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 Antibodies 1. 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 conjugate 6. 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 peptide anti-HIV antibody (Sample) anti-cat Ig-HRP

b. Sandwich ELISA (For measurement of antigens)  1 st Antibody Is the Capture Ab  2 nd Antibody is the Detection Ab

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 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.laboratory techniques 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).high performance liquid chromatography  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. proteins

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