Instruments for
measurement of AntigenAntibody interactions
Lab 12
Instruments for measurement of
Antigen-Antibody interactions
• Immunological tests are those in which
antigen-antibody interactions are used as
tools for diagnosis.
• Ag-Ab complexes that are formed are too
small to be observed directly, so indirect
methods must be used.
• Most use reagents that will induce
changes in light, radiation or solution color
as an indication of Ag-Ab interaction.
Immune complexes
May be
Detected
In the presence of
Indicator labels
Soluble immune complexes
That include
Nephelometry
Fluorochromes
Radioactive Isotopes
Enzymes
Such as
Such as
Turbidimetry
Such as
Alkaline phosphatase
Fluorescein
125
I, 131I, 3H
Substrate products
are either
Colored
Measured using
Chemiluminescent
Measured using
Measured using
Spectrofluorometer
Flow cytometer
Fluorescence microscope
Scintillation Counter
Luminometer
Measured using
Spectrophotometer
Labeled indicators
• Gamma ray emission, fluorescence and
chemiluminescence are all phenomena
that occur when unstable molecules or
electrons revert to a more stable state.
• Gamma radiation, fluorescence and visible
light are all the same phenomena that
differ only in their energy and hence their
wavelength.
Instrumentation for measuring color
intensity
• One of the ways to analyze the formation
of Ag-Ab complex is to label either Ag or
Ab with an enzyme.
• When a particular enzyme substrate is
added, the color is an indication of the no.
of Ag-Ab complexes formed.
• The presence of color provide a qualitative
test, while measuring the intensity of the
color formed can give quantitative data.
Spectrophotometer
Light
source
Prism
Wavelength
Sample
selector
Photomultiplier
tube
Digital
display
Microplate Reader
• Microplate reader is capable of measuring the absorbance of
96 samples in about 8 seconds.
• The basic design is the same as the spectrophotometer, a
selected wavelength of light passes through the samples and
a phototube measures the amount of light transmitted through
the sample, which the plate reader converts to absorbance.
• However, the samples are located in microwells that are
arranged in an 8 x 12 in one plastic plate.
• You can put your samples in any or all of the microwells.
• The plate is moved over an array of 8 fiber optics light
sources and 8 phototubes.
• Each row of 8 is scanned and then the plate advances by one
row and the process continues until all 12 rows are scanned.
• The absorbance data are then displayed on a screen in an 8 x
12 array.
• These data can be saved in the memory to be printed later.
Photomultiplier Tube (PMT)
• The photomultiplier was invented in 1936,
• PMT converts light signals into electrical
signals.
• It is also designed to amplify the signal so that
weak signal can be detected.
• PMT have a series of light sensitive metals
that eject electrons when light strikes the
surface.
• These ejected electrons cause more electrons
to be ejected causing a cascade response that
generates an electric current.
• This amplification creates a much stronger
signal for detection.
• The electric current which is generated is
detected to galvanometer and translated to
readable signal by a recording device and
then displayed.
Instrumentation for measuring
Chemiluminescence
• Chemiluminescence is the name given to the light emitted
•
•
•
•
•
from a chemical reaction.
It is similar to fluorescence but the stimulus that cause
the excitation molecules differs.
In fluorescence, the source of energy is external (light)
whereas in Chemiluminescence the stimulus is a
chemical reaction that results in the production of a
molecule that is luminescent.
This involves a chemical reaction in the presence of
enzyme where an intermediate molecule is produced.
The amount of light emitted from the intermediate is
proportional to the amount of luminescent material.
This in turn is proportional to the number of Ag-Ab
complexes present.
Instrumentation for measuring
Chemiluminescence
• Chemiluminescence is measured in a
luminometer.
• Because the emitted light is generated
from a chemical reaction the luminometer
must be designed so that no extraneous
light enters the chamber.
• Luminometer are relatively simple and
inexpensive instruments relative to
fluorometers.
Luminometer
• The components of the
luminometer include:
• A chamber for the sample
• May hold microtiter plate or test tubes
• A light detector.
• PMT
• A processor that handles the
signal.
• Monitor that displays the signal.
Instrumentation for measuring
Fluorescence
• Fluorochromes absorb light of a particular wavelength,
transiently excited and then emit light of a longer wavelength
as they return to their unexcited state.
• The emitted light is directly proportional to the amount of
fluorochrome present.
• Not all light energy absorbed by fluorochrome is emitted as
fluorescence because some of the energy dissipated as heat
or vibrational energy.
• The emitted light has less energy and so a longer wavelength.
• Different fluorochromes absorb light at different wavelength
and emit light at a different wavelength.
• Each fluorochrome has its own fingerprint of excitation
spectrum and emission spectrum.
• Fluorescence may be detected with spectrofluorometer, flow
cytometer or fluorescence microscope.
Schematic of a Fluorescence Detector
To avoid problems of differentiating between excitation and emission
fluorescence detector operates in “right angle configuration”
Radiation from a xenon lamp passes through an excitation filter, which
provides essentially monochromatic light of desired wavelength to excite
the sample.
Fluorescence Microscope
Fluorescence Microscope
Gamma Counter
• In radioimmunoassays,
radioisotopes that emit
gamma rays are frequently
used to detect Ag-Ab
interaction.
• Gamma radiation is
measured in a solid
scintillation counter,
commonly referred as
gamma counter.
Gamma Counter
• Essential components of gamma counter are:
• Fluorophore, well shaped for holding a sample
(sodium iodide crystal)
• Metal encasement that surrounds the crystal on
all but one side serves as a shield
• An open side (no lead shield) linked optically to
a photomultiplier tube
• A signal amplifier
• A counting device
• Monitor or printer
Gamma Counter
Sample with 125I
Sodium iodide
crystal
Lead shield
PMT
Gamma Counter
• To analyze the amount of radioactivity in a
sample, sample is placed in the crystal well.
• Gamma rays emitted from the sample will pass
through plastic or glass walls of the vial
containing the sample, and enter the crystal.
• When gamma rays interact with the crystal, their
energy is transferred to the crystal, causing
excitation of the atoms.
• Electrons within the crystal return to their original
energy level, light is emitted and detected with a
PMT.
Instrumentation for Detecting nonlabeled immune complexes
• When a biological sample containing an Ag of interest is
mixed with specific Ab for that Ag, immune complexes
will form.
• The size of the immune complex depends on the
concentration of the reactants.
• At the appropriate relative concentration, the complexes
will be sufficiently large that they precipitate out of
solution.
• When Ag or Ab is present in excess, immune complexes
form but they do not precipitate out of solution.
• These soluble immune complexes can be measured by
monitoring the effect of light on these complexes.
Instrumentation for Detecting
non-labeled immune complexes
•
When collimated light hits a particle ( immune
complexes) in solution, there are at lest four
effects, light is either:
1.
2.
3.
4.
absorbed, (measured by turbidimetry)
transmitted,
reflected
or scattered (measured by nephelometry)
Nephelometry
• Nephelometers are instruments that measure
the amount of light scatter caused by Ag-Ab
complexes in solution.
• This technique has largely replaced radial
immunodiffusion in clinical laboratory.
• Nephelometry measure soluble immune
complexes while immunodiffusion measures
precipitated immune complexes.
• Therefore, the two techniques target different
points in the precipitation curve.
Nephelometry
• In nephelometry the relative amount of antigen and
antibody must be such that small immune complexes
form, but precipitation does not occur.
• Therefore, this technique is only valid if the reaction is
carried out in antibody excess (when measuring Ag in
biological sample).
• If precipitation were to occur, the light scatter
measurement will be low, and this could lead to a false
negative result.
• Most assays that use nephelometry are available in kit
form.
• Kits available include those that measure albumin,
complements C3 & C4, IgG, IgA & IgM.
• Advantages is speed and precision.
Nephelometer
• The main components of
nephelometer are:
• Light source
• Collimating lens to focus
the light source
• Sample cell compartment
• Collecting lens
• And electronic detector
Quality Control in Nephelometry
• Nephelometry is based on measurement of the
scattered light.
• Therefore, presence of dust, dirty cuvettes and
contaminated reagents can affect the precision.
• Problems to background scatter arise from
proteins and lipids present in the sample.
• A high background value can be reduced by
dilution of the serum samples to at least 1:50
• And by also pretreatment of the sample with
polymers which tends to make these substances
less soluble and precipitate.
Turbidimetry
• A measure of the decrease in the transmission
of light caused by particle (immune complex)
formation in a solution.
• In turbidimetry, the amount of light transmitted
through the sample is the recorded measure.
• Turbidimetry can be measured in a
spectrophotometer.
• The sensitivity of a turbidimetric assay depend
on the ability of the instrument to detect small
changes in the light intensity.