LABELED IMMUNOASSAY

Characteristics of Labeled Assay

1.

2.
3. 4. 5. 6.

Competitive Noncompetitive Standards or calibrators Separation method solid phase Detection of label Quality control

RADIOIMMUNOASSAY - RIA
- Makes used of a radioactive substance 131I, 125I, 3H - It is easily incorporated into a protein molecule and it emits gamma radiation detected using a gamma counter - Thyroid function tests: TSH, FT3, FT4

RIA
Principle: Based on the principle of competitive binding where the unlabeled analyte being detected competes with a radiolabeled analyte for a limited number of binding sites on a solid-phase antibody

disadvantage: - health hazard of radioactive substance - Disposal problem - Expensive equipment

ELISA or EIA
Designed to detect antigen or antibody by

producing an enzyme-triggered color change It uses the catalytic properties of enzymes to detect and quantitate immunologic reactions Enzyme with its substrate detects the presence and quantity of antigen or antibody present in the patient specimen

Enzymes
Naturally occuring molecules that catalyze

biochemical reactions They react with substrate that allows breakdown of products which could be chromogenic, fluorogenic, luminescent Can be measured qualitatively: detects presence of antigen or antibody quantitatively: by measuring the actual concentration of an analyte spectrophotometrically

Various enzyme labels

Horseradish peroxidase

Alkaline phosphatase
Glucose-6-phosphate dehydrogenase B-galactosidase

Classification of EIA
Heterogeneous - requires step to physically separate free from bound analyte 2. Homogeneous - no separation step necessary because enzyme activity diminishes when binding of antigen and antibody occurs
1.

Heterogeneous EIA
Competitive b. Non-competitive c. Capture assay
a.

Non-competitive
Indirect ELISA

Has higher sensitivity

Antigen or antibody attached to a solid phase HIV screening, HAV, HCV

Principle of non-competitive EIA

1. 2. 3. 4. 5.

6.
7. 8.

Ag absorbed to the solid phase Serum that might contain the Ab is added Ag-Ab binding Washing Ab-enzyme conjugate added Enzyme-conjugated Ab will bind to the complex Washing Substrate is added

Note: Amount of enzyme label detected is proportional to the amount of antibody in the specimen

Indirect ELISA
1.

2.

3. 4. 5. 6.

7. 8.

Ag absorbed to the solid phase Serum that might contain the Ab is added Ag-Ab binding Washing Ab-enzyme conjugate added Enzyme-conjugated Ab will bind to the complex Washing Substrate is added

Competitive EIA
Based on RIA

Enzyme-labeled antigen competes with unlabeled

patient antigen for a limited binding sites on antibody attached on a solid phase Used for measuring small antigens that are relatively pure: insulin and estrogen

Principle of competitive EIA

1. Labeled antigen competes with patient unlabeled antigen for a limited number of binding sites on solid-phase antibody. 2. Washing removes unbound antigen. Enzyme activity is determined. 3. 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.

Capture Assay
Sandwich immunoassay

Antigen captured in this assay must have multiple

epitopes Antibody is bound to a solid phase Used in measurement of immunoglobulins: IgM, IgE

 Principle

Plate is coated with capture antibody 2. Sample is added and any antigen present binds to capture antibody 3. Enzyme-labeled secondary antibody is added and completes the sandwich 4. Substrate is added and is converted by enzyme to detectable form
1.

Note: Enzymatic activity is directly proportional to the amount of antigen in the test sample

CAPTURE ASSAY
Principle 1. Plate is coated with capture antibody 2. Sample is added and any antigen present binds to capture antibody 3. Enzyme-labeled secondary antibody is added and completes the sandwich 4. Substrate is added and is converted by enzyme to detectable form

FLUORESCENT IMMUNOASSAY
Makes use of fluorescent compounds:

fluorophores or fluorochrome These compounds absorb energy from an incident light source and convert that energy into light of a longer wavelength and lower energy Measured in nanoseconds 2 compounds used: fluorescein and rhodamine

1.

Fluoroscein - absorbs maximally at 490 495nm - emits green color at 517 520nm - has high intensity, good photostability

2. Tetramethylrhodamine absorbs at 550nm - emits red light at 580-585nm 3. Phycoerythrin 4. Europium 5. Lucifer yellow

Use of Fluorescent Immunoassay: Histochemical localization of antigens in tissues: Immunofluorescent Assay - qualitative observation: fluorescence microscope - antigens can be detected in fixed tissue sections or in live cell suspension - antigen is determines by the appearance of a localized color against a dark background - for rapid identification of microorganism in cell culture or infected tissue, tumor-specific antigen on neoplastic tissue

 Fluorescent Staining:

Direct immunofluorescent 2. Indirect immunofluorescent

1.

1.

Direct immunofluorescent - antibody that is conjugated with a fluorescent tag is added directly to unknown antigen fixed on a microscope slide incubation. Washing - slide is read using fluorescence microscope - antigen is visualized as bright green apple or orange-yellow objects against dark background - best suited to antigen detection in tissue or body fluids

2. Indirect immunofluorescent - involves 2 steps 1. incubation of patient serum with a known antigen attached to a solid phase washing. 2. antihuman immunoglobulin containing fluorescent tag is added

 Other Fluorescent Immunoassay

3. FIA heterogenous and homogenous - similar to EIA 4. FPIA Fluorescence Polarization Immunoassay - based on the change in polarization fluorescent light emitted from a labeled molecule when it is bound by antibody - similar to competitive EIA

CHEMILUMINESCENT IMMUNOASSAY
Chemiluminescence

- is the emission of light caused by a chemical reaction producing an excited molecule that decays back to its original ground state Substances used: luminol, acridinium esters, ruthenium derivatives, nitrophenyl oxalates
- excellent sensitivity compared to EIA, RIA - faster turnaround time - used for immunologic testing

 Principle:

Luminol or other substances used are oxidized using hydrogen peroxide and an enzyme.
Intermediates are produced that are of higher energy state They return to their original state giving off energy in the form of light

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