Competitive Inhibition 96-Well Microtiter Plate ELISA Development Quote

Introduction
     In addition to the MoAb-PoAb sandwich format, many immunoassays are structured in a competitive inhibition format.  Competitive inhibition assays are often used to measure small analytes because competitive inhibition assays only require the binding of 1 antibody rather than 2 as is used in standard ELISA formats.  Because of the high probability for steric hindrance occurring when 2 antibodies attempt to bind to a small molecule at the same time, a sandwich assay format may not be feasible, therefore a competitive inhibition assay would be preferable.  In a sequential competitive inhibition assay, the sample and conjugated analyte are added in steps like a sandwich assay, while in a classic competitive inhibition assay, these reagents are incubated together at the same time.
     In a sequential competitive inhibition assay format, a monoclonal antibody is coated onto a 96-well microtiter plate.  When the sample is added, the MoAb captures free analyte out of the sample. In the next step, a known amount of analyte labeled with either biotin or HRP is added.  The labeled analyte will then also attempt to bind to the MoAb adsorbed onto the plate, however, the labeled analyte is inhibited from binding to the MoAb by the presence of previously bound analyte from the sample. This means that the labeled analyte will not be bound by the monoclonal on the plate if the monoclonal has already bound unlabeled analyte from the sample. The amount of unlabeled analyte in the sample is inversely proportional to the signal generated by the labeled analyte. The lower the signal, the more unlabeled analyte there is in the sample.  A standard curve can be constructed using serial dilutions of an unlabeled analyte standard.  Subsequent sample values can then be read off the standard curve as is done in the sandwich ELISA formats. 
     The classic competitive inhibition assay format requires the simultaneous addition of labeled (conjugated analyte) and unlabeled analyte (from the sample).  Both labeled and unlabeled analyte then compete simultaneously for the binding site on the monoclonal capture antibody on the plate.  Like the sequential competitive inhibition format, the colored signal is inversely proportional to the concentration of unlabeled target analyte in the sample.   
     Detection of labeled analyte may be made by using a peroxidase substrate such as TMB,  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.  By using various components in the assay diluents, ICT will work to eliminate as many sample preparation steps as possible to make the ELISA simple and easy to run. Assay development will begin as soon as purified antigen and antibody are available.
     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 (for a detailed description of the reagents in an ELISA kit, see What Are the Typical Components in an ELISA?).  When stored at 2^o-8^o 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 (also see Kit Manufacturing).
     This sample proposal is meant to answer basic questions, and to act as a project guideline.  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.  All prices are in US dollars.

SAMPLE PROPOSAL
Competitive Inhibition 96-Well Microtiter Plate ELISA Development

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

Phase I - Reagent Preparation and Initial Assay Titration…………$
Time: 6-8 weeks

1. Selection of compatible monoclonal antibodies and labeled analyte.
2. Titration of capture antibody and labeled analyte to working dilutions.
3. Development and optimization of ELISA plate coating procedures for optimal antigen capture and competitive inhibition of labeled antigen binding to the plate-bound capture antibody.

1. Selection of proper diluents for sample and conjugate signal generation.
2. Development of a functional assay protocol within the target sensitivity range of the analyte.
3. Construction of a functional standard curve to mimic performance characteristics of the sample matrix.

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.

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

Total Assay Development Cost……………range of $30,000 - $100,000
Estimated Time: 21-27 weeks

Need to detect a molecule? 

Call us 800-829-3194

How do I have ICT develop an assay for me?

1. Read the background information on our website. 
2. Read and fill out our Assay Development Questionnaire as best you can.
3. Contact us to briefly discuss your project and determine how our capabilities can meet your needs ( Contact ICT , 1-800-829-3194) .
4. Request a confidentiality agreement (which we can email - fax - mail to you). Review, sign, and send it back to us, or send us your confidentiality agreement for us to review.
5. Discuss the project with us in further detail so we can prepare a reliable quote.
6. Accept the quote, pay the front fee, and we’ll get started as soon as possible!  

Questionnaire