Plate
Coating Buffers
The first step in making
a reliable ELISA is proper coating of the antibody
or antigen onto the plate. ICT has developed 2
coating buffers based on the type of ELISA being
made. ICT's 5x Universal Antibody Plate Coating Buffer (CB1) is a
unique buffer used to coat antibodies onto polystyrene microtiter ELISA plates, and can
be used for antibody-sandwich ELISAs as well as
antigen-down ELISAs. ICT's 5x
Antigen Coating Buffer (CB2) is designed for
antigen-down ELISAs. Both coating buffers
stabilize coated proteins by
maintaining their tertiary three-dimensional
structure, allowing for
greater binding reactivity with the detection
molecule, thereby enhancing the specific signal.
Blocking
and Stabilizer Buffers
Once the antibody or protein antigens have been
properly adsorbed onto the plate (we suggest using
ICT’s Plate Coating Buffer, CB1), the next
critical step in creating a reliable assay is the
blocking of the plate. Blocking is done to protect
the coated protein from harsh external conditions
while masking any uncoated regions on the plate.
As the blocking process has a profound effect on
both the specific and nonspecific signals
generated in the assay, the proper selection of
block buffer is a key element in the preparation
of a functional ELISA. The proper block buffer can
increase sensitivity, reduce nonspecific binding,
and lengthen the shelf-life of the coated plate.
Because ELISAs may be configured in several ways,
each with unique blocking requirements, ICT has
created 4 proprietary block buffer formulations
for use with sandwich and antigen-down ELISAs.
They are:
BB1
General Low-Level Blocker with
BSA.
BB2
Neptune Block
with Nonmammalian (fish) proteins, for extra
blocking strength.
BB3
SynBlock, based
on small synthetic non-protein blocking molecules,
for ELISAs that require extra blocking strength.
BB4
Phosph-Free
Blocker, does
not contain any phosphates nor any animal
proteins. It
is ideal for ELISAs using alkaline
phosphatase detection systems, and ELISA with
super sensitivity requirements.
If you would like
to test our first three blockers in your assay
system, just buy the
optimization pack: it includes 1 100mL bottle of
each BB1, BB2, and BB3.
HRP-Conjugate
Stabilizer Diluents
ICT has formulated a group of unique stabilizing
diluents designed to prolong the shelf-life of HRP-conjugated
proteins and enhance their utility in ELISA
applications. These conjugate stabilizer diluents
can be used to reconstitute lyophilized
conjugates, and to dilute concentrated conjugates
into the useful range of the assay. HRP-conjugated
antibodies are often unstable and can lose a
significant level of activity within a short time
after rehydration and dilution unless properly
stored. This loss of activity is based on two
factors: denaturization of the HRP-IgG protein
complex; and destabilization of the catalytic
region of the HRP. ICT’s proprietary
formulations preserve the integrity of the
antibody binding regions while maintaining the
enzymatic functionality of the HRP enzyme. ICT
has created 6 proprietary conjugate diluent
formulations for use with sandwich and
antigen-down ELISAs (all at 5x), and 1 conjugate
stabilizer for use with alkaline phosphatase
assays (at 1x):
CS1
Monoclonal/Polyclonal Conjugate Diluent &
Stabilizer.
CS2
Antigen-Down Conjugate Diluent & Stabilizer.
CS3
Polyclonal/Polyclonal Conjugate Diluent &
Stabilizer.
CS4
Monoclonal/Goat- Polyclonal Conjugate Diluent &
Stabilizer.
CS5
Monoclonal /Rabbit- Polyclonal Conjugate Diluent
& Stabilizer.
CS6
Conjugate Stock Stabilizing Reagent.
CS7
Alkaline Phosphatase Enzyme Conjugate Stabilizer. |
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Assay
Diluents
ICT’s assay diluents are proprietary buffer
formulations designed to equalize any differences
between the sample matrix (serum, plasma, urine,
cell culture fluid) and the diluent used to
generate the standard curve. These diluents also
reduce nonspecific interactions between the sample
matrix proteins and the plate surface which
translates into lower background noise.
Assay diluents are pipetted directly onto the
plate just prior to adding the samples, and may be
used with neat or diluted samples, depending on
the characteristics of the sample matrix and the
target range of the assay. Since different sample
matrices have unique characteristics when run in
an ELISA, they must be matched with specific assay
diluents. Use of an assay diluent reduces the
effects of the sample matrix and variation among
samples, without pre-dilution of the
samples. ICT has created 4 proprietary assay
diluent formulations for use with sandwich and
antigen-down ELISAs:
AD1
General Assay Diluent for Serum &
Plasma Samples.
AD2
IgM-Reducing (Positive
Interference) Assay Diluent for
Serum & Plasma Samples.
AD3
Neptune Assay Diluent for Serum &
Plasma samples.
AD4
Antigen-Down Assay Diluent for Serum and Plasma
samples.
If you would like to test
all four in your assay, buy the optimization pack:
it includes 1 100mL bottle of each AD.
Sample
Diluents
ICT’s unique serum sample diluents are
specifically formulated for dilution of animal and
bird serum samples in antigen-down ELISA formats.
Sample diluents are utilized to dilute serum or
antigen samples into the functional range of the
assay. Due to the finite binding capacity of the
coated ELISA plate surface, certain high samples
may overload this capacity, thus requiring
dilution prior to testing in the assay. Sample
diluents can also reduce background noise
associated with nonspecific bridging of
signal-generating conjugates to the plate
surface. Hyperimmunized serum samples
contain nonspecific IgG antibody that may
interfere with the titration signal. Proprietary
additives have been incorporated into these
diluents that minimize nonspecific binding
interactions of the nonspecific IgG in the
samples, thus decreasing background noise. These
sample diluents can be used to titrate rabbit,
mouse, goat, and chicken serum samples, along with
monoclonal cell culture supernatants.
Antigen samples may also be diluted with sample
diluents for evaluation in sandwich ELISA
formats. ICT has created 3 proprietary
sample diluent formulations for use with
antigen-down and some sandwich ELISA applications:
SD1
General Sample Diluent for Serum, Ascites, and Cell Culture Supernatant Samples.
SD2
Plasma Sample Diluent for Antigen-Down
testing of Plasma Samples.
SD3 Neptune Sample Diluent.
If you would like to test
all three in your assay, buy the optimization
pack: it includes 1 100mL bottle of each SD.
Wash
Buffer
ICT’s Universal ELISA Wash Buffer is a well-tested
formulation of buffers, salts, and detergents
designed to effectively remove excess material
from microtiter plates without disrupting ELISA
binding reactions. By maintaining the proper
buffering environment, unbound components can be
washed away without suppressing antigen-antibody
binding interactions, thereby reducing nonspecific
background noise and increasing the specific
signal. ICT’s Wash Buffer is compatible with all
routinely used conjugate components such as HRP,
AP, and avidin, among others.
PBS
ICT’s 10X Phosphate
Buffered Saline is a well-tested
formulation of buffers and salts
designed to effectively balance the pH without disrupting ELISA
binding reactions.
Also use it as a base to
create your own ELISA buffers or for other
applications in the lab like dialyzing proteins
and running samples over HPLC and Protein G
columns.
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