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CD ELISA Matched Pair Development

Matched pairs are the basis of many sandwich ELISAs, either in kits or for in house assay set up. The name refers to sets of antibodies which are known to be capable of detecting different epitopes on the same protein antigen, so they can be used together for the capture and detection of a single antigen in a sandwich ELISA or related immunoassay. Matched pairs can consist of two monoclonals, two polyclonals, or a combination of both. 

What we can do

Creative Diagnostics can develop the most suitable matched pairs for your ELISA assay. Projects can be initiated in conjunction with our custom hybridoma development services or antibodies can be supplied by customers for evaluation. We utilize the expertise of our own internal ELISA development team which supports a variety of ELISA products.

Antibody Testing:
ELISA assay/s are run to determine best antibody pair.
• Plates are coated with antibody and antigen to be tested.
• By the epitope mapping, each coating antibody is tested with antibody candidates. 
• A polyclonal Abs for the antigen of interest (if available) can be used as a coating or detection option.
• A 5-7 point standard curve of the antigen is typically used.
• A typical standard curve range is 6.8pg/ml - 5000pg/ml, plus a blank.
•Best pair/s is/are determined based on signal intensity, sensitivity, background and S/N.


ELISA optimization

• Optimizing capture antibody concentration
• Optimizing the blocking buffer
• Optimizing the standard diluent
• Optimizing sample concentration
• Optimizing the detection antibody concentration
• Optimizing the enzyme conjugate concentration
• Optimizing signal detection

 

Expertise

Ready to Use Antibody Pairs for ELISA

Creative Diagnostics provides a turnkey solution for quantitative measurement of your protein of interest. Leveraging our tested antibody pairs, Creative Diagnostics allows you to focus on more important parts of your research instead of the arduous task of searching and testing for the matching antibody components.
Search our collection of validated matched antibody pairs.

Antibody considerations

The antibodies used in ELISA assays can be monoclonal, polyclonal, or a combination of both. Each antibody type offers distinct advantages in the development of ELISAs, so it is important to appreciate the differences between them and how these can be used to advantage during ELISA development.

Monoclonal antibodies are homogeneous by definition, with specificity for a single epitope or small region of a protein. As a result, they are less likely to interact with closely-related proteins and are not generally expected to trigger non-specific signals in a given immunoassay.

Monoclonal antibodies can be used for all antibody-containing steps in all types of ELISAs. They are commonly used in sets as matched pairs in sandwich ELISAs, but can be used for capture or detection in conjunction with a polyclonal antibody to enhance signal or to provide a greater chance of capturing antigen from a complex solution.

Polyclonal antibodies are complex antibody pools which represent a collection of specificities to various epitopes found in a single antigen. Some epitopes predominate or there may be wide representation of the epitopes available in any given antigen. Polyclonals can very significantly from batch to batch, and must be tested and validated thoroughly.

As a result of their heterogeneity and the wide representation of epitopes present, polyclonal antibodies can be powerful tools for the thorough detection of an antigen, often yielding higher signal levels. It is also rare that they will fail to bind due to a single blocked antibody binding site, antigen configuration change, or misfolding. However, polyclonals are also more likely to share one or more epitopes with closely-related proteins, resulting in higher non-specific signal. One solution to reduce this problem is to use affinity purified or cross-absorbed polyclonal antibodies.

Sometimes the detection method for an ELISA is switched from direct to indirect detection, and thus from a monoclonal to a polyclonal in order to increase assay sensitivity due to higher levels of polyclonal antibody binding to the target antigen.

Creative Diagnostics always do our best to offer optimized ELISA reagent formats and services for your quantitation request.

Please feel free to contact us if you have any questions regarding our service.

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