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Microtiter Plate Antibody-Sandwich Sample Proposals

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:

  1. Analyte.
  2. Antibodies.
  3. Analyte negative samples.
  4. Analyte positive samples.

Phase I: Reagent Preparation and Initial Assay Titration

Time: 4-8 weeks

  1. Labeling of specific polyclonal antibody (with HRP, for example) for assay signal generation.
  2. Selection of compatible antibodies.
  3. Determination of a working titration of capture monoclonal antibody and detection polyclonal antibody conjugate.
  4. Development and optimization of ELISA plate coating procedures for optimal antigen capture.

Phase II: Assay Optimization

Time: 4-14 weeks

  1. Selection of proper diluents for sample and conjugate signal generation.
  2. Development of special additives to eliminate sample matrix effects such as interference and non-specific binding.
  3. Construction of a standard curve to mimic performance characteristics of the sample matrix.
  4. Development of a functional assay protocol within the target sensitivity range of the analyte.
  5. Elimination or modification of extraction or sample preparation steps.

Phase III: Assay Validation

Time: 4-5 weeks

  1. Definition and documentation of assay performance characteristics essential for optimal assay utility (such as sensitivity and precision).
  2. Documentation of diluent performance parameters to assure proper matrix composition (such as recovery and linearity).
  3. 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

  1. Quality control assessment of final components.
  2. Final assembly, packaging, and delivery of components for 25 ELISA kits.

Total Assay Development Cost $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:

  1. At least 25 mg of purified analyte.
  2. Antibodies.
  3. Analyte negative samples.
  4. 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.

  1. Affinity purification of polyclonal antibodies.
  2. Protein A purification of monoclonal antibodies.
  3. Conjugation of antibodies to alkaline phosphatase.
  4. Selection of compatible capture and detection antibodies.
  5. Titration of capture antibody and enzyme labeled antibody.
  6. 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.

  1. Selection of proper diluents for sample and conjugate signal generation.
  2. Development of special additives to eliminate sample matrix effects such as interference and non-specific binding.
  3. Construction of a standard curve to mimic performance characteristics of the sample matrix.
  4. Development of lyophilized standards to allow for long-term storage of ELISA kits at 4°C.
  5. 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.

  1. Definition and documentation of assay performance characteristics essential for optimal assay utility (such as sensitivity and precision).
  2. Documentation of diluent performance parameters to ensure proper matrix composition (such as recovery and linearity).
  3. Fine-tuning of specific assay components and incubation protocols to meet final performance requirements.
  4. 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

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