[M] Lec 03: Labeled Immunoassay Flashcards
Refers to:
- Rapid, specific, and sensitive assays to determine the presence of important biologically active molecules
- Designed for antigens and antibodies that may be small in size or present in very low concentration
Labeled Immunoassay
T or F: Labeled Immunoassays are only qualitative
False (can also be quantified)
T or F: Labeled Immunoassays can be adaptive for various types of samples like serum, urine, plasma
True
Refers to:
Substance being measured in immunoassay; can be antigen or antibody
Ligand
Refers to:
Immunoassay that uses radioisotope as label
Isotopic
Refers to:
Enzyme, fluorochrome, chemiluminscent
Non-isotopic
T or F: Non-isotopic immunoassay uses a radioisotope as label
False
Which between isotopic immunoassay and non-isotopic immunoassay uses a safer alternative for detecting biologically relevant substances?
Non-isotopic
Characteristics of labeled immunoassay
Competitive
Non-competitive
Heterogenous
Homogenous
Refers to:
Immunoassay in which patient Ag labeled reagent Ag compete for binding sites on reagent Ab
The antigens compete for very limited binding sites in the body
Competitive
Refers to:
Immunoassay that doesn’t involve competition for binding sites
Non-competitive
Refers to:
Immunoassay with a separation step to remove free from bound analyte
After binding reactions occur, a separation step is employed
Heterogenous
Refers to:
Immunoassay that doesn’t require a separation step; easier to automate
Homogenous
Refers to:
Unlabeled analytes that are made up in known concentrations of the substances to be measured
Standards (Calibrators)
Most immunoassays use a ______ vehicle for separation
Facilitates separation from bound and unbound analytes
Solid-phase
Refers to:
Polystyrene test tubes
Microtiter plates
Glass or polystyrene beads
Magnetic beads
Cellulose membranes
Solid vehicle for separation
Step that includes the physical means for separation
Washing
Detection of the label
- Typically a change in absorbance in a substrate is measured by spectrophotometry
- This involves a system for counting radioactivity
- Enzymes, fluorescence, chemiluminescence
- Radioimmunoassay
Quality control
- Run a blank tube, usually _________ saline with every test
- Any readings indicative of label in the blank are known as __________
- A negative control and a high and a low positive control should be _____ in addition
- All _______ and the ________ are usually run in duplicate
- Phosphate-buffered
- Background
- Run
- Control
- Patient sample
Refers to:
The first type of immunoassay, pioneered by Yalow and Berson in the late 1950s
Radioimmunoassay
Refers to:
A technoque used to measure small concentrations of an analyte, using a radioactive label on one of the immunologic reactants
Radioimmunoassay
Radioimmunoassay
- The amount of label in the bound phase is DIRECTLY proportional to the amount of patient antigen present
- The amount of label in the bound phase is INDIRECTLY proportional to the amount of patient antigen present
- Non-competitive binding assay
- Competitive binding assay
Enumerate the labels of radioimmunoassay
- 131I
- 125I
- Tritiated hydrogen or 3H
Used for detection in radioimmunoassay
Radioisotopes that emit radioactivity (deected through scintillation, gamma counter)
Radioimmunoassay labels
- Radioactive isotope of iodine with half-life of 8 days
- Label antibodies, Ag, and other biomolecules
- Label for steroids, vitamins, and drugs
- Isotope of iodine with a half-life of 59.4 days
- Radioactive isotope of hydrogen with a half-life of 12.3 years
- Commonly used to label proteins, peptides, and other molecules
- 131I
- 125I
- 3H
- 125I
- 3H
- 131I
Refers to:
- Most commonly used that detects and measures the radioactivity of scintillating materials like liquid scintillant or solid scintillators
Scintillation counters
Refers to:
- Instrument that specifically detects and measures the gamma radiation emitted by the radioisotopes like 131I and 125I
Gamma counter
The radioactivity of the sample is (directly/indirectly) proportional to the analyte present
Directly
Other types of RIA
- Noncompetitive / homogenous immunoassay
Example of non-competitive RIA
IRMA and Sandwich RIA
Noncompetitive
- Type of noncompetitive RIA that uses two antibodies to detect and quantify an antigen
Sandwich RIA
Application of competitive / heterogeneous assay
TSH Assay and Total IgE levels
Noncompetitive immunoassay
- Measures Ag conc directly
- Measures Ag conc indirectly
- IRMA
- Sandwich RIA
Enumerate the applications of RIA
- Radioallergosorbent Test (RAST)
- Radioimmunosorbent Test (RIST)
Match
- Detect allergenic antibodies IgE in the serum
- Measures the total IgE concentration in serum (allergic and parasitic response)
A. RIST
B. RAST
- B
- A
Refers to:
■ Employs a paper disc that is coated with allergen and is incubated with the patient’s serum
■ Washed with saline
■ Radioactively labeled anti-serum specific for human IgE is applied to the disc
■ Measure the IgE.
RAST
Applications of RIA
- Aids in the diagnosis of allergies, parasitic infections, and monitoring IgE levels in patients with allergic diseases.
- Aids in the diagnosis of allergies such as Hay Fever, asthma, and food allergies
- RIST
- RAST
Four enzyme labels of EIA
- Alkaline Phosphatase
- Horseradish Peroxidase
- B-D-galactosidase
- G6PD
Indentify the ff enzyme labels
- Widely used as an enzyme label that catalyzes the oxidation of substrates, resulting in a colored product
- Enzyme label that catalyzes the hydrolysis of β-D-galactosides, resulting in a colored product
- Enzyme label that catalyzes the oxidation of glucose-6-phosphate, resulting in a colored product
- Commonly used enzyme label that catalyzes the hydrolysis of phosphate esters
- Horseradish Peroxidase (HRP)
- β-D-galactosidase
- G6PD
- ALP
Types of EIA
- RAPID ELISA
- ENZYME-MULTIPLIED IMMUNOASSAY TECHNIQUE (EMIT)
EIA; refers to:
● Description: membrane-based
● Principle: uses the same principle of traditional ELISA but with a shorter incubation time and more sensitive detection
● Others: May have built-in control. Usually qualitative.
Rapid ELISA
EIA; refers to:
● Description: Homogeneous-based
● Principle: uses an enzyme-labeled antigen that competes with a sample antigen for binding to a specific antibody
ENZYME-MULTIPLIED IMMUNOASSAY TECHNIQUE (EMIT)
Detection
- Measures the absorbance of light by the colored product formed by the enzyme reaction
- Measure the fluorescence emitted by the product formed by the enzyme reaction
- Colorimetric
- Fluorometric
Familiarize the potential disadvantages of EIA
■ Matrix effects: The comparison of the specimen can affect the enzyme reaction, leading to an inaccurate result.
■ Enzyme instability: can be unstable, leading to
decreased activity over time
■ Reagent variability: The variability in reagent quality can affect the accuracy and precision of the EIA results.
■ Operator’s error: the EIA requires careful handling and processing of the specimen and reagents, and mistake can lead to inaccurate results.
Refers to:
○ An immunoassay that uses an enzyme as a label to detect and quantify a specific Ag or Ab
○ can be further classified into different types, such as ELISA (the popular format of EIA)
○ First type of EIA developed
Direct Enzyme Immunoassay
Refers to:
● PRINCIPLE: Color intensity is inversely proportional to the concentration of ligand in the specimen
● APPLICATION:
○ It is used to measure small, relatively pure Ag
○ Suitable for detecting and quantifying low concentrations of antigen in biological samples
● USEFUL
○ when working with a small, well-defined antigen
○ High sensitivity and specificity are required
Competitive EIA
Refers to:
● type of immunoassay that uses an enzyme-labeled secondary antibody to detect and quantify a specific antigen or antibody
● More commonly used than direct assays
Indirect Enzyme Immunoassay
Key features of Indirect EIA
- Enzyme-labeled antibody
- Substrate
- Enzyme activity measurement
Types of Indirect immunoassay
● ELISA (Enzyme-linked immunosorbent assay)
● Western Blot
● Immunohistochemistry
Refers to:
● Uses noncompetitive format
● There are two incubations and two washes to remove the unbound renOagents
● Enzyme-labeled secondary antibody
Non-competitive EIA
Refers to:
ADVANTAGES:
○ Higher sensitivity
○ Higher specificity
○ Can detect a small amount of antigen and antibody
○ Can be used in qualitative and quantitative tests
○ Suitable for large-scale screening and diagnostic testing
● PRINCIPLE: Color intensity is directly proportional to the
concentration of the antibody in the specimen.
● APPLICATION:
○ Cancer research
○ Diagnosis of infectious diseases
○ used to detect antibodies to viruses (HIV, HAV, HCV, EBV)
○ Immunological research and monitoring of immune response to infection or vaccination
Noncompetitive EIA
Refers to:
● Any immunoassay that uses an enzyme as a label
● A substrate is added to measure enzyme activity
* Uses solid phase vehicles
Solid Phase Enzyme Immunoassay
Examples of Solid Phase Enzyme Immunoassay
○ Enzyme-link immunosorbent assay (ELISA)
○ Enzyme immunoassay (EIA)
○ Immunoenzymatic assay (IEA)
Refers to:
● Principle: Enzyme activity is directly proportional to the amount of antigen in the sample
● Application: Used to measure immunoglobulins, hormones,
proteins, and detect tumor markers, viruses, parasites, and fungi.
Capture EIA
Refers to:
● A laboratory technique used to detect and quantify specific molecules such as antigens, antibodies, and hormones
● Detection method: fluorescence
Fluorescent Immunoassay
Labels used in fluorescent immunoassay
Fluorescein, rhodamine, texas red
Match
- Orange
- Green
- Red
A. Texas Red
B. Rhodamine
C. Fluorescein
- B
- C
- A
Match
- 550 nm
- 490-495 nm
- 600-620 nm
A. Texas Red
B. Rhodamine
C. Fluorescein
- B
- C
- A
Refers to:
- Labels that absorb energy from the light source; the absorbed energy is converted to a longer wavelength
- Emits light at the longer wavelength
Fluorochromes
Fluorescence Immunoassay types of assay
● Competitive
● Heterogeneous and homogeneous
Different methods of fluorescent immunoassay
- Direct fluorescent antibody (DFA) staining
- Indirect fluorescent antibody (IDA) staining
- Fluorescent polarization immunoassay (FPIA)
Different methods of fluorescent immunoassay
- Principle: The specimen on a glass slide overlaid with fluorescein-labeled antibody. If the corresponding antigen is present, the labeled antibody binds. Fluorescence is observed with a fluorescent microscope
- Principle: Labeled antigen competes with antigen in the specimen for sites on the reagent antibody. Free labeled antigen rotates rapidly and emits little polarized light. Bound labeled antigen rotates more slowly; it emits more polarized light. The amount of polarized light is inversely proportional to the concentration of Ag in the specimen
- Principle: Reagent antigen on a glass slide overlaid with patient serum. If the corresponding antibody is present in the serum, it attaches to the antigen. When fluorescein-
labeled anti-human globulin is added, it attaches to the antibody. Fluorescence is observed with a fluorescent microscope
- Direct Fluorescent Antibody Staining
- Fluorescent Polarization Immunoassay
- Indirect Fluorescent Antibody Staining
Principles involved in fluorescent immunoassay
- An antigen-antibody reaction
- Excitation/emission
Principle of fluorescent immunoassay
- The fluorescent dye is excited by a specific wavelength of light, and the emitted fluorescence is measured
- A specific antibody binds to the target antigen; A fluorescent labeling/dye is attached to the Ab/Ag
A. An Ag-Ab reaction
B, Excitation/emission
- B
- A
T or F: The degree of polarization is directly proportional to the amount of Ag-Ab complex formed.
False (indirectly)
Refers to:
● Based on principle of chemiluminescence
● 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
Chemiluminescent immunoassay
Labels in chemiluminescent immunoassay
Luminol, Acridinium esters, Ruthenium derivatives, Nitrophenyl oxalates
Chemiluminescent Immunoassay detection
Chemiluminescent molecules produce light from chemical reactions
Chemiluminescent Immunoassay type of assay
Competitive and non-competitive;
Heterogeneous and homogeneous
