Recombinant Protein Detection
by ELISA
Tanapat Palaga, PhD
Department of Microbiology
Faculty of Science
Chulalongkorn University
Topics
• Principle of recombinant protein detection
by antibody
• Principle of ELISA
• Detection of recombinant protein by ELISA
Structure of Antibody
From Kuby Immunology (6th Edition, 2006)
Application of Antibody-Antigen for
Detection
• Antigen-antibody has high affinity in the
range of 105 L/mol to 1012 L/mol (Ka)
• Affinity is influenced by pH, solvent and
temp.
• This high specificity can be applied in
detection of recombinant proteins and
biochemical analysis
Antigen-Antibody Interaction
From Kuby
Immunology (6th
Edition, 2006)
Antigen-antibody complex is held together by multiple non-covalent bonds
Principles of Detection for
Recombinant Proteins
• Recombinant protein as antigen
• Direct detection if antibody is available
• Indirect detection if antibody is not
available using antibody against epitope
tags
• Proper controls are needed to minimize
false positive
Purposes of Detecting
Recombinant Protein by ELISA
• To check the presence of recombinant
protein in samples
• To measure quantitatively the amount of
recombinant proteins
• To diagnose positivity of sera for certain
pathogens using recombinant antigens
Tagging Proteins
• Some epitope tags are useful for
– detection of proteins
– affinity column purification at later step
• Epitope tags can have undesirable effect
on biological function of recombinant
proteins
• Some commercialized tags can be
removed after purification by enzymatic
cleavage
Most Common Used Tags
Tag
Name
Source
Sequence
Antibodies
Reagent
Available
Myc
myc from
oncogene (Evan
et al. 1985)
EQKLISEEDDL
9E10
Antibody
Vectors
Peptides
HA
Flu HA-1 protein
(Niman, et al.,
1983)
YPYDVPDYA
12CA5
Antibody
Vectors
Peptides
FLAG
Novel (Hopp et
al., 1988)
DYKDDDDK
M1 and M2
Antibody
Vectors
Peptides
His
Designed
HHHHHH
6 His Ab
Antibody
Vectors
Crossreact??
Epitope Tagging of Recombinant
Protein
http://homepages.strath.ac.uk/~dfs99109/BB310/MGlect1.html
Enzyme Linked Immunosorbent
Assay (ELISA)
• Derived from principles of technique called
Radio Immunoassay (RIA) developed by Berson
and Yalow in 1960s
• Engvall & Perlmann (1971) and van Weemun &
Schurrs (1971)
• Using enzyme conjugated antibody and
enzyme-substrate reaction to detect antigens
(recombinant proteins)
• Can be used as quantitative or qualitative assay
Important Steps
•
•
•
•
•
Coating to fix antigen on solid support
Blocking to prevent non-specific binding
Washing to wash off non-specific binding
Direct or indirect method
Detection to detect the signal using
enzyme-substrate reaction
• Stopping to stop enzymatic reaction
Coating, Blocking and Washing
• Coating protein Ag or Ab onto solid support usually
is done in PBS (pH 7.4) or Na-carbonate buffer (pH
9.6) for O/N at 4°C or 37°C for 1 hr
• Blocking can be done using Casein, non-fat milk
powder or BSA in PBS
• Washing is accomplished by adding detergents such
as Tween 20 to PBS
13
Blocking Step
• Blotto- 5% nonfat dry milk in PBS with
0.2% Tween-20
• Bovine serum albumin (BSA)-3% BSA
(IgG free (Cohn fraction V)) in PBS
• Horse serum- 10% HS in PBS
Enzyme and Substrates
Commonly used enzymes for immunoassay are
horseradish peroxidase (HRP), alkaline phosphatase
(AP), beta galactosidase, and glucose oxidase
Enzyme activity can be measured by
- colorimetric
- fluorometric
- luminometric
Chromogenic Substrates for HRP in
Immunoassay
1. ABTS (2,2’-azinodi [ethylbenzthiazoline] sulfonate/H2O2
clear green 650 nm
2. OPD (o-Phenylene diamine)/H2O2
clear brown 492 nm
3. TMB (3,3’5,5’-Tetramethylbenzidine)/H2O2
clear yellow 450 nm
All water soluble products
TMB is the most sensitive substrate for detecting
HRP-labeled reagents
16
Antibody for ELISA
• Can be either monoclonal or polyclonal Ab
• Must recognize denatured proteins
• If polyclonal Ab is used, need to titer first to lower
background
• Use control Ab such as pre-immune sera or same
isotype of monoclonal Ab
Direct or Indirect Detection
• Antibody directly recognizing recombinant
protein is a primary antibody
• Antibody which recognizing primary
antibody is a secondary antibody
• If primary antibody is directly labeled (such
as being conjugated with enzyme direct
method
• If secondary antibody is needed to detect
the primary antibody indirect method
Secondary Reagent
• Secondary antibody must recognize part
of or whole molecule of primary antibody
• If primary antibody is conjugated with
biotin (biotinylated), strepavidin can be
used as secondary reagent
• Indirect method has advantage
in flexibility of primary
antibody usage and
signal amplification
Types of ELISA: Indirect ELISA
Sandwich ELISA
• Determine antigen concentration in
unknown samples
• Requires either 2 specific mAb binding to
nonoverlapping epitopes or purified
polyclonal antibodies
• One antibody is coated on solid support
• Another is used to detect antigens bound
to the coated antibody
• Can detect as low as 10 pg/mL-1 ng/mL
Sandwich ELISA
Competitive ELISA
Standard Curve
Appropriate Controls
• Recombinant protein:
– Crude preparation: similarly prepared
samples without recombinant proteins as
negative control
• If antibody specific for recombinant protein
is used: irrelevant isotype-matched mAb or
a non-immune serum should be included
• If antibody for peptide tags is used:
irrelevant protein with the same tag should
be included
Useful Tips
• Antigens can be immobilized at concentrations
ranging from a few ng to 10 µg/mL
• Modified plates to increase peptide binding
• If peroxidase is used as enzyme, buffer
containing sodium azide should not be used
• Incubation times for color development may
vary from 10 min-30 min or longer