ELISA Handbook

ELISA Handbook
Principle, Troubleshooting, Sample

Preparation and Assay Protocols ……
See All Boster Bio Products

Table of Contents Page
1. Introduction ………………………………………………………………………... 2
2. General ELISA Procedure …………………………………………………………... 2
3. ELISA Types ………………………………………………………………………… 3

• Direct ELISA ………………………………………………………………….… 4
• Indirect ELISA …………………………………………………………………… 4
• Sandwich ELISA ………………………………………………………………… 5
• Competitive ELISA ……………………………………………………………… 5
4. ELISA Data Interpretation ……………………………………………………………. 6
5. Sample Preparation …………………………………………………………………….7

• Cell Culture Supernatants …………………………………………………………. 7
• Cell Extracts ……………………………………………………………………… 7
• Conditioned Media ………………………………………………………………..7
• Tissue Extract ……………………………………………………………………...8
6. Recommended Protocols ……………………………………………………………….9

• Reagent Preparation ………………………………………………………………...9
• Sandwich ELISA …………………………………………………………………… 10
• Indirect ELISA …………………………………………………………………….12
• Direct ELISA ……………………………………………………………………… 14
• Competitive ELISA ………………………………………………………………… 16
7. Troubleshooting Guide ………………………………………………………………….. 19

• Weak or No Signal ………………………………………………………………….. 19
• Saturated Signal ……………………………………………………………………..20
• High Background ………………………………………………………………….... 21
• Low Sensitivity ……………………………………………………………………… 23
• Poor Standard Curve ………………………………………………………………... 24
• Poor Replicate Data …………………………………………………………………. 25
• Inconsistent Assay-to-Assay Results …………………………………………………. 26
• Slow Color Development ……………………………………………………………. 26
• Plate Imaging Problem ……………………………………………………………… 27
8. FAQs …………………………………………………………………………………….. 28
9. Ordering Information ……………………………………………………………………. 30
1
Introduction
ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and
quantifying peptides, proteins, antibodies and hormones. In an ELISA, an antigen must be immobilized to a
solid surface and then complexed with an antibody that is linked to a n enzyme. Detection is accomplished by
assessing the conjugated enzyme activity via incubation with a substrate to produce a measureable product.
The most crucial element of the detection strategy is a highly specific antibody-antigen interaction.
ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which will passively bind
antibodies and proteins. It is this binding and immobilization of reagents that makes ELISAs so easy to
design and perform. Having the reactants of the ELISA immobilized to the microplate surface makes it easy
to separate bound from non-bound material during the assay. This ability to wash away nonspecifically bound
materials makes the ELISA a powerful tool for measuring specific analytes within a crude preparation.
General ELISA Procedure

Unless you are using a kit with a plate that is pre-coated with antibody, an ELISA begins with a coating step, in
which the first layer, consisting of a target antigen or antibody, is adsorbed onto a 96-well polystyrene plate. This is
followed by a blocking step in which all unbound sites are coated with a blocking agent. Following a series of
washes, the plate is incubated with enzyme-conjugated antibody. Another series of washes removes all
unbound antibody. A substrate is then added, producing a calorimetric signal. Finally, the plate is
read.
Because the assay uses surface binding for separation, several washes are repeated in each ELISA step to
remove unbound material. During this process, it is essential that excess liquid is removed in order to prevent
the dilution of the solutions added in the next assay step. To ensure uniformity, specialized plate washers are
often used.
ELISAs can be quite complex and include multiple intervening steps, especially when measuring protein
concentration in heterogeneous samples such as blood. The most complex and varying step in the overall
process is detection, where multiple layers of antibodies can be used to amplify signal.
To be continued on next page
2
ELISA Types
ELISAs can be performed with a number of modifications to the basic procedure: direct, indirect,
sandwich or competitive. The key step, immobilization of the antigen of interest, can be accomplished by
direct adsorption to the assay plate or indirectly via a capture antibody that has been attached to the plate.
The antigen is then detected either directly (enzyme-labeled primary antibody) or indirectly (enzyme-labeled
secondary antibody). The detection antibodies are usually labeled with alkaline phosphatase (AP) or
horseradish peroxidase (HRP). A large selection of substrates is available for performing the ELISA with an
HRP or AP conjugate. The choice of substrate depends upon the required assay sensitivity and the
instrumentation available for signal-detection (spectrophotometer, fluorometer or luminometer).
Among the standard assay formats discussed and illustrated below, where differences in both capture and
detection were the concern, it is important to differentiate between the particular strategies that exist
specifically for the detection step. However an antigen is captured to the plate (by direct adsorption to the
surface or through a pre-coated "capture" antibody, as in a sandwich ELISA), it is the detection step (as either
direct or indirect detection) that largely determines the sensitivity of an ELISA.
3
1. Direct ELISA
For direct detection, an antigen coated to a multi-well plate is detected by an antibody that has been
directly conjugated to an enzyme. This detection method is a good option if there is no commercially
available ELISA kits for your target protein.
Advantages
• Quick because only one antibody and fewer steps are used.
• Cross-reactivity of secondary antibody is eliminated.
Disadvantages
• Immunoreactivity of the primary antibody might be adversely affected by labeling with enzymes or
tags.
• Labeling primary antibodies for each specific ELISA system is time-consuming and expensive.
• No flexibility in choice of primary antibody label from one experiment to another.
• Minimal signal amplification.
2. Indirect ELISA
For indirect detection, the antigen coated to a multi-well plate is detected in two stages or layers. First an
unlabeled primary antibody, which is specific for the antigen, is applied. Next, an enzyme-labeled
secondary antibody is bound to the first antibody. The secondary antibody is usually an anti-species
antibody and is often polyclonal. The indirect assay, the most popular format for ELISA, has the
advantages and disadvantages:
Advantages
• A wide variety of labeled secondary antibodies are available commercially.

• Versatile because many primary antibodies can be made in one species and the same labeled
secondary antibody can be used for detection.
• Maximum immunoreactivity of the primary antibody is retained because it is not labeled.
• Sensitivity is increased because each primary antibody contains several epitopes that can be bound by
the labeled secondary antibody, allowing for signal amplification.
Disadvantages
• Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal.
• An extra incubation step is required in the procedure.
4
3. Sandwich ELISA
Sandwich ELISAs typically require the use of matched antibody pairs, where each antibody is specific for
a different, non-overlapping part (epitope) of the antigen molecule. A first antibody (known as capture
antibody) is coated to the wells. The sample solution is then added to the well. A second antibody (known
as detection antibody) follows this step in order to measure the concentration of the sample. This type of
ELISA has the following advantages:
• High specificity: the antigen/analyte is specifically captured and detected
• Suitable for complex (or crude/impure) samples: the antigen does not require purification prior
to measurement
• Flexibility and sensitivity: both direct or indirect detection methods can be used
4. Competitive ELISA
The key event of competitive ELISA (also known as inhibition ELISA) is the process of competitive
reaction between the sample antigen and antigen bound to the wells of a microtiter plate with the primary
antibody. First, the primary antibody is incubated with the sample antigen and the resulting antibody–
antigen complexes are added to wells that have been coated with the same antigen. After an incubation
period, any unbound antibody is washed off. The more antigen in the sample, the more primary antibody
will be bound to the sample antigen. Therefore, there will be a smaller amount of primary antibody
available to bind to the antigen coated on the well, resulting in a signal reduction. The main advantage of
this type of ELISA arises from its high sensitivity to compositional differences in complex antigen
mixtures, even when the specific detecting antibody is present in relatively small amounts.
5
Summary of Key Steps in Different ELISA Types
Indirect Direct Sandwich Competitive

Capture Ab Coating X X √ X
Antigen Coating √ √ X √
Blocking √ √ √ √
Sample (Antigen) Incubation X X √ √
Primary Ab Incubation √ √ √ √
Secondary Ab Incubation √ X √ √
Substrate Prep √ √ √ √
Signal Detection √ √ √ √
Data Analysis √ √ √ √
ELISA Data Interpretation

The ELISA assay yields three different types of data output:
1) Quantitative: ELISA data can be interpreted in comparison to a standard curve (a serial dilution of a
known, purified antigen) in order to precisely calculate the concentrations of antigen in various samples.
2) Qualitative: ELISAs can also be used to achieve a yes or no answer indicating whether a particular antigen is
present in a sample, as compared to a blank well containing no antigen or an unrelated control antigen.
3) Semi-Quantitative: ELISAs can be used to compare the relative levels of antigen in assay samples, since
the intensity of signal will vary directly with antigen concentration.
ELISA data is typically graphed with optical density vs log concentration to produce a sigmoidal curve as
shown below. Known concentrations of antigen are used to produce a standard curve and then this data is
used to measure the concentration of unknown samples by comparison to the linear portion of the standard
curve. In fact, it is the relatively long linear region of the curve that makes the ELISA results accurate and
reproducible. The unknown concentration can be determined directly on the graph or with curve fitting
software which is typically found on ELISA plate readers.
6
Sample Preparation
The procedure below provides a general guidance for the preparation of commonly tested samples for use in
ELISA assays. At Boster, we are working on our detailed sample preparation protocols that cover more than 20 sample types
and expecting to update this handbook in the near future. Please check with the literature for experiments similar to
yours for your new assay development. Generally:
• Protein extract concentration is at least 1-2 mg/mL.

• Cell and tissue extracts are diluted by 50% with binding buffer.
• Samples are centrifuged at 10,000 rpm for 5 min at 4°C to remove any precipitate before use.
1. Cell Culture Supernatants
Centrifuge cell culture media at 1,500 rpm for 10 min at 4°C. Aliquot supernatant immediately and hold at
-80°C, avoiding freeze/thaw cycles.
2. Cell Extracts
Place tissue culture plates on ice. Remove the media and gently wash cells once with ice-cold PBS.
Remove the PBS and add 0.5 ml extraction buffer per 100 mm plate. Tilt the plate and scrape the cells
into a pre-chilled tube. Vortex briefly and incubate on ice for 15-30 min. Centrifuge at 13,000 rpm for 10
min at 4°C (this creates a pellet from the insoluble content). Aliquot the supernatant into clean, chilled
tubes (on ice) and store samples at -80°C, avoiding freeze/thaw cycles.
3. Conditioned Media
Plate the cells in complete growth media (with serum) until the desired level of confluence is achieved.
Remove the growth media and gently wash cells using 2- 3 mL of warm PBS. Repeat the wash step.
Remove the PBS and gently add serum-free growth media. Incubate for 1-2 days. Remove the media into
a centrifuge tube. Centrifuge at 1,500 rpm for 10 min at 4°C. Aliquot the supernatant and keep samples at
-80°C, avoiding freeze/thaw cycles.
7
4. Tissue Extract
Mince tissue on ice in ice-cold buffer, preferably in the presence of protease inhibitors. Place the tissue in
micro-centrifuge tubes and dip into liquid nitrogen to snap freeze. Keep samples at -80°C for later use or
keep on ice for immediate homogenization.
For every 5 mg of tissue, add 300 µL of extraction buffer to the tube and homogenize:
• 100 mM Tris, pH 7.4

• 150 mM NaCl
• 1 mM EGTA
• 1 mM EDTA
• 1% Triton X-100 0.5%
• 0.5% sodium deoxycholate
(This portion of the buffer can be prepared ahead of time and stored at 4°C. Immediately before use, the
buffer must be supplemented with phosphatase inhibitor cocktail [as directed by manufacturer], protease
inhibitor cocktail [as directed by manufacturer] and PMSF to 1 mM to generate a complete extraction
buffer solution.)
Rinse the blade of the homogenizer twice with 300 µL extraction buffer. Place the sample on a shaker at
4°C for 2 hours.
Centrifuge the sample for 20 min at 13,000 rpm at 4°C. Aliquot the supernatant into pre-chilled tubes
sitting in ice. Keep the samples at -80°C, avoiding freeze/thaw cycles.
Note: Lysis buffer volume must be determined according to the amount of tissue present. Typical
concentration of final protein extract is at least 1 mg/mL.
8
Recommended Protocols
Reagent Preparation
1. Standard Solutions
• 10,000 pg/mL: Add 1 mL of sample diluent buffer into one tube of standard (10 ng per tube) and mix
thoroughly. Note: Store this solution at 4°C for up to 12 hours (or -20°C for 48 hours) and avoid
freeze-thaw cycles.
• 5,000 pg/mL: Mix 0.3 mL of 10,000 pg/mL with 0.3 mL of sample diluent buffer and mix thoroughly.
• 2,500 pg/mL: Mix 0.3 mL of 5,000 pg/mL with 0.3 mL of sample diluent buffer and mix thoroughly.
• Perform similar dilutions until the standard solutions with these concentrations (pg/mL) are made:
1,250, 625, 312, 156 and 78.
• Add 100 µL of each of the diluted standard solutions to the appropriate empty wells. Repeat in
duplicate or triplicate for accuracy.
Note: The standard solutions are best used within 2 hours.
2. Biotinylated Antibody
• Calculate the total volume needed for the assay by multiplying 0.1 mL/well and the number of wells
required. Add 2-3 extra wells to the calculated number of wells to account for possible pipetting errors.
• Generate the required volume of diluted antibody by performing a 1:100 dilution (For each 1 µL
concentrated antibody, add 99 µL antibody dilution buffer) and mixing thoroughly.
3. Avidin-Biotin-Peroxidase (ABC)
• Calculate the total volume needed for the assay by multiplying 0.1 mL/well and the number of wells
required. Add 2-3 extra wells to the calculated number of wells to account for possible pipetting errors.
• Generate the required volume of diluted ABC solution by performing a 1:100 dilution (For each 1 µL
concentrated ABC solution, add 99 µL ABC dilution buffer) and mixing thoroughly.
Note: The diluted ABC solution should not be prepared more than 1 hour prior to the experiment.
9
Sandwich ELISA
All of the ELISA kits from Boster use the sandwich format and avidin-biotin chemistry. Our ELISA
assays require the dilutions of standard solutions, biotinylated antibody (detection antibody) and avidin-
biotin-peroxidase.
1. Capture Antibody Coating
(These steps are not required if the pre-adsorbed Picokine ELISA kits from Boster are used)
• Dilute the capture antibody to a final concentration of 1-10 μg/mL in bicarbonate/carbonate antigen-coating buffer (100 mM NaHCO3 in deionized water; pH adjusted to 9.6).
• Pipette 100 μL of diluted antibody to each well of a microtiter plate.

• Cover the plate with adhesive plastic and incubate at 4°C overnight (or 37°C for 30 min).
• Remove the coating solution and wash the plate 3X with 200 μL PBS (Phosphate Buffered Saline)
buffer (10 mM Na2HPO4 and 1.8 mM NaH2PO4 in deionized water with 0.2% Tween 20; pH Adjusted
to 7.4) with for 5 minutes each time. The coating/washing solutions can be removed by flicking the
plate over a sink. The remaining drops can be removed by patting the plate on a paper towel or by
aspiration. Do not allow the wells to dry out at any time.
2. Blocking
(These steps are not required if the pre-adsorbed Picokine ELISA kits from Boster are used)
• Pipette 200 μL blocking buffer (5% w/v non-fat dry milk in PBS buffer) per well to block residual protein-binding sites. Alternatively, BSA or BlockACE can be used to replace non-fat dry milk.
• Cover the plate with adhesive plastic and incubate for 1-2 hour(s) at 37°C (or at 4°C overnight).
• Remove the blocking solution and wash the plate 2X with 200 μL PBS for 5 minutes each time. Flick
the plate and pat the plate as described in the coating step.
3. Reagent Preparation
• Prepare for the diluted standard solutions, biotinylated antibody and ABC solutions as shown on p.9.
4. Sample (Antigen) Incubation

• Serially dilute the sample with blocking buffer immediately before use. The optimal dilution should be determined by a titration assay according to the antibody manufacturer.
• Pipette 100 μL of each of the diluted sample solutions and control to each empty well. Repeat in
duplicate or triplicate for accuracy. The negative control should be species- and isotype-matched as well
as non-specific immunoglobulin diluted in PBS buffer.
• Cover the plate with adhesive plastic and incubate for 2 hours at room temperature.
• Remove the content in the wells and wash them 3X with 200 μL PBS buffer for 5 minutes each time. Flick the plate and pat the plate as described in the coating step.
5. Biotinylated Antibody Incubation
• Pipette 100 μL of diluted antibody to the wells with control, standard solutions and diluted samples.
10
• Cover the plate with adhesive plastic and incubate for 1 hour at 37°C (or 2 hours at room temperature).
These incubation times should be sufficient to receive a strong signal. However, if a weak signal is
observed, perform incubation overnight at 4°C for a stronger signal.
• Remove the content in the wells and wash them 3X with 200 μL PBS for 5 min each time. Flick the
plate and pat the plate as described in the coating step.
6. ABC Incubation
• Pipette 100 μL of diluted ABC solution to the wells with control, standard solutions and diluted samples.
• Cover the plate with adhesive plastic and incubate for 0.5 hour at 37°C.
• Remove the content in the wells and wash them 3X with 200 μL PBS buffer for 5 min each time. Flick the plate and pat the plate as described in the coating step.
7. Substrate Preparation
Prepare the substrate solution immediately before use or bring the pre-made substrate to room
temperature. The two widely used enzymes for signal detection are horse radish peroxidase (HRP) and
alkaline phosphatase (AP), and their corresponding substrates, stopping solutions, detection absorbance
wavelengths and color developed are as follows:
Enzyme Substrate* Stop Solution Absorbance (nm) Color Developed
HRP TMB 2M H2SO4 450 Yellow
AP pNPP 0.75M NaOH 405 Yellow
* TMB: 3,3’,5,5’-tetramethylbenzidine; pNPP: p-nitrophenyl-phosphate
Note:
• The TMB substrate must be kept at 37°C for 30 min before use.
• Hydrogen peroxide can also act as a substrate for HRP.
• Sodium azide is an inhibitor of HRP. Do not include the azide in buffers or wash solutions if HRP-
labeled conjugate is used for detection.
8. Signal Detection
• Pipette 90 μL of substrate solution to the wells with the control, standard solutions and diluted samples.
• Incubate the plate at 37°C in the dark. If TMB is used, shades of blue will be observed in the wells with
the most concentrated solutions. Other wells may show no obvious color.
• Color should be developed in positive wells after 15 min. After sufficient color development, pipette
100 μL of stop solution to the appropriate wells (if necessary).
• Read the absorbance (OD: Optical Density) of each well with a plate reader.
9. Data Analysis
• Prepare a standard curve using the data produced from the diluted standard solutions. Use absorbance
on the Y-axis (linear) and concentration on the X-axis (log scale).
• Interpret the sample concentration from the standard curve.
11
ndirect ELISA
This is a general protocol in which antigen coating and blocking may not be required if the wells from the
manufacturer have been pre-adsorbed with the antigen.
1. Antigen Coating
• Dilute purified antigens to a final concentration of 1-10 μg/mL in bicarbonate/carbonate antigen-coating buffer (100 mM NaHCO3 in deionized water; pH adjusted to 9.6).
• Pipette 100 μL of diluted antigen to each well of a microtiter plate.

2. Blocking
• Remove the blocking solution and wash the plate 2X with 200 μL PBS for 5 minutes each time. Flick
the plate and pat the plate as described in the coating step.
• Prepare for the diluted standard solutions as shown on p.9.
4. Primary Antibody Incubation

• Serially dilute the primary antibody of choice with blocking buffer. The optimal dilution should be determined by a titration assay according to the antibody manufacturer.
• Pipette 100 μL of each diluted antibody per well. Repeat in duplicate or triplicate for accuracy. The
negative control should be species- and isotype-matched as well as non-specific immunoglobulin
diluted in PBS buffer.
• Remove the diluted antibody solution and wash the wells 3X with 200 μL PBS for 5 min each time.
Flick the plate and pat the plate as described in the coating step.
5. Secondary Antibody Incubation

• Serially dilute the conjugated secondary antibody with blocking buffer immediately before use. The optimal dilution should be determined by a titration assay according to the antibody manufacturer.
12
• Pipette 100 μL of diluted secondary antibody solution to each well.
Note:
7. Signal Detection
• Pipette 90 μL of substrate solution to the wells with the control and standard solutions.
• Color should be developed in positive wells after 15 min. After sufficient color development, pipette
100 μL of stop solution to the wells (if necessary).
8. Data Analysis
13
Direct ELISA
1. Antigen Coating
• Dilute purified antigens to a final concentration of 1-10 μg/ml in bicarbonate/carbonate antigen-coating buffer (100 mM NaHCO3 in deionized water; pH adjusted to 9.6).

• Cover the plate with adhesive plastic and incubate at 4°C overnight (or 37C for 30 min).
2. Blocking
• Remove the blocking solution and wash the plate 2X with 200 μL PBS for 5 min each time. Flick the

• Serially dilute the conjugated primary antibody with blocking buffer immediately before use. The optimal dilution should be determined by a titration assay according to the antibody manufacturer.

14
Note:
6. Signal Detection
• Pipette 90 μL of substrate solution to the wells with the control and standard solutions.
• Color should be developed in positive wells after 15 min. After sufficient color development, pipette 100
μL of stopping solution to the wells (if necessary).
7. Data Analysis
15
Competitive ELISA
1. Antigen Coating
• Dilute purified antigens to a final concentration of 20 μg/ml in bicarbonate/carbonate antigen-coating buffer (100 mM NaHCO3 in deionized water; pH adjusted to 9.6).

2. Blocking
• Remove the blocking solution and wash the plate 2X with 200 μL PBS for 5 min each time. Flick the
4. Sample (Antigen) Incubation

• Serially dilute the sample with blocking buffer immediately before use. The optimal dilution should be determined by a titration assay according to the antibody manufacturer.
• Pipette 100 μL of diluted sample to each well.

• Remove the content in the wells and wash them 3X with 200 μL PBS buffer for 5 minutes each time. Flick the plate and pat the plate as described in the coating step.

• Serially dilute the primary antibody of choice with blocking buffer. The optimal dilution should be determined by a titration assay according to the antibody manufacturer.
• Pipette 100 μL of each diluted antibody per well. Repeat in duplicate or triplicate for accuracy. The
negative control should be species- and isotype-matched as well as non-specific immunoglobulin diluted
in PBS buffer.
16
• Remove the diluted antibody solution and wash the wells 3X with 200 μL PBS for 5 min each time.
Flick the plate and pat the plate as described in the coating step.
6. Secondary Antibody Incubation

• Serially dilute the conjugated secondary antibody with blocking buffer immediately before use. The optimal dilution should be determined by a titration assay according to the antibody manufacturer.

Note:
8. Signal Detection
• Pipette 90 μL of substrate solution to the wells with the control, standard solutions and diluted samples.
• Incubate the plate at 37C in the dark. If TMB is used, shades of blue will be observed in the wells with
• Color should be developed in positive wells after 15 minutes. After sufficient color development, pipette
100 μL of stopping solution to the wells (if necessary).
17
9. Data Analysis
• Competitive ELISA yields an inverse curve: Higher values of antigen in the samples yield a smaller
amount of color change.
18
Troubleshooting Guide
The following guide serves as a checklist for the possible causes and solutions with respect to some of the
most commonly encountered problems from the ELISA assays.
1. Weak or No Signal
Possible Cause Solution

1 Blocking protein in coating Eliminate blocking protein from coating solution
solution
2 Capture antibody (or antigen) does Use ELISA plate, not tissue culture plate
not bind to plate
Try longer coating time
Increase concentration of coating components
3 Problem with the standard Use new sample
Check that the standard is appropriately handled
4 Incubation time too short Follow the manufacturer guideline (If the problem persists, try
incubating samples at 4°C overnight)
5 Incubation temperature too low Ensure incubations are done at correct temperature
Before proceeding, all reagents, including plate, should be at

room temperature or as recommended by the manufacturer
6 Incompatible sample type Use sample that the assay is known to detect a positive control
(Include such control in your experiment)
7 Incompatible assay buffer Ensure assay buffer is compatible with the target of interest
8 Target present below detection Decrease dilution factor or concentrate samples

limit
10 Incorrect/Insufficient/No Check the substrate identity
substrate
Increase concentration or amount of substrate
Follow manufacturer guidelines
11 Incorrect/Insufficient/No Check the antibody identity

antibody
Repeat the assay with higher antibody concentrations to find
the optimal one for your experiment
12 Antibody stored at 4°C for several Use fresh aliquot of antibody that has been stored at -20°C or
weeks or subjected to repeated below
freeze-thaw cycles
19
13 Incorrect reagents added/ Check protocol, ensure correct reagents are added in proper
prepared; Missing reagents order and prepared to correct concentrations (e.g. TMB for
HRP-labeled antibodies)
14 Expired/Contaminated reagents Make and use fresh/uncontaminated reagents
15 Enzyme inhibitor present Avoid sodium azide in HRP reactions
Avoid phosphate in AP reactions
16 Incorrect storage of components Double check storage conditions on kit level (Most kits need
to be stored at 4°C)
17 Ultra vigorous plate washing Gently pipette wash buffer (manual method)
Ensure correct pressure (automatic wash system)
18 Wells dry out Cover the plate using sealing film or tape for all incubations
19 Wells scratched with pipette or Carefully dispense/aspirate solutions into and out of wells
pipette tips
20 Plate read at incorrect detection Use recommended wavelength/filter
wavelength
Ensure plate reader is set correctly for type of substrate used
21 Slow color development Prepare substrate immediately before use
Allow longer incubation
Ensure stock solution is unexpired and uncontaminated
22 Epitope recognition impeded by Conjugate peptide to large carrier protein before coating onto
adsorption to plate plate
2. Saturated Signal

1 High sample concentration Use higher sample dilutions (Determine the optimal dilutions
by titration assay)
2 Excessive substrate Decrease concentration or amount of substrate: Follow

manufacturer guidelines (The substrate provided with the
ELISA kit might require further dilution)
3 Substrate color changed before use Make substrate immediately before use
4 Non-specific antibody binding Try different formulations in coating solutions
20
Ensure wells are pre-processed to prevent non-specific
binding
Use affinity-purified antibody and preferably one that is pre-

adsorbed.
Use serum (5-10%) from same species as secondary antibody

(bovine serum is also recommended)
5 Incubation time too long Follow the manufacturer guidelines (If the problem persists,
try incubating samples at 4°C overnight)
6 Excess antibody Repeat the assay with lower antibody concentrations to find
7 Contaminated buffers with metals Make and use fresh buffers

or HRP
9 Insufficient washing Follow the manufacturer guidelines
At the end of each washing step, flick the plate over a sink and
pat the plate on a paper towel
10 Plate sealers not used or re-used During incubations, cover plates with plate sealers.
Use a fresh sealer every time the used sealer is removed from
the plate
11 Plate read at incorrect detection Use recommended wavelength/filter

wavelength
12 Excess time before plate reading Read your plate within 30 minutes after adding the substrate
(If the reading is not performed within this time frame, add a
stopping solution after sufficient color is developed in the
plate)
3. High Background

2 Ineffective/Contaminated Try higher blocking protein concentration

blocking buffer
Increase blocking time
21
Use fresh buffer
3 Excess antibody Repeat the assay with lower antibody concentrations to find
4 Excess substrate Decrease concentration or amount of substrate
Follow manufacturer guidelines (Note: The substrate provided

with the ELISA kit might require further dilution)
5 Cross reactivity (Detection Run appropriate controls

antibody reacts with coating
antibody)
6 Non-specific antibody binding Try different formulations in coating solutions
Ensure wells are pre-processed to prevent non-specific

binding
Use affinity-purified antibody and preferably one that is pre-

adsorbed
Use serum (5-10%) from same species as secondary antibody

(bovine serum is also recommended)
7 Insufficient Tween in buffers Use PBS containing 0.05% Tween
8 Suboptimal salt concentration in Optimize salt concentration as high concentration can reduce
washing buffer non-specific interactions
9 Incubation temperature too high Optimize incubation temperature for your assay (antibodies
bind optimally at very specific temperature)
10 Reagents were not mixed properly Thoroughly mix all reagents and samples before pipetting
solutions into wells
11 Blanks contaminated with Change pipette tips when switching between blanks and
samples samples
Put a lid on pates to avoid any spilling between wells
12 Sample contaminated with Test samples with substrate alone to check for contaminating
enzymes enzymes
13 Contaminated TMB substrate Use a clean container to check that the substrate in not
contaminated (TMB substrate should be clear and colorless
before adding to wells)
22
14 Substrate incubation in light Carry out substrate incubation in dark or follow
recommendation from manufacturer
15 Uneven evaporation of solution Always incubate with a lid on the plate

from wells during incubation
16 Precipitate created in wells upon Increase dilution factor of sample or decrease concentration of
substrate addition substrate
17 Incubation time too long Follow the manufacturer guidelines (If the problem persists,
try incubating samples at 4°C overnight)
18 Incorrect standard curve dilutions Check pipetting techniques
Double check calculations
19 Plates stacked during incubations, Avoid stacking plates

leading to uneven temperature
distribution
20 Dirty or defective plates Clean the plate bottom
21 Unstopped color development Use Stopping solution to prevent over-development
22 Excess time before plate reading Read your plate within 30 minutes after adding the substrate
(If the reading is not performed within this time frame, add a
stopping solution after sufficient color is developed in the
plate)
Note: Color continues to develop even after adding the

stopping solution (although at a slower rate)
23 Incorrect plate reading setting Use recommended wavelength/filter
4. Low Sensitivity

1 Assay format not sensitive enough Switch to a more sensitive detection system (e.g. colorimetric
to chemiluminescence)
Switch to a more sensitive assay type (e.g. direct ELISA to

sandwich ELISA)
Increase incubation time and/or temperature
2 Improper storage of ELISA kit Store all reagents as recommended
23
Note: All reagents may not have identical storage requirements
3 Insufficient target Reduce sample dilution or concentrate sample
4 Inactive substrate Ensure reporter enzyme has the expected activity
5 Poor target adsorption to wells Covalently link target to wells
6 Insufficient substrate Increase concentration or amount of substrate
7 Incompatible sample type Use a sample that the assay is known to detect a positive
control
Include positive control in your experiment
8 Interfering ingredients in buffers Check reagents for any interfering chemicals, e.g. sodium azide
and sample in antibodies inhibit HRP enzyme; EDTA used as anti-
coagulant for plasma collection inhibits enzymatic reactions
9 Mixing or substituting reagents Avoid mixing components from different kits

from different kits
10 Incorrect plate reading setting Use recommended wavelength/filter
5. Poor Standard Curve

1 Improper standard solution Confirm dilutions are done correctly
Make new standard curve as appropriate
2 Standard improperly reconstituted Briefly spin vial before opening
Inspect for undissolved material after reconstituting
3 Standard degraded Store and handle standard as recommended
Prepare standards no more than two hours before use
4 Improper curve fitting Try plotting using different scales, e.g. log-log, 5-parameter
logistic curve fit
5 Pipetting error Use calibrated pipettes and proper pipetting technique
24
7 Poorly mixed reagents Thoroughly mix reagents
8 Poor/variable adsorption of Extend incubation time

reagents to plate
Check coating buffer
Use a different plate as appropriate
Check homogeneity of samples
9 Plates stacked during incubation Keep plates separated if not using rotating plates
10 Dirty or defective plates Clean the plate bottom
6. Poor Replicate Data

1 Bubble in wells Ensure no bubbles are present prior to reading plate
2 Insufficient washing of wells Carefully wash wells
Follow recommended protocols
Check that all ports of the plate washer are unobstructed
3 Incomplete reagent mixing Ensure all reagents are mixed thoroughly
4 Inconsistent pipetting Use calibrated pipettes and proper pipetting techniques
If a multi-channel pipette is used, ensure that all channels

deliver the same volume
5 Inconsistent sample prep or Ensure consistent sample prep and optimal sample storage
storage conditions (e.g. minimize freeze/thaw cycles)
6 Particulates in samples Remove the particulates by centrifugation
7 Plate sealers not used or re-used During incubations, cover plates with plate sealers
the plate
8 Cross-well contamination Ensure plate sealers and pipette tips are not contaminated with
reagents
25
9 Edge effect (higher or lower OD Ensure plates and reagents are kept at room temperature
in peripheral wells than in central before pipetting into wells unless otherwise instructed
wells)
During incubation, seal the plate completely with a plate sealer
and avoid stacking plates
7. Inconsistent Assay-to-Assay Results

1 Insufficient washing of wells Carefully wash wells
Follow recommended protocols
Check that all ports of the plate washer are unobstructed
2 Variation in incubation Adhere to recommended incubation temperature

temperature
Avoid incubating plates in area where environmental
conditions vary
3 Variation in protocol Adhere to the same protocol from run to run
4 Plate sealers not used or re-used During incubations, cover plates with plate sealers
the plate
5 Incorrect dilutions Confirm dilutions are done correctly for standard solutions, etc
Make new standard curve as appropriate
6 Contaminated buffers Make and use fresh buffers
7 Plates stacked during incubation Keep plates separated if not using rotating plates
8. Slow Color Development

1 Substrates too old, contaminated Make and use fresh substrates at correct pH: they should be
or used at incorrect pH prepared immediately before use
2 Expired/Contaminated solutions Make and use fresh reagents
3 Incorrect incubation temperature Ensure plates and reagents are kept at room temperature
before pipetting into wells unless otherwise instructed
26
During incubation, seal the plate completely with a plate sealer
and avoid stacking plates
4 Low antibody concentration Repeat the assay with higher antibody concentrations to find
5 Low substrate concentration Add more substrate to the wells
Make substrate no more than one hour before use
Note: Typical ELISA sensitivity is ~0.1 pg/mL with exact

value depends on antibody used.
9. Plate Imaging Problem

1 Oversaturated image after Use full resolution image to analyze results (Do not use jpeg
acquisition or other compressed formats)
2 Blurry spots in images Re-focus your camera before taking a new image
3 Repeated pixel values or Use lower bin size, higher image resolution and/or lossless file
rectangular spots type
4 Flat standard in images Reduce acquisition time
27
FAQs
1. The ELISA protocols do not recommend shaking during incubations. Have you tested shaking
and decided against it or is it unnecessary?
We tested our protocols with and without shaking during incubations and determined that there is no
difference between the two approaches. Therefore, we believe that shaking is not necessary.
2. Is your ELISA kit suitable for use with tissue lysates? If so, what are the protocols?
Theoretically, our ELISA kit can work with tissue lysates. Our general sample preparation protocol for
tissue lysates is as follows:
• Rinse the tissue with PBS to remove excess blood.

• Chop the tissue into 1-2 mm pieces.
• Using a tissue homogenizer, homogenize the samples in PBS or lysate solution such as the Mammal
Tissue Protein Extraction Reagent (Boster Bio Catalog Number AR0101) at a ratio of 10 mL lysate
solution to 1 g of tissue.
• Centrifuge the homogenates at approximately 5000 x g for 5 min.
• Assay immediately or store the homogenates at -20°C (avoid repeated freeze-thaw cycles).
3. My samples contain very low cytokine concentration. What is the minimum concentration that
can be measured with confidence using your ELISA kits?
Low cytokine concentrations are typical for many biological samples. When determining the minimum
concentration that can be reliably measured by ELISA, consider the following:
• The standard curve: As the concentration range of ELISA is typically 0 - 1000 pg/mL, the data points on
the standard curve for this range correspond to 0, 15.6, 31.25, 62.5, 125, 250, 500, and 1000 pg/mL.
• The assay sensitivity: Boster’s Picokine ELISA kits typically have a reported sensitivity of 10 pg/mL.
The concentration detected in many biological samples will fall between the 0 and 15.6 pg/mL data
points of the standard curve. As long as the value detected is above the statistical sensitivity of the
ELISA, (e.g., 5 pg/mL or greater), the value is statistically significant. Results below this detection
limit are of questionable validity.
4. Is your ELISA kit suitable for use with tissue homogenates?
For most cases, yes. If there is enough target protein present in the tissue of interest, the ELISA kit will
work. Also, if there are a known alternative processing of the protein in a specific tissue that results in
protein reactivity change to the kit, we will note it in our product datasheet.
5. Is your ELISA kit suitable for use with any non-validated sample types?
In order to use an ELISA kit with a non-validated sample type, it is necessary to perform a spike and
recovery study to determine if a non-validated sample type will work with a particular kit. To do this:
• Divide the sample into two aliquots.

• In one of the aliquots, you should “spike in” a known amount of the kit standard.
• Perform a dilution series to compare the spiked to the unspiked sample.
Generally, samples with expected recovery and linearity between 80-120% are acceptable. This method
can be used to validate any sample type that has not been previously evaluated by Boster.
28
6. Can I extend the standard curve?
No one can guarantee the assay accuracy once the concentrations outside the specified range within the
curve are used. A specific range is generated to provide the statistical confidence for the assay accuracy.
7. What causes high variability between sample duplicates?
The two main reasons for high sample variability in an assay are inconsistent pipetting and washing. Thus,
it is important to perfect these techniques. However, some of this variability is unavoidable — this is the
rationale for calculating the average results from sample duplicates. Another possible culprit for high
variability is the “edge effect” in which the outermost wells of the plate are more vulnerable to drying out
due to evaporation. Plate stacking will also cause variability because temperature will be unevenly
distributed across the plates.
8. What are the differences between the sandwich ELISA and competitive ELISA?
Sandwich ELISAs typically require the use of matched antibody pairs, where each antibody is specific for
a different, non-overlapping part (epitope) of the antigen molecule. A first antibody (known as capture
antibody) is coated to the wells. The sample solution is then added to the well. A second antibody (known
as detection antibody) follows this step in order to measure the concentration of the sample. Higher signal
output reflects higher concentration of the target antigen in the sample.
The key event of competitive ELISA is the process of competitive reaction between the sample antigen
and antigen bound to the wells of a microtiter plate with the primary antibody. First, the primary antibody
is incubated with the sample antigen and the resulting antibody–antigen complexes are added to wells that
have been coated with the same antigen. After an incubation period, any unbound antibody is washed off.
The more antigen in the sample, the more primary antibody will be bound to the sample antigen.
Therefore, there will be a smaller amount of primary antibody available to bind to the antigen coated on
the well, resulting in a signal reduction.
9. Why do my wells turn green after I add the stop solution?
The green color is a result of incomplete mixing between the substrate and stop solution. After adding the
stop solution, gently tap the plate or place it on a shaker until the mixture in the wells turns yellow.
10. Why does a brown or orange-brown precipitate appear in my wells after adding the stop solution?
How can I resolve this issue?
The precipitate is a result from insufficient washing after incubation with the HRP- labeled detection
antibody. To resolve this issue, perform a 30-second soak during each wash step followed by a complete
removal of all liquid in the wells.
11. If I don’t use all the wells from a microtiter plate for my current ELISA assay, how can I preserve
the unused wells for future use?
The microtiter plate typically has removable strips of wells. Unused wells may be removed from the plate,
returned to the foil pouch containing the desiccant pack and stored at 2-8°C for up to one month.
29
Ordering Information
With more than 20 years of experience and trust from 10,000+ scientists, Boster is proud of offering more
than 600 PicoKineTM ELISA kits that help accelerate scientific discovery in research areas including
immunology, neuroscience and cancer. Each of our ELISA kits has sufficient reagents for 96 tests per kit.
The table below shows some of the most commonly used ELISA kits.
Sample Types*
Target Species CCS Se P(h) P(e) P(c) U M CL T Cat. No.
Adiponectin Human √ √ √ √ √ √ EK0595
Angiopoietin-2 Mouse √ √ √ √ EK0938
BDNF Human √ √ √ √ √ EK0307
CRP Rat √ √ √ √ EK0978
CTLA4 Mouse √ √ √ EK0717
EGFR Human √ √ √ √ √ EK0327
FGF21 Human √ √ √ √ EK0994
IFN Gamma Human √ √ EK0373
IL-6 Human √ √ √ √ √ EK0410
IL-8 Human √ √ √ √ √ EK0413
IL-10 Human √ √ √ √ √ EK0416
Leptin Mouse √ √ √ √ EK0438
MCP-1 Rat √ √ EK0902
NGF Beta Rat √ √ EK0471
P53 Human √ EK0895
PD-1 Human √ EK0959
TGF Beta 1 Human √ √ √ √ EK0513
TNF Alpha Mouse √ √ √ √ EK0527
* Cell Culture Supernates [CCS], Serum [Se], Plasma: Heparin [P(h)], Plasma: EDTA [P(e)], Plasma: Citrate [P(c)],
Urine [U], Milk [M], Cell Lysate (CL), Tissue (T)
We also offer a variety of ELISA components that can be purchased separately from the kits:
• Lyophilized recombinant standard (1 ng to 100 ng)
• 96-well plate (No antibody pre-coated)
• Avidin-Biotin-Peroxidase Complex (ABC)
• Buffers (Sample diluent, antibody diluent, ABC diluent, PBS, TBS)
• TMB color developing agent and stop solution
• Biotinylated antibody
Contact Information
Boster Biological Technology
3942 Valley Ave., Suite B, Pleasanton, CA 94566
Phone: (888) 466-3604
Fax: (925) 485-4560
Sales and Customer Service: [email protected]

Product and Technical Support: [email protected]
Business Development: [email protected]
30

ELISA Handbook

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ELISA Handbook

Principle, Troubleshooting, Sample

See All Boster Bio Products

2. General ELISA Procedure …………………………………………………………... 2

3. ELISA Types ………………………………………………………………………… 3

4. ELISA Data Interpretation ……………………………………………………………. 6

5. Sample Preparation …………………………………………………………………….7

6. Recommended Protocols ……………………………………………………………….9

7. Troubleshooting Guide ………………………………………………………………….. 19

9. Ordering Information ……………………………………………………………………. 30

General ELISA Procedure

To be continued on next page

• A wide variety of labeled secondary antibodies are available commercially.

• High specificity: the antigen/analyte is specifically captured and detected

Indirect Direct Sandwich Competitive

ELISA Data Interpretation

• Protein extract concentration is at least 1-2 mg/mL.

1. Cell Culture Supernatants

To be continued on next page

• 100 mM Tris, pH 7.4

To be continued on next page

Note: The standard solutions are best used within 2 hours.

To be continued on next page

• Pipette 100 μL of diluted antibody to each well of a microtiter plate.

4. Sample (Antigen) Incubation

5. Biotinylated Antibody Incubation

• Pipette 100 μL of diluted antigen to each well of a microtiter plate.

• Prepare for the diluted standard solutions as shown on p.9.

4. Primary Antibody Incubation

5. Secondary Antibody Incubation

To be continued on next page

• Pipette 100 μL of diluted antigen to each well of a microtiter plate.

• Prepare for the diluted standard solutions as shown on p.9.

4. Primary Antibody Incubation

• Pipette 100 μL of diluted secondary antibody solution to each well.

To be continued on next page

• Pipette 100 μL of diluted antigen to each well of a microtiter plate.

• Prepare for the diluted standard solutions as shown on p.9.

4. Sample (Antigen) Incubation

• Pipette 100 μL of diluted sample to each well.

5. Primary Antibody Incubation

6. Secondary Antibody Incubation

• Pipette 100 μL of diluted secondary antibody solution to each well.

To be continued on next page

Possible Cause Solution

Increase concentration of coating components

3 Problem with the standard Use new sample

Check that the standard is appropriately handled

Before proceeding, all reagents, including plate, should be at

8 Target present below detection Decrease dilution factor or concentrate samples

Follow manufacturer guidelines

11 Incorrect/Insufficient/No Check the antibody identity

14 Expired/Contaminated reagents Make and use fresh/uncontaminated reagents

15 Enzyme inhibitor present Avoid sodium azide in HRP reactions

Avoid phosphate in AP reactions

Ensure correct pressure (automatic wash system)

21 Slow color development Prepare substrate immediately before use

Allow longer incubation

Ensure stock solution is unexpired and uncontaminated