pharos university faculty of allied medical science clinical laboratory instrumentation (meli-201)
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Pharos university Faculty of Allied Medical SCIENCE Clinical Laboratory Instrumentation (MELI-201). Dr. Tarek El Sewedy. Lecture 5. ELISA & Chromatography and Autoanalyzers. Intended Learning Outcomes. By the end of this lecture the student should learn the basics of the following: - PowerPoint PPT PresentationTRANSCRIPT
Pharos university Faculty of Allied Medical SCIENCE
Clinical Laboratory Instrumentation
(MELI-201)
Dr. Tarek El Sewedy
Lecture 5
ELISA & Chromatography and
Autoanalyzers
Intended Learning Outcomes
By the end of this lecture the student should learn the basics of the
following:
1. ELISA technique
2. ELISA Readers and washers
3. Chromatography
4. Auto analyzers
Lecture content
1. ELISA technique
2. ELISA Readers and washers
3. Chromatography
4. Auto analyzers
ELISA
Enzyme Linked Immunosorbent Assay (ELISA)
Can Be Used To Detect Both Antibody or Antigen
Very Sensitive, pg/mL
combines the specificity of antibodies with the
sensitivity of enzyme assays.
What ELISA measures ?
ELISA is a technique that measures
antigen or antibody concentration.
1. Detect antigens that are recognized by
an antibody.
2. Detect antibodies that bind to an
antigen.
ELISA is performed in 96 well plates
Types of ELISA
a) Indirect ELISA (used to measure antibodies)
b) Sandwich ELISA (used to measure antigens)
c) Competitive ELISA (used to measure Both)
ELISA Plates
96 well plate
Made of plastic on which protein can be adsorbed (bind) easily
a. Indirect ELISA For measurement of Antibodies1. Coat wells with antigen
2. Add test serum sample (containing antibodies)
4. Add antibody-enzyme conjugate specific for the immunoglobulin in the
test serum
5. Wash well to remove unbound conjugate6. Add chromogenic substrate for enzyme
7. Read absorbance on microplate reader
3. Wash well to remove unbound serum
Examples
Detection of HIV-specific antibody
viral peptideanti-HIV antibody (Sample)
anti-cat Ig-HRP
b. Sandwich ELISA (For measurement of antigens)
1st Antibody Is the Capture Ab
2nd Antibody is the Detection Ab
y = 0.027x + 0.1046R2 = 0.9879
0
0.05
0.1
0.15
0.2
0.25
0.3
0 2 4 6 8
Sandwich ELISA standard curve(Directly proportional)
ELISA reader
ELISA microplate reader
Microplate readers are special instruments designed to measure color
intensity in large number of chemical samples in a single procedure.
Microplate readers are valuable tools in medical laboratories where they
are used to analyze multiple samples of body fluids for disease.
A particular type of light is selected based on the type of analysis being
done (substrate used). Some chemicals absorb a particular color of
light. Their quantity can be determined by measuring how much of the
light is absorbed by the sample. This is called absorbance detection
ELISA microplate reader Some chemicals glow when exposed to a particular light. This is called
fluorescence detection. The amount of chemical is measured by the
intensity of glowing.
Microplate readers feed the absorbance or fluorescence measures into a
computer program that analyses the particular information being
collected.
Should have a wide wavelength measuring range 350 nm to 750 nm .
Should be able to accommodate different types of plates (24,48,96,384).
Should contain data analysis and curve fitting program.
ELISA washer
Elisa Washer specifications
Used To wash the ELISA plates
Should adapt different types of microplates.
Automatic buffer switching.
Should Create, edit, delete and save preset programs.
Programmable shaking duration and intensity option.
ELISA troubleshooting
1. Poor PrecisionIncomplete washing of wells
Inadequate aspiration of wells
Inadequate mixing of reagents in the wells
Pipetting error
Reused pipette tips or reagent reservoirs
Reused plate sealer
ELISA troubleshooting
2. Inadequate Signal Development
Incorrect preparation of substrate
Incorrect incubation times or temperatures
Conjugate or substrate reagent failure
Improper instrument settings
Chromatography Chromatography is the collective term for a set of laboratory techniques for the
separation of mixtures.
The mixture is dissolved in a fluid called the "mobile phase", which carries it through a
structure (column) holding another material called the stationary phase or matrix.
The various constituents of the mixture travel at different speeds, causing them to
separate depending on structure, charge, size and other characteristics.
Chromatography may be preparative or analytical:
Preparative chromatography is to separate the components of a mixture for more
advanced use (and is thus a form of purification).
Analytical chromatography is done normally with smaller amounts of material and is
for measuring the relative proportions of analytes in a mixture.
An immobilized phase is a stationary phase which is immobilized on the
inner wall of the column.
The mobile phase is the phase which moves carrying the mixture being
analysed or separated. It may be a liquid (LC) or a gas (GC).
Chromatography Types By physical state of mobile phase
Gas chromatography: mobile phase is a gas
Liquid Chromatography: mobile phase is a liquid (ex:
high performance liquid chromatography (HPLC).
Affinity chromatography: is based on selective non-covalent interaction between
an analyte (to be separated) and specific molecules that binds this analyte (packed
inside the column). It is often used in the purification of proteins bound to tags.
Chromatography
HPLC
Which separation technique for which compound
Some Applications of chromatography
Chromatography has many applications in biology:
It is used to separate and identify amino acids, carbohydrates, fatty acids,
hormones and other natural substances.
Environmental testing laboratories use chromatography to identify trace quantities
of contaminants in oil and pesticides such as DDT in groundwater. It is also used to
test air quality.
Pharmaceutical companies use chromatography to prepare quantities of extremely
pure materials.
The food industry uses chromatography to detect contaminants such as aflatoxin.
AUTOMATED CHEMICAL ANALYZERS
(Autoanalyzers)
An autoanalyzer sequentially measures blood
chemistry through a series of steps of mixing,
reagent reaction and colorimetric measurements .
The AutoAnalyzer profoundly changed the
character of the chemical testing laboratory by
allowing significant increases in the numbers of
samples that could be processed
Autoanalyzers main parts
Main Parts of autoanalyzers
Sampler: aspirates samples, standards, wash solutions into the system.
pump: It mixes samples with the reagents so that proper chemical color
reactions can take place, which are then read by the colorimeter.
Dialyzer: it controls selective passage of sample components through a semi
permeable membrane
Heating bath: The heating bath controls temperature (typically at 37 °C), as
temp is critical in color development
Colorimeter: It monitors the changes in optical density of the fluid stream
flowing through a tubular flow cell. Color intensities proportional to the
substance concentrations are converted to equivalent electrical voltages.
Recorder: The recorder displays the output information in a graphical form.
Assignment
Ramzy Asaad is selected to make the assignment Different applications of
Chromatography
The Assignment should be delivered before next lecture
Study questions
Mentions 3 different applications of PCR
Mention the main difference between different types of incubators
Suggesting reading
Encyclopedia of Medical Devices and Instrumentation, 2nd ed. New York: Wiley, 2006