ELISA Troubleshooting (No Color Development)

Sometimes there is no color development (no signal) in ELISA, and most of the case is because
of carelessness as to the possible causes. Just check or verify every step to troubleshoot the
unnecessary problem which can be completely avoided.
Possible causes Recommended troubleshooting
Reagents were used in the wrong
order or an assay step was omitted.
Check the package insert for the assay protocol and
repeat the assay.
Samples were not added to diluent
(indirect format only).
Verify that the samples were added to the diluent.
Wrong conjugate was used,
conjugate was prepared incorrectly
or has deteriorated.
Be sure that the conjugate used is the one that came with
the kit. All conjugates are kit- and lot-specific. If
preparation of a working conjugate is needed, be sure
that the concentrate and diluent are mixed in correct
volumes. Do not prepare the working solution too far in
advance and do not save any unused portion for future
use. If no conjugate preparation is necessary, be sure to
pour out only the amount required for immediate use
and do not return any unused portion to the stock bottle.
Incorrect or no detection antibody
was added.
Add appropriate detection antibody and continue.
Substrate solution was not added. Add substrate solution and continue.
Wash buffer contains sodium azide. Avoid sodium azide in the wash buffer.
ELISA Term Definition
Solid phase
Usually a microtiter plate
well. Specially prepared ELISA plates are commercially
available. These have an 8 12 well format and can be
used with a wide variety of specialized equipment
designed for rapid manipulation of samples including
multichannel pipets.
Adsorption
The process of adding an
antigen or antibody, diluted in buffer, so that it attaches
passively to the solid phase on incubation. This is a
simple way for immobilization of one of the reactants in
the ELISA and one of the main reasons for its success.
Washing
The simple flooding and emptying of the wells with a
buffered solution to separate bound (reacted) from
unbound (unreacted) reagents in the ELISA. Again, this
is a key element to the successful exploitation of the
ELISA.
Antigens
A protein or carbohydrate that when injected into
animals elicits the production of antibodies. Such
antibodies can react specifically with the antigen used
and therefore can be used to detect that antigen.
Antibodies
Produced in response to antigenic stimuli. These are
mainly protein in nature. In turn, antibodies are
antigenic.
Antispecies antibodies Produced when proteins (including antibodies) from one
species are injected into another species. Thus, guinea
pig serum injected into a rabbit elicits the production of
rabbit antiCguinea pig antibodies.
Enzyme
A substance that can react at low concentration as a
catalyst to promote a specific reaction. Several specific
enzymes are commonly used in ELISA with their
specific substrates.
Enzyme conjugate
An enzyme that is attached irreversibly to a protein,
usually an antibody. Thus, an example of antispecies
enzyme conjugate is rabbit antiguinea linked to
horseradish peroxidase.
Substrate
A chemical compound with which an enzyme reacts
specifically. This reaction is used, in some way, to
produce a signal that is read as a color reaction (directly
as a color change of the substrate or indirectly by its
effect on another chemical).
Chromophore
A chemical that alters color as a result of an enzyme
interaction with substrate.
Stopping
The process of stopping the action of an enzyme on a
substrate. It has the effect of stopping any further
change in color in the ELISA.
Reading
Measurement of color produced in the ELISA. This is
quantified using special spectrophotometers reading at
specific wavelengths for the specific colors obtained
with particular enzyme/chromophore systems. Tests can
be assessed by eye.

Chromogenic Assay

Chromogenic assays result in a colored reaction product that absorbs light in the visible range.
The antigen-antibody complex formed on the solid carrier is separated from other substances by
washing. The antibody was labeled by enzyme. After the substrate of the enzyme was added, the
substrate becomes a colored product under catalysis of the enzyme-catalyzed, which is directly
related to the amount of test substance. The optical density of the reaction product is typically
proportional to the amount of analyte being measured. Due to the very high efficiency of the
enzyme, the reaction can be greatly enlarged with a high sensitivity.

Principle of optical absorption
Lambert-Beer Law: logI
0
/ I = C b. Absorbance also called optical density values (OD) is
proportional to the concentration.

The two most popular enzymes labeling the antibodies are alkaline phosphatase (AP) and
horseradish peroxidase HRP
1 AP
Alkaline phosphatase extracted from Escherichia coli is approximately double the size of
peroxidase (MW ~80kD) with optimum pH of 8.0. The MW of calf intestinal extracted AP is
100kD and the optimal pH is 9.6.This means that one will typically see a lower enzyme to
antibody conjugation ratio. It also means that the larger molecular size of alkaline phosphatase
can cause steric hindrance issues due to close packed antigen-antibody complexes. This can
result in lower activity than expected for the estimated number of bound enzyme molecules
(which is sometimes considered responsible for the high dose hook phenomenon). Alkaline
phosphatase is slightly more expensive than peroxidase, but is considered to be more stable.
Substrates for alkaline phosphatase range from soluble to insoluble; many can be signal
enhanced to increase sensitivity. The most common substrate that produces an insoluble reaction
product is BCIP/NBT (5-bromo- 4-chloro-3-indolyl-phosphate/nitroblue tetrazolium). It is
recognized as the most effective substrate for immunoblots due to its stability and resistance to
fading when exposed to light. The most widely used substrate that produces a soluble reaction
product is p-NPP (p-nitrophenylphosphate). It produces an intense yellow color measurable at
405 to 410 nm. An advantage of this substrate is that it can be allowed to develop for extended
periods to obtain a corresponding increase in sensitivity. Normally p-NPP has a slow reaction
rate which should be allowed 30 to 60 minutes to reach optimal color development before being
stopped with 1N NaOH. It is not recommended for kinetic analysis. The chemical reaction is as
follows:

The major disadvantage associated with using alkaline phosphatase is that it is inactivated by
chelating agents, acidic pH (< 4.5), or inorganic phosphates. This means that buffers must be
specific for alkaline phosphatase, and one cannot use standard assay phosphate buffered saline 2
solutions as diluents or wash solutions that come in contact with the enzyme during an assay.
However, chelators (EDTA) and acidic pH are typically used as convenient and inexpensive
stopping reagents for alkaline phosphatase reactions

Properties: low background; high sensitivity; but more expensive

2 HRP
Peroxidase is a glycoprotein with sugar about 18% (MW~44kD) that can usually be conjugated o
an antibody in a 4:1 ratio. It is a combination of a porphyrin protein composed of a apoenzyme
(enzyme) and a prosthetic group (heme).The apoenzyme is a colorless glycoprotein with a
maximum absorption peak at 275 nm. The prosthetic group is a dark brown iron porphyrin ring
and has a maximum absorption peak at 403 nm. Due to its small size, it rarely causes steric
hindrance problems with antibody/antigen complexes bound on a surface. Peroxidase is very
inexpensive compared to alkaline phosphatase. Several substrates, yielding either soluble or
insoluble reaction products, are commercially available for peroxidase. Since all peroxidase
reactions require hydrogen peroxide, purchasing commercially available substrates is
recommended because these preparations contain stabilized hydrogen peroxide which adds to
their value and usefulness.
The major disadvantage associated with peroxidase is that it is incompatible with many
preservatives, such as sodium azide, that are used to reduce microbial contamination in many
biological buffer solutions. Sodium azide, even in low concentrations, inactivates peroxidase
activity. Other compounds or elements that interfere with peroxidase activity are metals found in
water and endogenous peroxidases found in biological specimens. These disadvantages can be
overcome by using sterile buffers without preservatives, using reagent grade type II water, and
pretreating specimens suspected of having high peroxidase levels with hydrogen peroxide prior
to use in an assay. Typically, nonbound biological components are washed away prior to the
addition of the enzyme, so endogenous peroxidase activity is usually not an issue.
The three most common substrates that produce an insoluble product are TMB (3,3',5,5'
tetramethylbenzidine),DAB (3,3',4,4' diaminobenzidine), and 4CN (4-chloro-1-naphthol). The
most common substrates that produce soluble reaction products are TMB (dual function
substrate), ABTS (2,2'-azino-di [3-ethylbenzthiazoline] sulfonate), and OPD (o-
phenylenediamine). TMB is a highly sensitive substrate. Due to its rapid reaction rate, it is
ideally suited for on-line kinetic analysis. It produces a blue color measurable at a wavelength of
650 nm. TMB can also be used in endpoint assays by stopping the reaction with 1M phosphoric
acid. A yellow reaction product is formed upon acidification that is measurable at 450 nm. ABTS
is considered an all-purpose substrate. Although it is less sensitive than either TMB or OPD, it
has the widest working range of any substrate currently available for peroxidase or alkaline
phosphatase. The reaction product for ABTS is a blue-green compound measurable at 405 to 410
nm. Its reaction rate is suitable for endpoint assays and is easily stopped with 1% sodium
dodecyl sulfate (SDS), which does not change the color or the absorbance of the reaction
product. OPD was once the most popular substrate for peroxidase. It is slightly less sensitive
than TMB. Its reaction product is yellow and can be read at 490 nm. The chemical reaction is as
follows:

Properties: Commonly used; very simple; inexpensive; but less sensitive or specific than AP
system

Direct ELISA

Direct ELISA, Simple and Time-Saving
Initially in a direct ELISA test which is considered to be the simplest type of ELISA the antigen
is adsorbed to a plastic plate, then an excess of another protein (normally bovine serum albumin)
is added to block all the other binding sites. While an enzyme is linked to an antibody in a
separate reaction, the enzyme-antibody complex is applied to adsorb to the antigen. After excess
enzyme-antibody complex is washed off, enzyme-antibody bound to antigen is left. By adding in
the enzyme's substrate, the enzyme is detected illustrating the signal of the antigen.
However, in terms of direct ELISA versus indirect ELISA, in an indirect ELISA, the steps are
similar, but with important differences and an additional step. After the antigen is adsorbed to the
plate (and after the BSA step), the next antibody to be added is the antibody that recognizes the
antigen (this antibody does not have the enzyme attached to it). Then, an enzyme-antibody
conjugate is prepared, which is added to the plate and detects the antibody that is adsorbed to the
antigen (in a direct ELISA, the enzyme-antibody conjugate directly adsorbs to the antigen), then
the substrate is added which detects the presence of the enzyme and thus the antigen. So, in the
indirect ELISA, the enzyme-antibody conjugate uses an antibody against the type of antibody
that is used to detect the antigen, kind of like a sandwich. For instance, if the antigen is HIV-1
gp120, then an anti-HIV antibody (HIV-1 gp120 Antibody) is prepared (let's say from a mouse).
Then, in a separate reaction, an enzyme is attached to an anti-mouse antibody. So, in order to
detect the HIV in the assay, an anti-mouse antibody is used to detect the antibody attached to the
antigen.

Direct ELISA Schema Indirect ELISA Schema
Direct ELISA, when compared to other forms of ELISA testing, is performed faster because only
one antibody is being used and fewer steps are required. This can be used to test specific
antibody-to-antigen reactions, and helps to eliminate cross-reactivity between other antibodies.
Disadvantages of direct ELISA
The primary antibody must be labeled individually, which can be time-consuming and inflexible
when performing multiple experiments. Also, the signal is less amplified in direct ELISA, which
means a lower sensitivity and could be viewed as a disadvantage to some.
Direct ELISA protocol is shown elsewhere.
Please click direct ELISA appllication in monoclonal antibody screening to get more
information.

Indirect ELISA, conventional but efficient

Indirect ELISA is a two-step ELISA which involves two binding process of primary antibody
and labeled secondary antibody. The primary antibody is incubated with the antigen followed by
the incubation with the secondary antibody. However, this may lead to nonspecific signals
because of cross-reaction that the secondary antibody may bring about.
1. Micro-well plates are incubated with antigens, washed up and blocked with BSA.
2. Samples with antibodies are added and washed.
3. Enzyme linked secondary antibody are added and washed.
4. A substrate is added, and enzymes on the antibody elicit a chromogenic or fluorescent signal.
Learn more about indirect ELISA protocol
Indirect ELISA advantages
:
High sensitivity: More than one labeled antibody is bound per antigen molecule;
Flexible: Different primary detection antibodies can be used with a single labeled secondary
antibody;
Cost-saving: Fewer labeled antibodies are required.
In the indirect ELISA test, the sample antibody is sandwiched between the antigen coated on the
plate and an enzyme-labeled, anti-species globulin conjugate. The addition of an enzyme
substrate-chromogen reagent causes color to develop. This color is directly proportional to the
amount of bound sample antibody. The more antibody present in the sample, the stronger the
color development in the test wells. This format of indirect ELISA is suitable for determining
total antibody level in samples (Newcastle disease virus, B. abortus, etc.). Detailed information
about indirect ELISA application in the determination of antibody titer and procedures of
antibody concentration determination are discussed in the following section of ELISA
applications.
ELISA Advantages

Compared to other immunoassay methods, there are many advantages of ELISA. ELISA tests
are more accurate. They are considered highly sensitive, specific and compare favorably with
other methods used to detect substances in the body, such as radioimmune assay (RIA) tests.
ELISA possesses the added advantages of not needing radioisotopes (radioactive substances) or a
costly radiation counter (a radiation-counting apparatus).
1.High Sensitivity
The high sensitivity of ELISA, comes from the enzyme as a reporting group. As is known to all,
the enzyme is an organic catalyst, a small amount of which could induce a large span of catalytic
reactions to produce observable chromogenic reaction phenomenon. Therefore, this system is
often taken as the amplification system of enzyme. By ELISA, a tracer of the antigen or antibody
is achieved in the cell or subcellular level, also, antigen or antibody quantification can be done in
the microgram or even nanogram levels.
2.Strong Specificity
Specificity of ELISA is because of the selectivity of the antibody or antigen. Actually, the
binding of antigen or antibody only occurs in the epitope of an antigen or antigen-binding site of
an antibody. Since, there is a complementary relationship between epitope and antigen-binding
site both in chemical structure and spatial configuration, the reaction between antigen and
antibody shows a strong specifity.
These advantages of ELISA make it an useful biotechnical tool with many appllicaitons, either in
scientific research or clinical diagnosis of diseases or conditions.