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HCP Assay Development


 

HCP Assay Development Overview

 

After polyclonal antibody generation, Rockland performs assay development for clients who choose to add that service.  Assay development is customized to each individual client. Client can decide to stop the project at the end of multiple project phases.  Below is an example of an assay development project for a capture sandwich ELISA that was performed recently by Rockland Immunochemicals.  
 
Assay development Step I:  Assay Configuration using Soluble Extract diluted in PBS.
 

  • In this project the same antibody may be used unconjugated as the capture antibody and either biotin orperoxidase conjugated as the detection antibody  

 

  • Peroxidase conjugation of purified antibody and reagent qualification by ELISA and Western Blotting 

 

  • Optimize capture antibody concentration / optimize concentration of detection antibody

 

  • Develop assay standards for ELISA

 

  • Assess optimized reagents for detection of the HCP extract in phosphate buffered saline (PBS) 

 

  • Assess antibody combinations using the HCP soluble extract in dilute buffer (containing BSA or similarblocking agent) to determine initial working dilution ranges of antibody and antigen.

 

Conditions varied to obtain optimum results:

 

 

Concentration

Time    

Temperature

Blocking

Buffer    

Detergents    

Ionic Strength

Conjugate Reporter

 

 

Assay development Step II:  Limit of Detection (LOD) and Assay Validation

  • Optimized antibodies (PAb-PAbpair) as determined in Step I will be applied to micro titer plates todetermine reactive to proteins at concentrations in the predetermined detectionrange. Alternative labeling approaches, such as the use of biotin/streptavidin, luminescent substrates, and fluorescent substrates are available.

 

  • Analysis of Matrix effects

 

  • Analysis of assay accuracy and precision

 

  • Antigen Limit of Detection (LOD) of configured assay

 

  • Full protocol and reporting are included for HCP assay development projects


This assay development strategy is but one example of the work that we have performed at Rockland. The client has the ability to manage the project which includes several defined go/stop decision points.    We can do as little or as much as the client needs, and we frequently work directly with client laboratories to complete the project under strict timelines.  Please inquire if you have further questions about assay development.
 
We have completed HCP antibody reagent generation for more than 12 years and have compelling data to support our established procedures.  With all of our projects we provide documentation and traceability to satisfy FDA requirements.

 

HCP Basic Schematic

 

Related Links:

 

Host Cell Protein


 

 Standard HCP Reagent Development


 

 Cascade Antibody Generation


 

 1D Analysis


 

 2D Analysis