| Monoclonal
(MoAb) - Polyclonal (PoAb) Sandwich Assay Sample Quotes
- MoAb-PoAb
Sandwich Assay Devp
-
High-Sensitivity
MoAb-PoAb Sandwich Assay Devp
Introduction
These sample
proposals are based on the development of a monoclonal -
polyclonal sandwich ELISA. For some analytes, this
combination provides an assay with decreased nonspecific
binding interference and increased sensitivity. In this
format, the monoclonal antibody is coated onto a 96-well
microtiter plate to capture the analyte from the sample.
Once the analyte is captured, the HRP-labeled polyclonal
is added to bind to the analyte (thereby
‘sandwiching’ it between the two antibodies). The
HRP generates a signal for detection, which can be read
on a microtiter plate reader. With a standard curve (1
blank and 7 standards) and 3 controls, a 96-well
microtiter plate format can test 21 samples in
triplicate and 37 samples in duplicate (for a detailed
description of the reagents in an ELISA kit, see What
Are the Typical Components in an ELISA?
In
some cases, often when a MoAb is unavailable, a
polyclonal antibody is coated onto the plate, and
another polyclonal is used for detection.
Depending on availability, different pairs of antibodies
will be investigated for each assay. ICT will also
work to eliminate as many sample preparation steps as
possible to make the ELISA simple and easy to run.
This is often accomplished by using various components
in the assay diluents. Assay development will
begin as soon as the antigen and antibodies are ready.
If
the target analyte and necessary antibodies are
available, assay development generally costs between
$30,000 - $100,000 and usually takes 4-8 months.
Proposals are often broken down into 4 phases, which
detail time and cost estimates. Payments are often
structured with a front fee of 30% of the estimated
total, and milestone payments disbursed thereafter,
which can be paid after each phase or monthly.
When
the assay has been developed, we normally supply
customers with reagents for 25 immunoassay kits. They
can be supplied as individually packaged kits or in bulk
packaging. When stored at 2o-8o C,
each kit may have a shelf life of at least 6 months.
If you need more kits, ICT will manufacture the
components as you order them (this usually requires a
3-6 week lead-time). Once developed, the cost for
additional kits typically ranges from $50 to $300 each
(with a minimum manufacturing charge of $2,000).
This price will be determined during development and
depends on the expense of the kit components, the type
of packaging required, and the volume ordered. If
you will be testing samples at different locations, we
can ship the assay kits directly to the other
laboratories.
These
sample proposals are meant to answer basic questions,
and to act as project guidelines. Depending on
your specific project, the amount of antigen required
may vary, conjugations may be necessary, the antibody
may need to be purified, sample preparation steps may be
necessary, and the length of time for the project will
vary. In order to answer some of your
questions, or to prepare a specific quote for your
project, please review the Assay
Development Questionnaire, and then contact us.
SAMPLE
PROPOSAL 1
Antibody Sandwich Assay Development
Required materials and
reagents to begin assay development:
- Analyte.
- Antibodies.
- Analyte negative
samples.
- Analyte positive
samples.
Phase I: Reagent
Preparation and Initial Assay
Titration..................$
Time: 4-8 weeks
- Labeling of specific
polyclonal antibody (with HRP, for example) for
assay signal generation.
- Selection of
compatible antibodies.
- Determination of a
working titration of capture monoclonal antibody and
detection polyclonal antibody conjugate.
- Development and
optimization of ELISA plate coating procedures for
optimal antigen capture.
Phase II: Assay
Optimization...............................................................$
Time: 4-14 weeks
- Selection of proper
diluents for sample and conjugate signal generation.
- Development of special
additives to eliminate sample matrix effects such as
interference and non-specific binding.
- Construction of a
standard curve to mimic performance characteristics
of the sample matrix.
- Development of a
functional assay protocol within the target
sensitivity range of the analyte.
- Elimination or
modification of extraction or sample preparation
steps.
Phase III: Assay
Validation...................................................................$
Time: 4-5 weeks
- Definition and
documentation of assay performance characteristics
essential for optimal assay utility (such as
sensitivity and precision).
- Documentation of diluent
performance parameters to assure proper matrix
composition (such as recovery and linearity).
- Fine-tuning of specific
assay components and incubation protocols to meet
final performance requirements.
Phase IV: Production of
25 Finished
Kits..............................................$
Time: 3-4 weeks
- Quality control
assessment of final components.
- Final assembly,
packaging, and delivery of components for 25 ELISA
kits.
Total Assay Development
Cost ...................range of $50,000 - $100,000
Estimated Time: 15-31 weeks
SAMPLE
PROPOSAL 2
Development of a High Sensitivity MoAb-PoAb Sandwich
ELISA using an Amplified Substrate System
and GMP Documentation
Required materials and
reagents required to begin assay development:
- At least 25 mg of
purified analyte.
- Antibodies.
- Analyte negative
samples.
- Analyte positive
samples.
Phase I: Reagent Preparation and
Initial Assay Titration..................$
Time: 5-8 weeks At the
completion of Phase I, the general configuration of the
assay will be set. The following items will have been
selected: antibody or antibody cocktails to capture and
detect the analyte; and an appropriate signal
amplification system (beyond normal conjugation
techniques and substrates) which will be necessary to
reach the desired sensitivity.
- Affinity purification
of polyclonal antibodies.
- Protein A purification
of monoclonal antibodies.
- Conjugation of
antibodies to alkaline phosphatase.
- Selection of
compatible capture and detection antibodies.
- Titration of capture
antibody and enzyme labeled antibody.
- Development and
optimization of ELISA plate coating procedures for
optimal antigen capture and signal generation.
Phase II: Assay
Optimization.............................................................$
Time: 4-8 weeks Upon
completion of Phase II, development of the assay and
assay protocol will be complete. Unless further changes
are necessary for validation, the assay will be in its
final form.
- Selection of proper
diluents for sample and conjugate signal generation.
- Development of special
additives to eliminate sample matrix effects such as
interference and non-specific binding.
- Construction of a
standard curve to mimic performance characteristics
of the sample matrix.
- Development of
lyophilized standards to allow for long-term storage
of ELISA kits at 4°C.
- Development of a
functional assay protocol within the target
sensitivity range of the analyte.
Phase III: Assay
Validation................................................................$
Time: 4-6 weeks
During Phase III, enough plates and reagents will be
manufactured to complete our in-house validation (this
is not as thorough as the validation required for FDA
approval). ICT will also supply the client with 25 ELISA
test kits for their evaluation.
- Definition and
documentation of assay performance characteristics
essential for optimal assay utility (such as
sensitivity and precision).
- Documentation of
diluent performance parameters to ensure proper
matrix composition (such as recovery and linearity).
- Fine-tuning of
specific assay components and incubation protocols
to meet final performance requirements.
- Set performance
criteria.
Phase IV: Preparation of
GMP Level Documents...............................$
Time: 4 weeks
Upon completion of Phase IV, all GMP level documents for
manufacturing and quality control for release of ELISA
kits will be completed. At this time, ICT will provide a
firm quote for additional kits and can begin
manufacturing.
Total Assay Development
and GMP Documentation..$100,000-$350,000
Total time: 17-26 weeks |
