Optimization of all the critical factors for successful development of an ELISA kit.

1. Critical Success Factors for Optimization of
Enzyme Linked Immunosorbent Assay
Dr. Chayanika Das
Ph.D
VETERINARY MICROBIOLOGY

2. The Past-
How do you rapidly, cheaply and easily detect a single
analyte present in a complex heterogeneous mixture (e.g.,
blood, soil, etc.) ?
Use a naturally occurring or synthetic analog of a molecule (antibody,
aptamer, etc.) that has a high affinity to a specific ligand (or analyte)

3. ELISA-The Past
Developed by Peter Perlmann and Eva Engvall
of Stockholm University, Sweden
First paper in 1971
Quantitative measurement of IgG in
rabbit serum
Reporter label: Alkaline phosphatase
Source: Clinical Chemistry 51:12
2415-2418 (2005)

4. Enzyme Linked Immunosorbent Assay
Ag of interest is adsorbed on to plastic surface (Sorbent)
Ag is recognized by Ab (Immuno)
This Ab is recognized by second antibody
(immuno) which has enzynme attached
(Enzyme linked)
Substrate reacts with enzyme to produce
product (Colour)

5. Surface
Sorbent
ELISA
Immune
Antigen/antibody
complex
Source: www.corning.com/lifesciences

6. Critical Success Factors for
Optimization of Enzyme Linked
Immunosorbent Assay

7. INTERFACE-
SOLID-LIQUID
LIQUID-GAS
ASSAY PLATE: Surface and its interaction
with biomolecules
Structure of polystyrene
Long carbon chain with benzene
ring on every other carbon
 A very hydrophobic compound
96/384/1536 well
plate
Source: www.corning.com/lifesciences

8. Surface flavors
feature hydrophobic characteristics
to introduce a defined number of
hydrophilic groups to the plate
surface
Passive adsorption
Non-treated
polystyrene plates
Treated
polystyrene plates
Hydrophobic
interactions
Hydrophilic
interactions
H-bonding
PRINCIPLE IN ADSORPTION TO POLYSTYRENE
Source: www.gbo.com/bioscience

9. Determine which surface is best suited for a
specific application
Surface flavors
Contd...
Source: www.gbo.com/bioscience

10. Pigment
Well shape
“Eased” edged
Flat bottom Round bottom
Transparent (colorimetric
signals)
Black opaque ( fluorescent signals) white opaque (chemiluminescent)
Source: www.corning.com/lifesciences

11. Coating Buffer Antibody
Wash Buffer Analyte
Blocking Buffer Standard
Citrate Buffer Conjugate
Substrate
Stopper
Reagents for ELISA

12. Factors that affect coating
• Concentration of Ag/Ab to be coated (1-10µg/ml)
• Coating buffer : composition and pH
• Time & temperature
• It is imperative that no other proteins are included in
the coating buffer
• Maintain the plates in a moist environment

13. Coating Buffer
For adsorptive immobilization of proteins and antibodies in solid surfaces
Commonly used coating buffers
-Carbonate-bicarbonate buffer, pH 9.6
- PBS, pH 7.4-8
-TBS, pH 8.0
-0.15 M NaCl, pH 8.0
Always store stock solution (10X)
Before preparing working solution-
-Warm up the stock solution to room temperature
-Incubation period should also be optimized
pH
- NaOH, pH 13 for NDV Ag coating (Botus and Oncescu, 2006)

14. Influence of incubation time on coating process
• At higher temperature Ag/Ab are adsorbed faster but with
non-specific binding
• Longer incubation at 40C presents the lowest N/P ratio
Negative/Positive Ratio
Dilution of coating reagent
1:100 1:200 1:300 1:400 1:500 1:600
N/P Ratio (1Hr at 370C) H H M M H H
N/P Ratio (2Hr at 370C) H H M M H H
N/P Ratio(16 Hr at 40C) H H L M H H
**H: high, M: medium, L: low
L
N/P ratio

15. Hook effect
Caused by very high concentrations of a
particular analyte or antibody
Both the capture and detection antibodies become
saturated by the high analyte concentration
A C
B

16. Wash Buffer & Plate Washing
(To separate bound and unbound reagents)
- Factors associated with effectiveness of the washing step:
- Wash Volume:
- Wash Volume > Coating volume
- Too little: Unbound molecules may remain,
More background signal
- Too much: Stripping specifically bound
- Should not cause overflow into other wells
- Number of wash cycle: 3-6
- Too few wash cycles: High background
- Too many wash cycle: Reduce signal strength
- Aspiration Parameters:
- Aspiration height
- Aspiration head
- Location of aspiration tips
- Shape of the well
Individual well
of a 96 well
microtitre plate
can hold 330 to
460 µl
Why PBST?

17. Blocking : A compromise between low background and high
sensitivity
Donot take active part in the specific assay reaction
Two major classes
Protein blockers Detergent blockers
•Block non-occupied sites on the
surface
•Stabilise biomolecules bound to
the surface to reduce steric
hindrance
•Eg. Bovine serum albumin (1-
3%), Non-Fat Dry Milk (0.1-0.5%),
Whole normal sera (10%), etc.
•Useful in washing solution (their
presence blocks areas on the surface
that may be physically stripped of
specifically bound biomolecules during
the wash step)
•Help dislodge loosely bound
biomolecules that are physically trapped
in corners
•Eg. Tween 20 (0.01-0.10%), Triton X-
100, etc
Ionic Non-ionic

18. PROTEIN
BLOCKERS BSA Non-fat dry milk Whole normal serum
Advantages
•Inexpensive
•Effective at
concentrations 1-3%
•Inexpensive
•Effective at
concentrations 0.1-0.5%
•Highly stable in dry
form
•More effective at
blocking covalent
interactions
•Effective at blocking
all nonspecific
interactions
•Acts as protein
stabilizer
Disadvantages
•High lot-to-lot variability
due to variable fatty acid
content
•May cross-react with
some classes of antibodies
•Less effective at blocking
covalent interactions
•May cross-react with
phospho-specific
antibodies
•Incompatible with
alkaline phosphatase
•May cause overall
higher background
•Cross-reacts with
anti-IgG antibodies
•Expensive
•Requires up to 10%
concentration

19. Antibody diluent buffer
• Phosphate buffered saline and Tris buffered saline are the most
often used
• Optionally, can be supplemented with blocking and stabilizing
molecules such as Bovine Serum Albumin (BSA) to prevent
background reactions
• When diluted antibodies have to be stored (at 4ºC), dilution
buffers should also contain Sodium azide or Thimerosal as
preservatives.
Source: http://www.scientistsolutions.com ,
http://www.ihcworld.com

20.  Primary antibodies (conjugated or not)
(a) 0.01 M PBS with 0.05% Tween 20, pH 7.2 supplemented with
1% BSA
(b) 0.01 M PBS with 0.05% Tween 20, pH 7.2 supplemented with
1% BSA and normal serum (non immune serum up to 5% final)
(c) 0.01 M PBS with 0.05% Tween 20 , pH 7.2 supplemented with
1% BSA and sodium azide or thimerosal
 Secondary antibodies
0.01M PBS, pH 7.2-7.4 with sodium azide or thimerosal
For peroxidase labeled reagents thimerosal is used instead of azide
BSA or other serum containing reagents should not be used to dilute secondary
Abs since they may bind to BSA or serum therefore reducing antibody affinity
Source: http://www.scientistsolutions.com ,
http://www.ihcworld.com

21. Substrate Buffer (Acetate/ Citrate Buffer)
PH- 5 ?
Oxidized chromogen is unstabilised by pH increase
Optimal reaction rate is reached at pH 4.2 for 2mM ABTS
concentration
1
Peroxidase affinity α -------------
pH
- Enzymatic transformation of substrate increases proportionately at first
10-15 minutes, then a plateau is reached
- Absorbance for positive & negative control are into optimal limits
- After 15 minutes : Absorbance of positive control will be almost same
(Ag-Ab complex is already formed)
Absorbance of negative control will be increased
Why 15 minutes? Absorbances Vs. Time
Source: Botus & Onsescu, 2006; Optimizing immunoenzymatic reactions (ELISA) for
the detection of antibody against ndv virus
Acetate/ Citrate Buffer

22. Stopping Solution
Reaction inhibitor
Change the pH of the medium
Enzyme present in the solution do no act on its substrate
Every enzyme need optimum pH range to
perform catalysis

23. ANTIBODY: Selection of Right Clonality
Antibodies are produced and purified in two basic forms for use as reagents-
polyclonal and monoclonal
Polyclonal Antibody
 Recognize multiple epitopes, making them less sensitive to minor
antigen changes
 May be generated in a variety of species, including rabbit, goat, sheep,
donkey, chicken, and others, giving the users many options in
experimental design
 Relatively easy to generate, less complex, and are more cost-effective
 Target multiple epitopes on the same protein and thus usually provide
more robust detection
 Can amplify signals from a target protein with low expression levels,
as the target protein will bind more than one antibody molecule

24. ANTIBODY: Selection of right clonality Contd...
Monoclonal Antibody
• Highly specific and detect only one
epitope on the antigen
• Excellent as the primary antibody and
often minimize background signal and
eliminate cross-reactivity
• Two very closely related antigens can be
distinguished from each other
• Homogeneity of monoclonal antibodies is
very high and they provide consistent,
reproducible results if experimental
conditions are kept constant
• Hybridoma cells can serve as an infinite
source of the monoclonal antibody.

25. ELISA Ab concentration optimization
Checkerboard titration experiment
To optimize two components simultaneously
Direct assessment of the activity at the range of concentration

26. Figure: Example of a checkerboard titration experiment to optimize two ELISA
parameters at once. This example shows primary antibody vs. detection antibody with all
other reagents constant
Checkerboard titration experiment

27. STANDARDS
All quantitative ELISA systems are calibrated by the
use of standards.
Enables quantification of analytes in a sample

28. ANALYTES
The claimed analyte of an ELISA could be
Protein molecule
Carbohydrate molecule
Microorganisms
Allergens
Viruses, etc
Concentration is unknown

29. SAMPLE MATRIX
Components of the sample other than the analyte of interest
Matrix effect
A common approach for accounting matrix effect
Calibration Curve/ Standard Curve
Sensitivity
Specificity
Robustness
Sample
preparation
Reduced amount
of interfering
compounds
Reduced
complexity
Source: Bioanalysis (2015) 7(17), 2133–2134
pH, detergents, organic
solvents, high protein
concentration, high buffer salt
concentrations

30. STANDARD CURVE
•Serial dilution of standard with known concentration
•Determination of OD value of both standard and analyte
•Plotting OD values (Y axis) against concentrations (pg/ml) of standard (X axis)
Concentration of target protein in the sample
To determine the concentration of target protein concentration in
each sample, first find the mean absorbance value of the sample.
concentration
OD
value

31. At the point of intersection, extend a
vertical line to the X axis and read the
corresponding concentration (b)
From the Y axis of the standard curve graph,
extend a horizontal line from this absorbance
value to the standard curve.
Source : http://www.abcam.com/protocols/calculating-and-evaluating-elisa-data

32. Spike recovery & Dilutional Linearity
• Known amount of analyte is added to the sample matrix and a standard
diluent
• The two sets of responses are compared based on values calculated
from a standard curve
Source : http://www.abcam.com/protocols/calculating-and-evaluating-elisa-data
SPIKE RECOVERY
Recovery observed for the
analyte prepared in sample
matrix
Recovery obtained for the
analyte prepared in standard
diluent
Identical

33. LINEARITY OF DILUTION
 The Meaning and Purpose of Linearity-of-Dilution
Assessment
Linearity is defined relative to the calculated amount of analyte based on
the standard curve
If the linearity is good over a wide range of dilution, then the assay
method provides flexibility to assay samples with different levels of
analyte
 Performing a Linearity-of-Dilution Experiment
Using a low-level sample containing a known spike of analyte
Testing several different dilutions of that sample in the chosen sample
diluent
Comparison of observed vs. expected values based neat (undiluted)
samples.

34.  Interpreting Linearity-of-Dilution Results
Source:
http://www.woongbee.com/0NewHome/RnD/ELISA_HA/Duoset_link/spike_recovery.pdf
Poor linearity of dilution
Natural sample matrix, the sample
diluent and/or standard diluent
affect analyte detectability differently

35. Conjugate: Key substance in ELISA
Possess-
1. Catalytic activity of enzyme
• Always prepare working conjugate as per manufacturers
recommendation just before use.
• Don’t save leftover working solution for future use.
+
Ab linked with enzyme
2. Immunological competence of Ab

36. SECONDARY ANTIBODY- Selection And Choosing Suitable Enzyme Conjugate
To successfully choose a secondary antibody, consider
the following factors:
•Host and target species
•Targeted reactivity
•Purification
•Cross-adsorption
•Antibody class and subclass
Selecting the right secondary antibody
Source: https://www.thermofisher.com

37. Selecting the appropriate enzyme label
CRITERIA
 Stability at typical assay temperature: 4°C, 25°C, 37°C
 Greater than 6 month self life when stored at 4°c
 Commercially available
 Capable of being conjugated to an Ag or Ab
 Easily measured
 Unaffected by biological component of the assay

38. SUBSTRATE
Types-
• Colorimetric (ELISA)
• Chemiluminescent (LIA)
• Fluorescent (FIA)
Colorimetric (ELISA)

39. ENZYME SUBSTRATE REACTION COLOUR Light absorbance
Nonstopped Stopped
Alkaline
phosphatase
PNPP (p-Nitrophenyl
Phosphate, Disodium Salt)
yellow water-soluble
reaction
405 nm 405 nm
HRP
ABTS (2,2'-Azinobis [3-
ethylbenzothiazoline-6-
sulfonic acid]-
diammonium salt)
water-soluble green
end reaction
410 nm 650 nm
OPD (o-phenylenediamine
dihydrochloride)
yellow-orange
reaction
450 nm 492 nm
TMB (3,3',5,5'-
tetramethylbenzidine)
blue color ; changes to
yellow with the
addition of stopping
solution
630 nm 450 nm
Enzyme specific substrates
Source: http://info.gbiosciences.com/

40. Principle of Colour Development
Insoluble
coloured reaction
product
Soluble coloured
reaction product
SUBSTRATE
Membrane-based Assay
ELISA
Eg. OPD
OPD + H2O2 2,3-diaminophenazine + 2 H2O
HRP

41. Colour development will not be observed if
enzyme specific substrate is not used
Unexpectedly higher (or lower) optical densities (O.D.) are
measured in the peripheral wells than in the central wells
Edge effect
Causes: Temperature variation among wells, Light exposure
Since polystyrene is a poor conductor of heat, incubations
performed in incubator usually results in edge effect

42. For intra-plate homogeneity the coefficient of variation (CV) must not
exceed 5% for colorimetric assays
•The CV is a measure of the variation of the optical density values
in different wells.
•The lower the CV the more consistent are the binding properties
of the tested surfaces.
Cv= σ
µ
or
Coefficient of variation
Indicates any inconsistencies and inaccuracies in the results

43. Coefficient of variation
High CV can be caused by:
 Inaccurate pipetting
 Splashing of reagents between wells
 Bacterial/fungal contamination of either screen samples or
reagents
 Cross contamination between reagents
 Temperature variations across the plate
 Some of the wells drying out

44. Conclusion
Still the most used platform for the evaluation of
various research and diagnostic targets
Development of ELISA and its transfer to the
counterpart laboratories is the key element of the
works under R&D division of diagnostic centres
To develop an in-house ELISA with high
specificity, sensitivity, robustness and
reproducibility, optimization of all the critical
success factors followed by validation is
mandatory
Still the most used platform for the evaluation of
various research and diagnostic targets
Development of ELISA and its transfer to the
counterpart laboratories is the key element of the
works under R&D division of diagnostic centres
To develop an in-house ELISA with high
specificity, sensitivity, robustness and
reproducibility, optimization of all the critical
success factors followed by validation is
mandatory