Labelled Immunoassay

LABELLED IMMUNOASSAY

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 antibodies is determined indirectly by using a LABELED REACTANT to detect whether or not specific binding has taken place.

Analyte substance to be measured are bound by molecules that react

specifically with them(antibody)

Examples: bacterial antigens, hormones, drugs,

tumor markers, specific immunoglobulins, and many other substances.

Importance of Labelled Immunoassays:

rapid quantitative measurement

ability to detect very small quantities of antigen or antibody

CHARACTERISTICS OF LABELED ASSAYS Current techniques include these

labels: Fluorescent Radioactive Chemiluminescent Enzyme

There are two major formats for all labeled assays: competitive noncompetitive

Competitive immunoassay

Reactants are mixed together Labeled antigen competes with unlabeled

patient antigen for a limited number of antibody-binding sites.

Interpretation: The amount of bound label is inversely

proportional to the concentration of the labeled antigen.

This means that the more label detected, the less there is of patient antigen.

Non-competitive Immunoassay The antibody, often called capture antibody, is

first passively absorbed to a solid phase. Unknown patient antigen is then allowed to

react with and be captured by the antibody. After washing to remove unbound antigen, a

second antibody with a label is added to the reaction.

Interpretation: The amount of label measured is directly

proportional to the amount of patient antigen.

Antibodies

The higher the affinity of antibody for antigen, the larger the amount of antigen bound to antibody and the more ACCURATELY specific binding can be measured.

The ultimate sensitivity of the immunoassay, in fact, depends largely on the magnitude of affinity.

Standards or Calibrators

are unlabeled analytes that are made up in known concentrations of the substance to be measured.

They are used to establish a relationship between the labeled analyte measured and any unlabeled analyte that might be present in patient specimens.

Separation Methods

a partitioning step, or a way of separating reacted from unreacted analyte

If a separation step is employed in an assay, the efficiency of the separation is critical to the accuracy of the results.

The bound and unbound fractions are usually separated by physical means including: Decanting Centrifugation Filtration Washing

Great care must be taken to perform this correctly, because incomplete washing leads to incomplete removal of the labeled analyte and inaccurate results.

solid-phase vehicles used for separation: polystyrene test tubes microtiter plates glass or polystyrene beads Magnetic beads cellulose membranes

Terminology

Heterogeneous or Homogeneous assays:

Heterogeneous assays called separation assays Involve a solid phase Require multiple steps Careful washing of surface to remove

unbound reagents and samples.

Homogeneous assays do NOT require a separation step. Consists only of a liquid phase, no

washing required Mix reagents and patient sample. Measure the labeled product. Easier and faster to perform

Detection of the Label

The last step common to all immunoassays Radioimmunoassays: involves a system for

counting radioactivity other labels such as enzymes, fluorescence,

or chemiluminescence: change in absorbance in a substrate is measured by spectrophotometry.

All systems must use stringent quality controls.

Quality Control

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.

Negative control Positive control(low and high)

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 in duplicate.

If any controls are out of range, test values should not be reported.

Radioimmunoassay(RIA)

radioactive substance--label is used as a label. Radioactive elements have nuclei that decay

spontaneously, emitting matter and energy. Several radioactive labels includes:

131I 125I tritiated hydrogen(3H)

125I is the most popular. has a half-life of 60 days has a higher counting rate than that of

3H, the total counting time is less. easily incorporated into protein

molecules, and it emits gamma radiation, which is detected by a gamma counter

Competitive Binding Assays pioneered by Yalow and Berson in the late

1950s. First used to determine the level of insulin–anti-

insulin complexes in diabetic patients.

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.

Interpretation: The amount of label in the bound phase

is INDIRECTLY PROPORTIONAL to the amount of patient antigen present.

Examples of substances that are measured by RIA include: TSH(thyroid-stimulating hormone) total serum IgE

Disadvantages: health hazard involved in working with

radioactive substances disposal problems short shelf life need for expensive equipment

ENZYME IMMUNOASSAY

Enzymes are naturally occurring molecules that

catalyze certain biochemical reactions. react with suitable substrates to produce

breakdown products that may be chromogenic, fluorogenic, or luminescent.

Spectroscopy—used for measurement

Advantages: cheap and readily available long shelf life easily adapted to automation can be measured using inexpensive

equipment. without disposal problems or the health

hazards of radiation

Enzyme labels can either be used: Qualitatively

to determine the presence of an antigen or antibody

Quantitatively to determine the actual concentration of

an analyte in an unknown specimen.

Typical enzymes that have been used as labels in colorimetric reactions include: horseradish peroxidase glucose-6-phosphate dehydrogenase alkaline phosphatase β-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

HETEROGENEOUS ENZYME IMMUNOASSAYS

Competitive EIA

Steps: 1. Enzyme-labeled antigen competes with

un-labeled patient antigen for a limited number of binding sites on antibody molecules that are attached to a solid phase.

2. WASHING to remove Solid-phase antibody labeled and specific binding any non-specifically bound antigen

3. Measurement of enzyme activity

Interpretation: Enzyme activity is inversely proportional to the

concentration of the test substance. The more patient antigen is bound, the less enzyme-

labeled antigen can attach.

In this manner, a sensitivity of nanograms (10-9 g)/mL can be achieved.

Used for measuring small antigens that are relatively pure such as: Insulin Estrogen

Non-competitive EIA

are often referred to as indirect Enzyme-linked Immunosorbent Assays (ELISA)

The enzyme-labeled reagent does not participate in the initial antigen–antibody binding reaction.

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.

solid-phase supports used includes: microtiter plates nitrocellulose membranes 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

This technique remains the preferred screening method for detecting antibody to: HIV Hepatitis A Hepatitis C

Also used to identify Epstein-Barr–specific antibodies produced in infectious mononucleosis.

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

Capture/Sandwich immunoassay

Capture assays are best suited to antigens that have multiple determinants: Antibodies polypeptide hormones Proteins tumor markers 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.

Clinical applications: Detection of Rotavirus in stool and

respiratory syncytial virus in respiratory tract secretions

useful for identifying fungi such as Aspergillus, Candida, and Cryptococcus

Measurement of immunoglobulins, especially those of certain classes

Types of ELISA

RAPID IMMUNOASSAY

Membrane-Based Cassette Assays are a relatively new type of enzyme

immunoassay They are rapid, easy to perform, and

give reproducible results. for point-of-care or home testing use can be made semi-quantitative for

use in a clinical laboratory designed as single-use, disposable

assays in a plastic cartridge

Nitrocellulose membrane 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

Interpreted by presence of a colored reaction product

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.

This may be in the form of a plus sign.

Immunochromatography

Apply sample to one end, migrates forward.

Sample dissolves labeled antigen or antibody to which it binds.

Migrates towards detection zone where it will bind to immobilized antigen or antibody.

Color change occurs.

Clinical application: Identification of Streptococcus pyogenes

and Streptococcus agalactiae Pregnancy test Troponin in a heart attack Hepatitis B surface antigen screening

test

Test results are most often qualitative rather than quantitative.

Homogeneous Enzyme Immunoassay

no separation step is necessary. 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.

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.

Sensitivity:micrograms per milliliter far less than that achievable by heterogeneous

enzyme assays

Disadvantages of Enzyme Immunoassays: some specimens may contain natural

inhibitors size of the enzyme label may be a

limiting factor in the design of some assays

Nonspecific protein binding

FLUORESCENCE IMMUNOASSAYS

1941, Albert Coons

Fluorophores or Fluorochromes 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.

are typically organic molecules with a ring structure, and each has a characteristic optimum absorption range.

The time interval between absorption of energy and emission of fluorescence is very short and can be measured in nanoseconds.

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. Tetramethylrhodamine

absorbs at 550 nm and emits red light at 580 to 585 nm.

Other compounds used are: Phycoerythrin europium (β-naphthyl trifluoroacetone) lucifer yellow VS

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.

Direct Immunofluorescent Assays 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.

best suited to antigen detection in tissue or body fluids, while indirect assays

Examples of antigens detected by this method include:

Legionella pneumophila Pneumocystis carinii Chlamydia trachomatis respiratory syncytial virus (RSV)

Indirect Immunofluorescent Assays involve two steps:

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.

result in increased staining, because multiple molecules can bind to each primary molecule, thus making this a more sensitive technique.

can be used for both antigen and antibody identification.

Fluorescent Immunoassay

Complex must form for fluorescence to occur.

Fluorescent Immunoassay

Antibodies and bacteria are fixed on a glass-plate. The surplus i.e. non-bounded antibodies are washed out,

antibody-bacteria-complexes ("sandwiches") remain. The "sandwich" becomes visible by adding fluorescent anti

bovine immunoglobulin which can be seen as green light in the fluorescence microscope.

Fluorescent Immunoassay

Direct immunofluorescence Tagged antibody added to unknown antigen

fixed to slide If patient antigen present = fluorescence

Indirect immunofluorescence – sandwich assay Patient plus known fixed antigen Allow to react and wash off unbound

reactants Add tagged anti-antibody Fluorescence

Fluorescent Immunoassay

Positive Immunofluorescence

Cryptosporidium parvum oocysts Photo Credit: H.D.A Lindquist, U.S. EPA

Clinical application: used to detect treponema, antinuclear,

chlamydial, and toxoplasma antibodies, as well as antibodies to such viruses such as herpes simplex, Epstein- Barr, and cytomegalovirus.

Direct and indirect Immunofluorescence

OTHER FLOURESCENT 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.

Homogeneous Fluorescent Immunoassay 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.

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.

Fluorescence polarization immunoassay (FPIA) 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

Advantages: high sensitivity and versatility Simple methodology No problem with disposal of hazardous waste

Disadvantages: separation of the signal on the label from

autofluorescence produced by different organic substances normally present in serum.

nonspecific binding to substances in serum can cause quenching or diminishing of the signal and change the amount of fluorescence generated.

CHEMILUMINESCENT IMMUNOASSAYS It is the emission of light caused by a

chemical reaction, typically an oxidation reaction, producing an excited molecule that decays back to its original ground state.

most common chemiluminescent substances used: Luminol acridinium esters ruthenium derivatives nitrophenyl oxalates

Catalyst used: Hydrogen peroxide Enzymes

These intermediates are produced and 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 ester--emit a quick burst or flash of light Luminol and dioxetane--light remains for a longer time

Different types of instrumentation are necessary for each kind of emission.

Chemiluminescent Immunoassays The process of chemiluminescence occurs when

energy in the form of light is released from matter during a chemical reaction. 

Chemiluminescent Immunoassays

Large number of molecules capable of chemiluminescence Luminol Acridium esters Ruthenium derivatives Nitrophenyl oxalates

Use sodium hydroxide as a catalyst Light emission ranges from quick burst or

flash to light which remains for a longer time. Different types of instruments are required

based on emission.

Chemiluminescent Immunoassays

Can be used for heterogeneous or homogeneous assays.

Can attach label to antigen or antibody.

Heterogeneous assays use competitive and sandwich assay.

Competitive assays used to measure smaller analytes.

Sandwich assays are used to measure larger analytes.

Chemiluminescent Immunoassay

Many applications. Can measure antigen or antibody. Add chemiluminescently tagged analyte. Measure light which is emitted which is directly related to

concentration although competitive binding assays are available.

Chemiluminescent Immunoassays

Best known application of chemiluminescense is luminol

Luminol reacts with the iron in blood hemoglobin.

Advantages: excellent sensitivity Stable and nontoxic reagents inexpensive to perform High speed Inexpensive instrumentation