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Microtitre ELISA plate

Immunoassay and ELISA Optimisation

Once a prototype assay format has been selected an extensive optimisation program will typically follow, aiming to achieve the product performance requirements identified at the start of the project. There are many variables that can be modified to give you the best possible assay, including:

  • Antibody and conjugate concentrations

  • Buffers, diluents and additives

  • Conjugate incorporation levels

  • Number of assay steps 

  • Wash protocols

  • Mixing conditions

  • Times and temperatures


Basic assay performance targets such as range, sensitivity and precision can often be achieved quite quickly, but Fleet’s expertise will ensure that other critical factors are adequately addressed. These may include:

  • Specificity vs. potential cross-reactants

  • Resistance to interfering factors

  • Robustness to assay, process and raw material variables

  • Sample dilution constraints

  • Initial stability information


Without careful attention to such factors, it is easy for a superficially acceptable assay design to go “off the rails” later in the program, wasting valuable time and resources! For additional information, see our blog post "8 critical tips to consider when developing and optimising an immunoassay or ELISA."


Once optimisation is complete, a design review will confirm that performance meets the customer’s requirements and that any conflicts between design input and output have been satisfactorily resolved – typically paving the way for formal performance verification studies.


Sometimes however the development program may stop at this point – for example where the overall aim is to develop your “bench” assay to the point where it will attract buyers, collaborators or investors. Such an exercise is far more likely to succeed with a well-optimised assay design. Data presentation is important too: with our industry background we know what real diagnostics players are looking for at this stage – and that it isn’t always what they’re given! You’ll probably only get one chance to put your data in front of the right people, and Fleet can help make sure you don’t blow it…

Automated analyser
Quality by design
Design of experiments

Example Projects

Optimisation of a range of allergen assays

Having optimised the generic allergy assay format for an automated analyser, Fleet went on to optimise over fifty individual assays for the platform. These assays have since been successfully launched globally.

Optimisation of auto-antibody ELISA panel with complex and demanding requirements

A panel of auto-antibody assays were developed at Fleet. These assays required careful optimisation to ensure that an equivalent response and sensitivity was seen across the entire panel. It was also necessary to overcome significant non-specific binding issues.

Having accomplished these aims, the resulting assay revealed differences between geographic populations which required extension of the panel with additional antigens. These further assays needed to be optimised to match the initial panel.

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