Week 2: Electrophoresis, Chromatography & Immunoassay

Place the electrophoretic fractions in the correct order from cathode to anode and vice versa: gamma, alpha-2, alpha-1, albumin, beta

(+) - Albumin > alpha-1 > alpha-2 > beta > gamma (-)

Compare competitive versus noncompetitive reaction formats

- Competition between the patient Ag and the reagent Ag-L (labeled) for the reagent Ab sites. --Inverse Relationship between Signal Strength & Concentration. Decreased Signal = Increased [Patient Ag]. - Noncompetitive or Sandwich Assay Reagent Ab attached to well ├Ab. Patient Ag is added forming ├Ab-Ag. - Second Reagent Ab-L is added forming ├Ab-Ag-Ab-L. - Label is Activated and Read. --Direct Relationship between Signal Strength & Concentration. Increased Signal = Increased [Patient Ag].

Basic procedure and utility for Fluorescent Polarization Immunoassay (FPIA)

- Competitive - Quantitative - Inversely Proportional - Increase in Patient Ag = Decrease in Signal - One Step Procedure - Add Reagent Ab + Fluorescein-labeled Reagent Ag-F + Analyte Ag - Light Source at 420nm causes Bound Ab-Ag-F to emit fluorescent light - Polarized Filter Light detector - Free Ag-F emits Lower Polarization Signal - Bound Patient Ab-Ag-F emits Higher Polarization Signal - Used in ID & Quantification of drugs in serum & urine eg. cannabinoids, cocaine, PCP & opiates

Enzyme Multiplied Immunoassay Technique (EMIT) Principle, Procedure & Application

- Competitive Method - Quantitative - 2 step procedure - Step 1 Reagent Ag-L + Reagent Ab - Step 2 Add Patient Ag (analyte) - Less Patient Ag = More Reagent Ag-L will bind with Reagent Ab Enzyme Label Inactivation Lower Enzyme Signal - More Patient Ag = Higher Enzyme Signal - Directly Proportional - Used for ID & Quantification of drugs in serum and urine

Discuss the basic principle, method, and risks for RIA

- Competitive Reaction - Antigen labeled with radioactive C- isotope to a specific known Ab - Patient Ag is added - Released/Labeled Ag is Proportional to Patient Ag - Radioactive Waste

Explain how chromatography is used to separate compounds contained in a mixture

- Does not rely upon an electric current to separate components, as does electrophoresis. - Consists of two phases: mobile and stationary. - Samples are introduced into a mobile phase, usually a flowing stream of liquid or gas, which then passes through a bed of support medium or particles (stationary phase) that facilitates separation. - Components of the mixture travel at different speeds, causing them to separate. Separation is based on differential partitioning between the mobile and stationary phases.

Immunoelectrophoresis (IEP) Principles, Procedures & Applications

- Noncompetitive - Qualitative present/not present, not amount - Gel - Patient Ag in wells - Electrical Field moves Patient Ag horizontal - Reagent Ab in trough Diffuses toward Separated Patient Ag vertically - Precipitin bands form where Ab-Ag complexes form in optimal proportions

Immunofixation Electrophoresis (IFE) Principles, Procedures & Applications

- Noncompetitive - Qualitative present/not present, not amount - Used to separate, ID & Quantify Immunoglobulins only - Specifically looks for M-protein (abnormal Ig produced by plasma cells in Multiple Myeloma) - 2 steps - Step 1 Electrophoresis separates serum/urine proteins by Mass & Charge into 5 fractions (Albumin, α1, α2, β & γ) - Step 2 Immunofixation using specific Ig antisera (anti-IgG, anti-IgA, Anti-IgM, anti-κ & anti-λ) in each migration lane. - Current is applied to move the γ-fraction horizontally across the Ig antisera migration lanes. - Only the specific Ig in the serum/urine are captured by the anti-Ig so that If IgG and κ light chains are present (MM), those are the only migration lanes that will show up.

Enzyme-Linked Immunosorbent Assay (ELISA) Principle, Method & Applications

- Quantitative - Most common used immunoassay in the lab - Solid phase with adsorbed ligands (Ab or Ag) - Patient Ag or Ab are added with a label

Isoelectric Focusing (IEF) Principles

- Sample molecules migrate through a pH gradient. - Used when the size and net charge of the solutes to be separated are similar. - The pH gradient is created with the aid of a group of low molecular weight compounds called ampholytes. - When current is applied, the carrier ampholytes migrate to a zone along the medium where their net charge is zero (pI). - Specimen proteins move more slowly and stop when they reach the zone where their net charge is zero. - The high buffering capacity of the ampholytes creates stable pH zones. - Protein bands tend to be very sharp because the pI of a protein is confined to a very narrow pH range.

capillary electrophoresis (CE) principles

- Separates molecules using a capillary tube (acts as support medium) connected to a high voltage power source. - Once introduced into the system, the sample analytes separate due to their individual electrophoretic mobility and travel through the capillary tube carried by the electro-osmotic flow. - The electroosmotic flow is created by interaction between negatively charged functional groups attached to the interior surface of the capillary and positively-charged ions in the buffer. - A detection system is placed near the cathode end of the system and data collected is sent to a computer as the sample and buffer flows from the anode (+) cathode (-) and into the destination reservoir. - Records time vs charge

Principle of Western Blotting

- Separation of proteins by electrophoresis - Followed by transfer (blotting) of the separated proteins to a sturdier medium. - Separated proteins are blotted onto another medium such as nitrocellulose that is stronger and can be treated in some way. - Labeled antibodies are applied to the medium to visualize the proteins.

isoelectric focusing (IEF) applications

- When molecules are very similar - Some isoenzymes and variant hemoglobins in prenatal screening are separated with IEF. - Detection of oligoclonal bands in gamma-globulin is a newer use - commonly used as one of the separations in two-dimensional electrophoresis.

Calculate Rf where: origin to spot = 39.43nm origin to solvent front = 53.97nm to the nearest hundredth.

0.73

Outline the components of an electrophoretic system

1. A hydrated support medium is placed in between the buffer chambers 2. The buffer chambers are filled 3. Samples are applied to wells in the support medium at a point of application 4. A cover is placed over support medium 5. The current is applied for an appropriate time 6. At the end of timed electrophoresis, the support medium is removed 7. A stain is applied to the fixed support medium to visualize protein bands that represent the various proteins within the sample

isoelectric focusing (IEF) components

1. Add ampholytes to gel to create a pH gradient from (+) 3-9 (-) 2. Pipette serum protein mix to application well of the support medium 3. Apply current 4. Proteins will move until they reach the zone where the pI = 0

Describe the general procedure of electrophoretic separation

1. Set up apparatus, fill chamber with appropriate buffer, place support medium & add sample 2. Attach electrodes, cover and run gel 3. Stain gel (Amido Black B or Coomassie Brilliant Blue) 4. Read stained gel with densitometer from anode (+) to cathode (-) direction

What isolation technique(s) employ precipitation

Ag-Ab matrixes form and precipitate at the zone of equivalence in Immunoelectrophoresis (IEP)

Electrophoresis Limitations of Voltage

Buffer issue. Excess voltage may cause proteins to migrate faster, creating greater distances between bands or loss of bands off the end of the gel. This may make the interpretation of the banding pattern difficult.

Power Supply, Anode, Cathode, Capillary Tube, Buffer, Detector, Source Reservoir, Destination Reservoir & Sample.

Capillary Electrophoresis (CE) Components

internal standards used in chromatography

Continuous flow with sample(s) Used when sample sizes are low and/or volatile Measure change in mass peak

What technique allows us to quantify proteins on a stained support medium? a Chromatography b Densitometry c Nephelometry d Spectrophotometry

Densitometry

Net electrical charge and migration rate

Direct effect. The higher the net charge, the faster the migration rate.

4 Types of ELISA

Direct, Indirect, Sandwich & Competitive

What is the most commonly used immunoassay used in the lab?

ELISA

basic theory of electrophoretic separation

Electrophoresis is a technique which induces the migration of charged solutes or particles under the influence of an external electrical field. It is a separation technique based on the concept that different molecules move at different speeds in an electric field. When oppositely charged electrodes are placed in a solution, ions have an affinity for and travel toward the electrode of opposite charge. A cation is a positively charged ion that will travel toward the negatively charged cathode. Anions (negatively charged molecules) travel toward the anode.

Identify the various types of labels used in immunoassays

Enzymes, Isotopes, Fluorescence

Two-Dimensional Electrophoresis (2-DE) Advantages

Expands the number of proteins that can be separated and identified. It is very unlikely that two molecules will be similar in both properties.

IFE Pros

Faster & More Sensitive than protein electrophoresis

Types of Columnar Chromatography

Gas (GC) Gas Chromatography-Mass Spectrometry (GC-MS) Liquid (LC) High Performance/Pressure Liquid (HPLC)

Strength of the electrical field and migration rate

Have variable effects on electrophoresis. Distortions in migration may occur if the strength of the electrical field is too high. Typically, stronger electrical fields cause faster migrations.

PAGE Pros

High Resolution (Distinct Banding), separation based on charge: mass ratio AND molecular size (molecular sieving), increased sensitivity on similar molecules

What immunoglobulin is used in immunoassays?

IgG

What serum protein fractions are found in Multiple Myeloma

IgG & κ-light chains (M-protein)

Gas (GC-HPLC) Mobile Phase

Inert gas eg Nitrogen, Argon or Helium + Heat Block to vaporize sample Separates compounds based on vapor pressure

Which of the following processes separate amphoteric molecules by creating pH gradients in an electric field? a Western blotting b Isoelectric focusing c Capillary electrophoresis d Potentiometry

Isoelectric Focusing

PAGE Cons

Long Run Time & Carcinogenic

Agarose Gel Cons

Lower resolution, Low Protein Affinity & Electroendosmosis

GC-MS Detection Device

Mass Spectrophotometer

Summarize the procedure for the quantitation of the electrophoretic fractions using a densitometer

Measures degree of darkness or optical density (OD) by passing a light through the support medium as it moves from anode (+) to cathode (+) direction of the support medium. The amount light absorbed depends on the stain density. The non-absorbed light penetrates the translucent layer. The information is graphed as OD x distance

Cellulose Acetate Uses

Most Commonly Used. Serum Protein (SPEP), Urine Protein, Hgb & Isoenzymes

cathode

Negative charged & attracts positively charged cations

Factors which influence the rate of migration in electrophoresis

Net electrical charge, size & shape of the molecule, Strength of the Electrical Field, Supporting Medium Properties and Temperature

IFE Cons

Only specific to Ig proteins & More expensive than protein electrophoresis

Types of Planar Chromatography

Paper & Thin Layer (TLC)

Thin-Layer (TLC) Detection Method

Phosphor stain with UV Light

Which of the following is the positive electrode in an electrophoretic unit? a anode b cathode

Positive = anode Negative = Cathode

anode

Positive charged & attracts negatively charged anions

Commonly used to analyze mixtures of two properties in 2D respectively. - Proteins are first separated by their pI using IEF method. - Proteins are then separated by molecular weight by SDS-PAGE method in a 90˚ direction from the first phase.

Principle of Two-Dimensional Electrophoresis (2-DE)

Resolution

Quantitative measure of the separation of chromatographic peaks between two molecules. R = (2 x change in retention time of peak 1 & 2 )/ (baseline widths of peak 1 + peak 2)

In TLC, the distance of leading edge of component / distance of solvent front. Compare to standards to ID sample(s) in mixture.

Retention Factor (Rf)

Agarose Gel Uses

Routine Serum Proteins, Lipoproteins & Hemoglobin

Process of Serum Protein Electrophoresis (SPEP)

Separates proteins based on net charge, size and shape. Used to identify diseases such as Multiple Myeloma or other gammopathies.

Serum Proteins Analyzed in Western Blot

Serum Albumin (smaller/faster) & Serum Globulins

High Performance/Pressure Liquid Chromatography (HPLC)

Solvent Reservoir, Pump, Sample Injector, Column, Detector, Read-out Device & Waste Receptacle

cross-reactivity

The ability of antibodies to react with other compounds other than the target antigen that have a similar structure

What isolation technique(s) employ solid phase isolation and how

Thin Layer Chromatography & Liquid HPLC - use silica or diatomaceous earth in columns or beads - isolate viral (alkaline lysis) or bacterial DNA from serum, plasma or CSF - isolate cellular DNA for genetics & oncology studies

Southern Blot Method

To Identify dsDNA 1. Agarose Gel that has been pretreated to depurination, denatured & neutralized 2. Digested Sample DNA cleaved into fragments by Restriction Enzymes 3. Electrophoresis 4. Blot onto Nitrocellulose Membrane (capillary action) 5. Apply Non-/Radioactive Probes of Single Stranded DNA & allow to hybridize. 6. Measure membrane with X-rays, Chemiluminiescence or Calorimetrics to ID the size of the DNA fragment

Western Blot Method (Immunoblot)

To identify Protein 1. Polyacrylamide Gel 2. Denature (unfold) Protein 3. Electrophoresis 4. Blot onto Nitrocellulose Membrane (or PVDF Membrane if hydrophobic proteins) via Electrophoresis from Cathode (-) to Anode (+) direction 5. Apply Antibody Probes, then apply Labeled Markers to the antibody probes 6. Measure using X-rays, Chemiluminiescence or Calorimetrics & ID the proteins

Northern Blot Method

To identify ssRNA 1. Formaldehyde Agarose Gel 2. RNA Extraction 3. Electrophoresis 4. Blot onto Nitrocellulose Membrane (capillary action) 5. Apply Non-/Radioactive Probes of cDNA/cRNA (Single Stranded Complimentary DNA/RNA) & allow to hybridize. 6. Measure membrane with X-rays, Chemiluminiescence or Calorimetrics to ID the size of the RNA fragment

external standards in chromatography

Used prior to running a patient sample

Direct ELISA Principle, Method & Applications

Used to Detect Patient Ag eg. viral particles to HIV or WNV in an acute case. Indirect is used to detect Patient Ab eg. Abs to HIV or WNV in a convalescent case - Step 1 Place patient serum in well; adsorption via charge interaction with plastic well - Step 2 Add enzyme-labeled reagent Ab (Ab-L) - Wash - Add substrate --> Color change - Measure with Spectrophotometry - Directly Proportional; Increase in absorbance = [Patient Ag]

Describe the procedure and application for chemiluminescent immunoassays

Uses luminol label Used in forensic blood analysis

Mobile phase

a liquid or gas that travels through the stationary phase, carrying the mixture components with it at varying speeds based on the affinity to the stationary phase

hapten

a low molecular weight molecule that can stimulate production of antibody only when coupled to a larger carrier molecule. Some examples of haptens include some steroid hormones, drugs, and drug metabolites.

antigen

a molecule that can combine with antibody to form an immune complex (Ag-Ab).

antibody

a protein produced by plasma cells in response to exposure to an immunogen. Antibodies combine with antigens to form immune complexes.

Stationary phase

a solid, or a liquid supported on a solid backing; bed of support medium or particles that facilitates separation.

immunogen

a substance that may elicit an immune (antibody) response and can be either a protein or substance coupled to a carrier.

epitope

antigenic determinate, is a site on an antigen that combines with an antibody. There is often more than one region on an antigen.

Gel precipitation in immune reaction assays

based on the interaction of antibodies and antigens. They are based on two soluble reactants that come together to make one insoluble product, the precipitate. eg. Immunoelectrophoresis

Electrophoresis Limitations of Buffer Contamination

buffer contamination of bacteria and fungi. Refrigerate buffer & only reuse those specified for that use

wicking system

carries buffer across support or the support dips into the buffer at each end

Electrophoresis Limitations of Interfering Substances

cause false +/-. Hemolysis of sample causes hemoglobin proteins that add unexpected contaminating bands. Plasma contains fibrinogen which can cause difficulty in interpreting fractions. Only use serum. Albumin-bound drugs can make the albumin band less distinct.

support media types

cellulose acetate, agarose gels, and polyacrylamide gel electrophoresis (PAGE)

Temperature effect on migration rate

critical factor in electrophoresis. High temperatures may cause major distortions such as wick flow. Wick flow is the increased evaporation of the buffer solution causing an unintended flow of the buffer solution. This buffer movement may then affect the direction and speed of the migrating protein

What does Retention Factor (Rf) Measure?

distance traveled by sample/distance traveled by the solvent

Electrophoresis Limitations of Wick Flow and Buffer

evaporation due to excess voltage heat causing buffer to move from reservoirs to gel center changing the rate of movement of the molecule(s) of interest. Use lowest power required.

Capillary Electrophoresis (CE) Advantages

faster method than traditional zone electrophoresis, uses a smaller sample size and fewer reagents, and is adaptable to automation

PAGE Uses

genetic variation in proteins, isoenzymes or specific individual proteins, RNA & DNA. Note utilizes molecular sieving - charge to mass ratio + molecular size

migration rate

how fast a protein moves

Retention time (Rt)

in HPLC how long (min) to Peak Volume (mV)

Retention Time (Rt)

in HPLC, the amount of time the molecule stayed in the column from the time of entry.

Ion Exchange (HPLC) Stationary Phase

ions

Molecular size and shape's effect on migration rate

key factors that will affect how the molecule moves through pores of the test medium. If pores are comparatively small, large proteins will have trouble migrating and be slowed. Therefore, the smaller proteins migrate faster than the larger.

polyclonal antibodies

mixture of antibodies directed against different epitopes on a specific target molecule. A different plasma cell clone produces each of the antibodies in the mixture.

ligand

molecules that form a specific complex with another molecule. In immunology, a ligand usually refers to the immunogen.

Electrophoresis Limitations of Electroendosmosis/Endosmosis

occurs when positively charged buffer ions that are moving towards the cathode "pull" more weakly charged gamma globulins toward the cathode. This issue occurs more commonly with cellulose acetate and agarose media.

Gas (GC-HPLC) Stationary Phase

packed column or capillary

Liquid (HPLC) Stationary Phase

particles or silica gel in a column

Cellulose Acetate Pros

pliable when soaked in buffer, requires a small sample size, fast run time (<60min), thin membrane = greater sensitivity, durable and stores well

Properties of supporting medium effect on migration rate

pore size, charge, and impurities may influence movement of molecules through the medium.

amphoteric

proteins function as either an acid or base and may have a net +/- charge

buffer purpose

provides ions that act as carriers for the current and help to maintain the pH.

Agarose Gel Pros

small sample size, separation is based only on charge-mass ratio, available in different thicknesses, easily read by instruments, moderate run time (30-90min)

Thin-Layer (TLC) Mobile Phase

solvent carried by capillary action

Liquid (HPLC) Mobile Phase

solvent pumped through column at a constant rate

isoelectric point (pI/IEP)

the pH at which the positive and negative charges are equal. At the isoelectric point, there will be no net charge on the protein so it will not be able to move in an electric field. pI = (pka1 + pka2) / 2

monoclonal antibodies

the product of a single clone of plasma cells. One monoclonal antibody is one that has been produced by a single B-cell clone. These are homogenous antibodies that will react with only one epitope.

Explain the principles of antigen-antibody interactions and the role of the law of mass action in antigen-antibody binding

the rate of a reaction is proportional to the masses of the reacting substances.

affinity

the strength of attraction of a molecule, atom or receptor to bind to a ligand. Measured/Reported as the equilibrium dissociation constant KD.

Thin-Layer (TLC) Stationary Phase

thin layer of a adsorbent (silica gel, alumina or cellulose) has been spread evenly on a glass, plastic, or aluminum plate

Cellulose Acetate Cons

time to soak in buffer & lower resolution

What isolation technique(s) employ adsorption

used in Thin Layer Chromatography (TLC) and ELISA classified into physisorption (weak van der Waals forces) and chemisorption (covalent bonding). It can also be caused by electrostatic attraction. The molecules are held loosely on the surface of the adsorbent and can be easily removed

electrophoresis

uses molecular charge and mass to separate molecules in an electrical field

Explain the basic principles of electrophoresis

• If a protein is placed in an alkaline solution, fewer H+ are available, causing groups to release protons, and leaving the protein with a net negative charge. • If the pH is acidic, there will be many charged hydrogen ions available that will attach to the groups giving a net positive charge. • The isoelectric point of a protein is the pH at which the positive and negative charges are equal. At the isoelectric point, there will be no net charge on the protein so it will not be able to move in an electric field.