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
sensitivity of enzyme assays.
with the
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 Antibodies
1. Coat wells with antigen
Examples
2. Add test serum sample (containing antibodies)
3. Wash well to remove unbound serum
4. Add antibody-enzyme conjugate specific
for the immunoglobulin in the test serum
5. Wash well to remove unbound conjugate
6. Add chromogenic substrate for enzyme
7. Read absorbance on microplate reader
Detection of HIV-specific antibody
anti-cat Ig-HRP
anti-HIV antibody (Sample)
viral peptide
b. Sandwich ELISA (For measurement of antigens)
 1st Antibody Is the Capture Ab
 2nd Antibody is the Detection Ab
Sandwich ELISA standard curve
(Directly proportional)
y = 0.027x + 0.1046
R2 = 0.9879
0.3
0.25
0.2
0.15
0.1
0.05
0
0
2
4
6
8
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 Precision
Incomplete 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