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