ELISAs, SDSPAGE and Western blotting

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ELISAs,SDS-PAGE andWestern blotting
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Solid-phase binding assays

• ELISAs and Western blots are solid-phase or
  _____________ binding assays
• ELISAs
• Wells of plastic microtiter plates
• Western _____________
• Nitrocellulose or other membrane
  (PVDF, nylon)
• Polystyrene and nitrocellulose
  bind proteins, carbohydrates and DNA
• Surfaces can be chemically modified to
  increase binding
  

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ELISA

• Stands for Enzyme-Linked Immunosorbent Assay
• Also abbreviated to EIA
• Enzyme-linked refers to the secondary antibody
  which is linked (_____________) to an
  ______________
• Immunosorbent refers to the fact that the
  ______________ (immuno-) bind to the antigen on
  the solid support (-sorbent)

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ELISAs

• The _____________ ELISA is the most common method
  used in clinical labs to detect patient
  antibodies
• ELISAs are _________________
• Detects low levels of antibodies or antigens
• Inexpensive, rapid, quantitative, ____________
• Expensive equipment is not required
• More accurate results are obtained using a
  special spectrophotometer to determine the OD of
  the substrate
• ELISAs can be automated

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ELISA method

• The basis for the indirect ELISA is as follows
• __________ is applied to the wells of a
  microtiter plate
• Additional reactive sites on the plastic are
  blocked with an irrelevant protein (often BSA)
• Dilutions of ____________________ are added
• If specific antibodies are present they will bind
  to the antigen
• A secondary, conjugated antibody is added
• If specific antibodies are present, the conjugate
  will bind
• A colorimetric __________________ is added
• If the conjugate is present, the enzyme will
  react with the substrate, changing it from
  colorless to colored
• Washing between steps removes unbound components

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ELISA method negative reaction

• Antigen is added
• The well is blocked
• Serum is added
• There is no specific antibody no binding
• Conjugate is added no binding
• Substrate is added no enzyme, no color change

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ELISA method positive reaction

• Antigen is added
• The well is blocked
• Serum is added
• Specific antibody binds to the antigen
• Conjugate binds to the specific antibody
• The enzyme on the conjugate reacts with the
  substrate resulting in a color change

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ELISAs are quantitative

• The amount of antibody conjugate bound is
  _________ ___________ to the amount of primary
  antibody bound
• The intensity of the substrate _________ (as
  measured by optical density) is proportional to
  the amount of antibody conjugate bound

More color more antibodies
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Antibody conjugates

• Also referred to as _____________ antibodies
• The primary antibodies are the test serum
• Consist of an antibody raised against ________ of
  the primary antibody species
• Rabbit anti-mouse IgG/IgM/IgA
• Pig anti-donkey IgG
• Donkey IgG is recognized as foreign in the pig,
  so the pig mounts an _____________________

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Antibody conjugates

• Why are these secondary antibodies called
  conjugates?
• The antibody molecule is conjugated to another
  _________________
• Conjugation is a __________________ linkage of
  the antibody to this other molecule
• Common types of molecules used in conjugates
• Enzymes
• __________________
• Gold beads
• Biotin, etc...

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Antibody conjugates

• ________________
• The two most common are horseradish peroxidase
  (HRP) and alkaline phosphatase (AP)
• ________________
• Fluorescent dyes are often used
• The dye is detected using a fluorescent
  microscopy

Enzyme interacts with substrate
The dye emits light in the UV range
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Types of enzyme substrates

• Horseradish _______________
• Cleaves peroxide into H2 and O2
• The O2 reacts with the chromagen in the
  substrate, and this reaction results in a
  ________________
• 4-chloro-1-naphthol colorless purple
• 3,3,5,5-tetramethylbenzidine colorless blue
• Alkaline _____________________
• Cleaves a phosphate monoester to release
  phosphate
• The phosphate reacts with the chromagen to cause
  the color change
• Fast Red colorless pink

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Why use antibody conjugates?

• Too __________/time consuming to label each
  individual patient serum with an enzyme or a dye
• A single human-specific antibody conjugate can be
  used for all patient sera
• Depending on the immunizing antigen (IgG, IgM,
  total Ig), the antibody conjugate can be specific
  to all immunoglobulins or just a single _________
• IgM indicates a (very) recent infection
• IgG indicates a past infection
• Allows signal ____________________

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Signal amplification

• With the enzyme conjugated to the primary
  antibody, there is a signal
• Amplification occurs because the secondary
  antibody binds to more than one site on the
  primary antibody

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Types of ELISAs

• ____________ ELISA
• Coat with antigen, detect antibodies
• Most common type in clinical use
• ____________ ELISA
• Also known as the sandwich ELISA
• Uses an antibody to capture an ________________
• Other types
• Not discussed

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Capture ELISA

• Used to __________ the presence of antigen in a
  patients sample
• A ______________ protein/carbohydrate/toxin
• Indicates infection (Rapid Strep A test)
• A host molecule such as a hormone or other
  molecules
• Pregnancy or fertility testing
• _____________________
• Method
• Specific antibody is used to coat the plate
• Sample is added and if antigen is present, it
  binds
• Antigen binding is detected with a conjugated
  antibody directed against the antigen

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Types of ELISAs in clinical use

• Infectious diseases
• Tests for antigen capture and detection of
  antibodies
• Autoimmune diseases
• Detects autoimmune antibodies
• Hormone levels (thyroid function, fertility)
• Antigen capture
• Drug screening
• Antigen capture
• Other weird, biological stuff
• Identification of snake venom, meat, exotic
  animal bits

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Infectious disease ELISAs

• _______________ antibodies
• To determine immune status after vaccination
• Chlamydia antibodies
• Lyme disease antibodies Borrelia burgdorferi
• Organisms are difficult to grow
• Clostridium difficile ________________ in feces
• Antigen capture not all C. difficile are
  toxigenic
• __________________
• Antibodies to measure exposure
• Antigen capture to measure viral load

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Infectious disease ELISAs

• Animals
• __________ virus antigen capture
• Rotavirus antigen capture in feces
• Porcine influenza antibodies
• West Nile Virus _______ detection
• Plants antigen capture (no _____________)
• Bacterial tomato canker
• Soybean root rot
• Bacterial geranium blight

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SDS-PAGE andWestern blotting
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SDS-PAGE

• Sodium dodecyl sulfate-polyacrylamide gel
  electrophoresis
• A procedure to electrophoretically __________
  antigens in a mixture
• For our purposes we will only talk about protein
  antigens
• Can also be used to determine the __________
  _____________ of proteins by comparison with
  protein standards

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Electrophoresis

• Essentially, an electrical field pushes
  proteins through a _______________
  (polyacrylamide)
• The ________________ is carried by glycine
  molecules in the electrophoresis buffer
• Using a glycine buffer, the flow of the current
  is from the cathode (-) to the anode ()
• Electrophoresis of DNA through an agarose gel
  uses essentially the same principles

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Electrophoresis

• Migration of a protein is dependent on its
  _______, shape and ___________________
• Size is determined by how many amino acids the
  protein contains (primary structure)
• Shape is determined by the secondary, tertiary
  and quaternary structure of the proteins
• Net charge is determined by the types of
  _____________________

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Protein structure

• Primary structure
• Secondary structure
• Tertiary structure
• Quaternary structure

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Sample preparation

• Protein samples are placed into sample buffer
  containing SDS and BME and are boiled for 5-10
  minutes
• Both these chemicals ________________ the protein
  shape from influencing migration through the gel
• SDS also prevents the net charge of the protein
  from influencing migration through the gel
• SDS also protects the ________________ structure
  of the protein during ________________
• Boiling is done essentially to speed up the
  effect of SDS and BME on the protein

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Sample preparation

• Sodium dodecyl sulfate SDS
• A negatively charged _______________ which
  disrupts secondary and tertiary protein
  structures by breaking hydrogen bonds and
  unfolding the proteins
• Also, disrupts quaternary structure preventing
  protein aggregation
• _______________ the charges on proteins
• The proteins now have a net negative charge
• ß-mercaptoethanol BME
• Reducing agent which disrupts secondary and
  tertiary protein structures by breaking
  ____________________

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Protein samples
SDS/BME/boiling
Proteins are loaded into the wells
An electric field is applied and the negatively
charged proteins migrate to the anode ()
Polyacrylamide gel
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After SDS-PAGE...

• The gel can be _________ with a number of stains
  that allow _________________ of the proteins
• Coomassie blue staining
• Quick, easy, moderate sensitivity
• Silver staining
• Slow, laborious, very sensitive
• Or... the proteins in the gel can be transferred
  to a ______________ Western blotting
• This allows subsequent detection of the proteins
  with specific antibodies

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Protein size standards (markers)

• Proteins of known mass are used as
  ________________ to determine the size of
  ________ __________ proteins
• Mobility on gels is defined as Rf
• Rf distance the dye front migrated/distance the
  protein migrated
• Rf x/y
• Plot the values on semi-log paper

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Western blotting

• Proteins are electrophoretically transferred from
  the gel onto ____________________ membrane
• Following transfer, the membrane becomes the ____
  surface on which the antibody binding is done
• Detection of ______________ is performed exactly
  the same as for an indirect ELISA
• Block (buffer containing skim milk)
• Add primary antibody
• Wash/ add secondary antibody
• Wash/ add substrate

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Western blotting in research
E. coli cpb2
E. coli cpb2
Markers
E. coli
E. coli

• Western blotting can be used as a tool in
  research to look at gene expression

Coomassie blue Western blot
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Western blots in clinical use

• Detection of Herpes Simplex 1 and 2 antibodies in
  serum
• HIV antibodies
• Can determine the response to _____________
  antigens on one test strip

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ELISA vs. Western blots

• Advantages of the ELISA over the Western blot
• Can process _______________ in a shorter amount
  of time
• Can be automated using robotic systems
• The data obtained is less _______________ if
  using a spectrophotometer to determine the
  intensity of the substrate color
• The test uses native (non-denatured) antigen can
  detect antibodies to conformational epitopes
• Can be _____________ than Western blots developed
  with chromagenic (color-changing) substrates

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ELISA vs. Western blots

• Disadvantage of the ELISA over the Western blot
• To determine a specific response, antigen must be
  purified before using it in the ELISA
• Advantages of the Western blot over the ELISA
• As antigens are separated during SDS-PAGE, you
  can measure antibody levels to _________________
  in one test
• HIV antibodies
• Can be ___________________ than the ELISA, if
  luminescent (light-emitting) substrates are used

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ELISA vs. Western blots

• Disadvantages of the Western blot over the ELISA
• Acrylamide is used in the SDS-PAGE gel
• Acrylamide is a potent neurotoxin and possible
  carcinogen
• Requires ____________________ equipment
• Not an issue if Western blots are purchased from
  a commercial supplier
• More laborious, therefore, more _________________
• Generally not quantitative
• Not as good at ____________________

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What you need to know

• How the ELISA ___________, steps involved and why
  they are performed
• Positive vs. negative reactions
• What an antibody conjugate is and how it is used
• Species and isotype specificity of antibody
  conjugates
• The difference between an ______________ and a
  capture ELISA
• How ELISAs are _________________
• Diagnosis of infectious disease drug testing
  measurement of hormones

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What you need to know

• The steps involved in SDS-PAGE and why they are
  performed
• Sample preparation
• Understand the steps involved in developing a
  Western blot and why they are performed
• Know how to __________________ molecular weight
  of a protein
• ______________ and ________________ of the two
  methods
• ELISAs vs. Western blots