1. Instruments for measurement of Antigen-Antibody interactions
Lab 12

2. 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.

3. Immune complexes Chemiluminescent Turbidimetry Nephelometry
Soluble immune complexes
Indicator labels
Fluorochromes
Such as
Fluorescein
Measured using
Spectrofluorometer
Flow cytometer
Fluorescence microscope
Colored
Alkaline phosphatase
Scintillation Counter
125I, 131I, 3H
Enzymes
Radioactive Isotopes
Immune complexes
Detected
In the presence of
Spectrophotometer
Luminometer
Substrate products
are either
May be
That include
A scintillation counter measures ionizing radiation. The sensor, called a scintillator, consists of a transparent crystal, usually phosphor, that fluoresces when struck by ionizing radiation.

4. 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.

5. 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.

6. Spectrophotometer Light source Digital display Photomultiplier tube
Sample
Wavelength
selector
Prism

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.

12. 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.

13. 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.

14. Fluorescence Microscope
A dichroic filter or thin-film filter is a very accurate color filter used to selectively pass light of a small range of colors while reflecting other colors.

15. Fluorescence Microscope

16. 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.

17. 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

18. Gamma Counter Y Y Y PMT Sodium iodide crystal Lead shield
Sample with 125I Y Y
Sodium iodide
crystal Y
Lead shield
PMT

19. 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.

20. Instrumentation for Detecting non-labeled 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.

21. 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:
absorbed, (measured by turbidimetry)
transmitted,
reflected
or scattered (measured by nephelometry)

22. 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.

23. 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.

24. Nephelometer The main components of nephelometer are: Light source
Collimating lens to focus the light source
Sample cell compartment
Collecting lens
And electronic detector

25. 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.

26. 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.