a label can be
applied to either antigen or antibody.
•Solid-phase heterogeneous fluorescent assays
 Have been developed for the identification of antibodies to
nuclear antigen, toxoplasma antigen, rubella virus, and
numerous other virus antigens.
•In addition, fluorescent assays
 Are used to detect such important biological compounds as
cortisol, progesterone, and serum thyroxine (T4).
•Homogeneous FIA,
 Just like the corresponding EIAs, requires no separation
procedure, so it is rapid and simple to perform.
 There is only one incubation step and no wash step, and
usually competitive binding is involved.
•The basis for this technique is the change that occurs in the
fluorescent label on antigen when it binds to specific antibody.
 Such changes may be related to wavelength emission,
rotation freedom, polarity, or dielectric strength.
•There is a direct relationship between the amount of fluorescence
measured and the amount of antigen in the patient sample. As binding
of patient antigen increases, binding of the fluorescent analyte
decreases, and hence more fluorescence is observed.
•One of the most popular techniques developed is fluorescence
polarization immunoassay (FPIA),
 which is based on the change in polarization of fluorescent
light emitted from a labeled molecule when it is bound by
antibody.
•Incident light directed at the specimen is polarized with a lens or prism
so the waves are aligned in one plane. If a molecule is small and
rotates quickly enough, the emitted light is unpolarized after it is excited
by polarized light.
•If, however, the labeled molecule is bound to antibody, the molecule is
unable to tumble as rapidly, and it emits an increased amount of
polarized light.
•Thus, the degree of polarized light reflects the amount of labeled
analyte that is bound.
•In FPIA,
 labeled antigens compete with unlabeled antigen in the
patient sample for a limited number of antibody binding sites.
•The more antigen that is present in the patient sample, the less the
fluorescence-labeled antigen is bound and the less the polarization that
will be detected.
•Hence, the degree of fluorescence polarization is inversely proportional
to concentration of the analyte (Fig. 10–7).
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X.0Y Antigen Antibody Reaction (Part 3) LE 2 TRANS 08
•This technique is limited to small molecules that tumble freely in •In heterogeneous assays,
solution, usually those analytes with a molecular weight under 2000 d.  competitive and sandwich formats are the ones most often
•An additional consideration is nonspecific binding of the labeled used.
conjugate toother proteins in serum. Binding to these molecules would •Smaller analytes such as therapeutic drugs and steroid hormones are
increase polarization, thus falsely decreasing values. measured using competitive assays,
•FPIA  while the sandwich format is used for larger analytes such
 has been used mainly to determine concentrations of as protein hormones.
therapeutic drugs and hormones. A. Advantages and Disadvantages of Chemiluminescent
XV. Advantages and Disadvantages of Fluorescent Assays
Immunoassay •Chemiluminescent assays
•In principle, the use of fluorescence has the potential for high  have an excellent sensitivity, comparable to EIA and RIA, and
sensitivity and versatility. the reagents are stable and relatively nontoxic.
•The methodology is fairly simple, and there is no need to deal with and •The sensitivity of some assays has been reported to be in the range of
dispose of hazardous substances. attamoles (10-18 mol) to zeptomoles (10-21 mol).
•The main problem, however, has been separation of the signal on the •The relatively high speed of detection also means a faster turnaround
label from auto fluorescence produced by different organic substances time.
normally present in serum.  Detection systems basically consist of photomultiplier tubes,
•Another difficulty encountered is the which are simple and relatively inexpensive.
 fact that nonspecific binding to substances in serum can •However, false results may be obtained if there is lack of precision in
cause quenching or diminishing of the signal and change the injection of the hydrogen peroxide or if some biological materials such
amount of fluorescence generated. as urine or plasma cause quenching of the light emission.
•Fluorescence polarization
 has been developed to overcome some of these problems,
and this technique has seen more widespread use.
•It does, however, require expensive dedicated instrumentation, which REFERENCES
may limit its use in smaller laboratories. •Immunology&SerologyinLaboratoryMedicine–5thEditionBy:
XVI. CHEMILUMINESCENT IMMUNOASSAYS MaryLouiseTurgeon,EdD,MLS(ASCP)CM
•Chemiluminescence •ClinicalImmunology&Serology–ALaboratoryPerspective3rdEditionBy:
 is another technique employed to follow antigen–antibody ChristineDorresteynStevens,EdD,MT(ASCP)
combination.
 It is the emission of light caused by a chemical reaction,
typically an oxidation reaction, producing an excited molecule END OF TRANSCRIPTION
that decays back to its original ground state.
•A large number of molecules are capable of chemiluminescence, but
some of the most common substances used are luminol, acridinium
esters, ruthenium derivatives, and nitrophenyl oxalates.
•When these substances are oxidized, typically using hydrogen
peroxide and an enzyme for a catalyst, intermediates are produced that
are of a higher energy state. These intermediates spontaneously return
to their original state, giving off energy in the form of light.
•Light emissions range from a rapid flash of light to a more continuous
glow that can last for hours.
 Acridinium esters, for example, emit a quick burst or flash of
light, while the light remains for a longer time with luminol and
dioxetane.
•This type of labeling can be used for heterogeneous and
homogeneous assays, because labels can be attached to either antigen
or antibody.

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