LABEL IMMUNOASSAY

Clinical Serology-Immunology Lecture

Ag and Ab in small concentrations needs labeled molecules for

quantitation

LABELED IMMUNOASSAYS

Indicator labels

ANALYTE

substance to be measured bound by molecules that react

specifically to them 

Constituents of Labeled Immunoassays

Labeled Analyte

Antibodies

Standards/CalibratorsSeparation

Detection

LABELED ANALYTE

Radioactive Isotopes

125I

131I

Fluorochromes

absorb light

Fluorescence

spectrofluorometer or flow cytometer

Enzyme

Alkaline phosphatase

horseradish peroxidase

antigen: spectrophotometer

antibody: luminometer

ANTIBODIES

sensitivity depends on the affinity

specificity of antigen to antibody is

also important

STANDARDS OR CALIBRATORS

unlabeled analytes

to establish a relationship between the labeled analyte

SEPARATION

Centrifugation or filtration

Precipitation

sandwich technique

Solid-phase separation

DETECTION Presence of labeled analyte

Immunoassays

Ab-Ag interaction ImmunoassaysImmunoprecipitation

Detected

using

Indicator Labels

RadioactiveIsotope

Enzymes Fluorochrome

Which may be

Are based on

RADIO IMMUNOASSAY

LABELED IMMUNOASSAY

RADIOIMMUNOASSAY (RIA)

Developed by Yalow and Berson

Direct binding assay

131I, 3H, 125I

ADVANTAGES

Can measure hCG, FSH, gastrin, insulin, CEA, thyroxine, TSH, estrogens, androgens, and IgE

Extremely sensitive and precise technique

Can determine small or trace amounts of analytes that are small in size

DISADVANTAGES

Health hazard involved in working with radioactive substances

More difficult and expensive to maintain a laboratory license in compliance with federal law

Disposal problems

Short shelf life

Expensive equipment

Competitive binding assay

PRINCIPLELabeled and

unlabeled antigens can

compete equally for the same binding

site on the antibody

INDICATOR LABEL

Radioactive labelsEx. 125I

PURPOSETo determine the presence of antigen or antibody in

the biological sample

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Radiolabeled antigen mixed with a known amount of antibody

two chemically bind to one another

Competitive binding assay

•unknown quantity of that same antigen is added

•unlabeled antigen compete with the radiolabeled antigen for antibody binding sites

•unlabeled antigen will displace the radiolabeled variant

ratio of Ab - bound radiolabeled antigen to free radiolabeled

antigen will be reduced

bound antigens are separated from the

unbound ones

radioactivity of the free antigen

remaining in the supernatant is

measured

The amount of label in the

bound phase is indirectly

proportional to the amount of patient antigen

present.

As the amount of patient antigen increases, fewer binding sites will be occupied by labeled antigen.

IMMUNORADIOMETRIC ASSAY (IRMA)

Noncompetitive Immunoradiometric Assay

Uses labeled antibody that is present in excess

the reversible and non-covalent binding of antigen by a specific

labeled antibody

•The supernatant containing the bound complexes is counted

•The amount of bound labeled antibody is in direct proportion to the amount of patient analyte

Advantage

Faster reaction rate

Increased sensitivity

Excess antibody allows all of the unknown analyte to be involved in the reaction

Disadvantage

Loss of specificity

Increase in antibody concentration can result in cross reactivity with other

antigens

Immunoradiometric Assay (IRMA)

ENZYME LABEL IMMUNOASSAY

LABELED IMMUNOASSAY

ENZYME LABEL IMMUNOASSAY

Non isotopic Label

Naturally occurring =

CHEAP

Quali and Quanti Spectro

HOMO or HETERO

Table 1. Examples of Enzyme Immunoassay

Borrelia burgdorferi (IgG and IgM)

Cytomegalovirus (IgG and IgM Ab)

Cytomegalovirus (Ag)

Hepatitis A (total Ab)

Hepatitis B: Anti-HBs Anti-HBc Anti-HBc Anti-HBc (IgM) HBs Ag HBe Ag

Hepatitis delta Virus (total Ab)

Hepatitis non-A and non-B

HIV Ab

HIV Ag

HTLV-I Ab

HTLV-II Ab

Human B lymphocytic virus Ab

Rubella Virus (IgG and IgM Ab)

Toxoplasma gondii (IgG and IgM Ab)

Table 2. Enzymes used in Enzyme Immunoassay

ENZYME SOURCE

Acetylcholine Esterase

Electrophorous electicus

Alkaline Phosphatase

Escherichia coli

Beta-Galactosidas

e

Escherichia coli

Glucose Oxidase

Aspergillis niqer

G6PD Leuconostoc mesenteroides

Lysozyme Egg white

Malate dehydrogena

se

Pig Heart

Peroxidase Horse Radish

HETERO

Separation Step

ELISA and Capture assays

HETEROGENOUS ENZYME

IMMUNOASSAY

ELISA

COMPETITIVE

Competition Bet. Enzyme Labeled Ag and unlabeled Ag on the Ab

attached to a solid phase

Insulin and estrogen

COMPETITIVE

Competitive assay, high concentration of analyte

COMPETITIVE

Competitive assay, low concentration of analyte

COMPETITIVE

Color 1/α to amount of analyte present

NON COMPETITIVE

•Antibody bound to a solid phase

• Antigens are captured• Multiple epitopes

• Enzyme-labeled antibody

•Enzymatic activity directly proportional to the amount of antigen present

CAPTURE ASSAYS

Solid-phase antibody

Patient’s antigen

Incubate

Enzyme-labeled antibody

Colored reaction

Spectrophotometer

CAPTURE ASSAYS

Antigens

Antibodies

Polypeptide hormones

Proteins

Tumor markers

Microorganisms

CAPTURE ASSAYS

CAPTURE ASSAYS

Remember:

• Capture antibody• High specificity• High affinity

CAPTURE ASSAYS

Qualitative > quantitative

Rapid and easy to perform

Gives instant results

Home and point-of-care testing

Single use

Disposable plastic cartridge

MEMBRANE-BASED CASSETTE ASSAYS

MEMBRANE-BASED CASSETTE ASSAYS

Nitrocellulose membrane

MEMBRANE-BASED CASSETTE ASSAYS

MEMBRANE-BASED CASSETTE ASSAYS

Separate type

• Separate addition of:• Patient’s sample• Wash reagent• Labeled antigen or antibody• Substrate

MEMBRANE-BASED CASSETTE ASSAYS

Combined type/ Immunochromatography

• All requirements are combined already in the cassette

•Labeled zone•Analyte combines with the labeled antigen or antibody

•Detection zone•Captures the immune complex•Formation of colored line or a plus sign (+)

MEMBRANE-BASED CASSETTE ASSAYS

MEMBRANE-BASED CASSETTE ASSAYS

MEMBRANE-BASED CASSETTE ASSAYS

HOMOGENOUS ENZYME

IMMUNOASSAY

Antigen-antibody system in which no separation step is needed

Less sensitive than Heterogeneous assay

No washing step necessary

Major use

Determination of low molecular weight analytes

Horomones Threpeutic drugs Drugs of abuse

Principle

Change in enzyme activity as specific antigen-antibody combination occurs

Antigen labeled with enzyme tag

Antibody binds to determinant

Enzyme active site blocked

Competitive assay

Enzyme

activity

Concentration of

antigen

proportional

Detectability(enzyme activity)

Change in activity

Strength of binding(antibody)

Susceptibility(interference)

sensitivity

INTERFERENCES:

a. Endogenous enzyme activity

b. Cross reacting antigens

c. Enzyme inhibitors

CLONED ENZYME DONOR IMMUNOASSAY

GENETIC ENGINEERING

β-GALACTOSIDASE

TWO SUBUNITS

Subunits

A. Large polypeptide- enzyme acceptor

B. Smaller subunit- enzyme donor

• Small piece- attached as label to antigen

• The complex will compete with patients antigen

Ab

Labeled

Ag

Enzyme

Activity

reduced

Ab

Patient

Ag

Enzyme

activity

greater

ADVANTAGES AND DISADVANTAGES OF ENZYME IMMUNOASSAY

ADVANTAGE DISADVANTAGE

High sensitivity Inhibitors

Cheap instrumentation Size of enzyme label

Cheap and long lasting reagents

Non specific protein binding

Requires no separation(homogeneous)

Sensitivity of enzyme to temperature

FLOURESCENCE IMMUNOASSAY

LABELED IMMUNOASSAY

• ALBERT COONS –

developed the fluorescent method of labeling proteins, a significant tool for the study of infection in human beings.

• Antibodies could be labeled with molecules that fluoresce called Fluorochrome / Fluorophores

•sensitive technique

• measurement of many compounds, including drugs, hormones, and proteins;

•Identification of antibodies

• Quantification of antigens

Fluorochrome orFluorophores

RHODAMINEFLUORESCEI

N

ISOTHIOCYANATES

FLUORESCEIN ISOTHIOCYANATE

Absorbs maximally at 490 – 495

GREEN color at 517 nm

Highly sensitive

Good photostability

TETRAMETHYLRHODAMINE

Absorbs at 550 nm

RED light at 580 – 585 nm

FITC TAMRA

USED TOGETH

ER

PHYCOBILIPROTEIN

Emits red flourescence

At over 600 nm

Newer compound used

FLUORESCENCE MICROSCOPY

LIGHT SOURCE – EMITS LIGHT IN THE APPROPRIATE WAVELENGTH TO EXCITE THE FLUOROCHROME

HIGH INTENSITY LIGHT SOURCES SUCH AS:

TUNGSTEN HALOGENMERCURY VAPOR ARC

FIRST•Between light source and specimen•Remove uneccessary wavelengths

SECOND

•Between specimen and ocular lens•Screens out light other than that produced by the fluorchrom

FILTERS

FLUORESCENT STAINING

DIRECT

INDIRECT

Antigen Detection

Ag and AbDetection

DIRECT IMMUNOFLUORESCENT ASSAYAb conjugated

with fluorescent tag

Added directly to Ag fixed on

slide

Incubate and wash

Viewed using Fluorescence Microscope

Ag appear BRIGHT APPLE GREEN or

ORANGE-YELLOW OBJECTS against dark

background

DIRECT IMMUNOFLUORESCENT ASSAY

Best suited for Antigen Detection of:

Legionella pneumophilia

Pneumocystitis carinii

Best suited for Antigen Detection of:

Chlamydia trachomatis

Respiratory Syncytial virus

DIRECT IMMUNOFLUORESCENT ASSAY

INDIRECT IMMUNOFLURESCENT ASSAYPatient Serum + Known Ag

Wash and an Anti-Human Immunoglobulin containing fluorescent

tag is added

Fluorescence is determined

Amount of fluorescence is directly proportional to

amount of patient antibody present

INDIRECT IMMUNOFLURESCENT ASSAYUseful in antibody

detection of

Treponema

Antinuclear

Chlamydia

Toxoplasma

Herpes simplex virus

Epstein – Barr virus

Cytomegalo virus

HETEROGENOUS FLUORESCENTIMMUNOASSAY

• Require a Separation Step

•Includes : • Indirect Assays• Competitive Assays• Sandwich or Capture Assays

•Based on the principles of Enzyme Immunoassay but the label is Fluorescent that can be applied to either Antigen or Antibody.

• Used to Detect compounds such as:• Cortisol• Progesterone• Serum Thyroxine (T4)

• Solid Phase Fluorescent Assays identifies:• Ab to Nuclear Ag• Toxoplasma Ag• Rubella virus• Other virus Ags

HETEROGENOUS FLUORESCENTIMMUNOASSAY

Microbeads are used

Ag or Ab attaches to the

beads

React with analyte and a

fluorescent labeled analyte

Mixture is centrifuged;

supernantant discarded

Analyzed for fluorescence

SOLID PHASE SEPARATION

microbeads

SOLID PHASE SEPARATION

Dipstick Coated with Ag or Ab

Reacted with Patient

Sample

Labeled Ab is then added

One side of the stick is not coated (serves as

control)

Dipstick

HOMOGENEOUS ASSAYSNo Separation step ; Only one incubation step and no wash step

Basis: Change that occurs in the Fluorescent label on Ag when it binds to specific Ab

Such changes may be related to: Wavelength emission, Rotation freedom, Polarity or Dielectric strength

Amount of fluorescence is directly proportional to amount of Antigen

Not Sensitive

FLUORESCENCE POLARIZATIONIMMUNOASSAY

Based on change in polarization of fluorescent light

More Ag present, Less fluorescent Ab is bound and less polarization is detected

Degree of polarization is inversely proportional to concentration of analyte

IF LABELED MOLECULE IS BOUND TO AN ANTIBODY, THE MOLECULE IS UNABLE TO TUMBLE AS RAPIDLY AND IT EMITS AN INCREASED AMOUNT OF POLARIZED LIGHT,

DEGREE OF POLARIZED LIGHT REFLECTS AMOUNT OF LABELED ANALYTE THAT IS BOUND

FLUORESCENT IMMUNOASSAYS

Advantages

• More sensitive than radiolabels and enzyme reactions

• Simple and no hazardous wastes

Disadvantages

• Non specific binding can cause change in fluorescence

• Bilirubin or Hemoglobin present can absorb either excitation or emission energy

• Expensive

CHEMILUMINESCEN

CE IMMUNOASSAY

LABELED IMMUNOASSAY

Chemiluminescent Immunoassay

Chemiluminescence - emission of light caused by a chemical reaction

- produces an excited molecule that decays back to its original ground state - measured using a luminometer.

Common Chemiluminescent substances

Luminal

Acridium esters

Peroxyoxalates

Ruthenium derivatives

dioxetanes

Chemiluminescent Immunoassay

Chemiluminescent substances

Oxidized(hydrogen peroxide + enzyme)

Intermediates (higher energy state)

Chemiluminescent Immunoassay

Heterogenous assays

*competitive assay

* Sandwich format

Homogeneous assays

** labels can be attached either to the antigen or antibody**

Chemiluminescent Immunoassay

Advantages

Have excellent sensitivity

Reagents are stable and relatively non-toxic

LABELED IMMUNOASSAY

CLINICAL SEROLOGY AND IMMUNOLOGY LECTURE

AREVALO-GALANG-CERVANTES-CABANAG-CRUZ-CARLA-KHAN-BERNARDES-MARIANO-BAUTISTA-ZABALLERO-MOLANO

3HMT

Assoc. Prof. Jennifer Tiburcio