Label Immunoassay
-
Upload
cherry-reyes -
Category
Documents
-
view
1.788 -
download
3
Transcript of Label Immunoassay
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
K
n
o
w
n
q
u
a
n
tit
y
o
f
a
n
a
n
ti
g
e
n
i
s
m
a
d
e
r
a
d
i
o
a
c
ti
v
e
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