HuCAL® Antibodies Technical Manual

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The Human Combinatorial Antibody Library (HuCAL) is one of the most powerful synthetic antibody libraries ever created. It is a highly sophisticated technology that is widely used in basic research, target validation, bioanalytical assay development and for analysis of novel proteins. Using the HuCAL technology, we provide a custom service for the development of novel antibody specificities for research and diagnostic applications.

Discover the benefits of this cutting-edge in vitro technology through the pages of our HuCAL antibodies technical manual, which describes the HuCAL technology and its advantages over conventional antibody production methods, and provides resources and protocols for using HuCAL antibodies in a range of applications, including flow cytometry, immunofluorescence and ELISA.

Introduction to Recombinant Antibodies

Recombinant antibody technology, such as HuCAL, is a rapidly evolving field with a number of major benefits over conventional antibody generation methods. This chapter provides an overview of recombinant antibody technology and outlines the methods used for generating recombinant antibodies, their advantages and important considerations for their use.

HuCAL Technology

HuCAL is designed to represent the essential features of the natural human antibody repertoire. In this chapter we describe how the HuCAL library was generated, its benefits and its applications. We present the process for generating HuCAL antibodies as well as the guided selection strategies for identifying antibodies to certain epitopes or epitope areas.

Applications: ELISA

All HuCAL antibodies are routinely tested by indirect ELISA and can be used in all other types of ELISA. In addition to indirect ELISA, in this chapter we provide standardized protocols for the use of HuCAL antibodies in direct, sandwich, bridging (ADA and PK), and competition/inhibition ELISA. We also provide sources for the recommended antibodies and reagents used in the protocols described and a troubleshooting guide to ensure optimal results.

Applications: Flow Cytometry

HuCAL antibodies are well-suited for use in flow cytometry primarily because of their small size and lack of an Fc region. This chapter provides a standardized protocol for flow cytometry using HuCAL antibodies, including appropriate cell preparation techniques based on cell type, recommended controls and examples of our data analysis. A troubleshooting guide and the source of antibodies and reagents used in the suggested protocol are also provided.

Applications: Immunofluorescence

The high specificity of HuCAL antibodies makes them ideal for immunofluorescence analysis. Furthermore, using bivalent Fab formats for immunofluorescence improves target recognition due to avidity effects. Accordingly, both bivalent and full-length HuCAL antibody formats can be used in immunofluorescence assays in the same manner as conventional antibodies. This chapter provides detailed immunofluorescence protocols as well as examples of data generated using HuCAL antibodies.

Applications: Immunoprecipitation

In this chapter we highlight methods and protocols for immunoprecipitation using HuCAL generated antibodies. For antibodies in full immunoglobulin format we recommend Bio-Rad SureBeads™, Protein A or Protein G Magnetic Beads. We provide alternative instructions for Fab format antibodies, which require a different strategy since they lack the Fc region. Example data and troubleshooting guidance are provided.

Applications: Western Blotting

HuCAL antibodies can be used for western blotting following the same general protocols used for conventional antibodies. The selection of the secondary antibody for the experiment needs additional attention because HuCAL antibodies are fully human in sequence, and Fab format antibodies lack the Fc region. This chapter provides a general protocol and experimental tips, plus a list of recommended HRP conjugated secondary antibodies, including those specifically for use with the different epitope tags available on HuCAL Fab format antibodies.

Measuring antibody affinity is a useful tool for characterizing, evaluating and ranking antibodies, and provides information to aid assay development and optimization. Bio-Rad uses bio-layer interferometry to carry out real time, label-free affinity determination and off-rate ranking for HuCAL generated antibodies. The protocols we work with are described in this chapter.

Antibody Conjugation

For applications such as immunofluorescence and flow cytometry, using a conjugated primary antibody reduces the risk of non-specific background staining from cross-reactive secondary antibodies. HuCAL primary antibodies can be directly conjugated to biotin or various fluorescent dyes in the same manner as traditional antibodies. This chapter provides detailed protocols for the conjugation of HRP, fluorescent dyes or biotin to HuCAL antibodies including buffer recipes, recommended sources for the reagents used and a troubleshooting guide.

Choosing the Best HuCAL Antibody Format

Selection of the optimal antibody format is an important step in HuCAL antibody generation projects, and is necessary for ensuring success in your desired research application. This chapter provides recommended antibody formats for use in a wide range of applications, as well as details on the composition and molecular weights of the various epitope tags.

Reagents to Support HuCAL Assay Development

HuCAL antibodies can be used in a wide range of immunoassays such as western blotting, immunohistochemistry, ELISA and flow cytometry. In this chapter, we provide a collection of reagents that have been tested and qualified in-house for use with HuCAL antibodies. These include secondary antibodies of various specificities, negative control antibodies and kits for antibody conjugation.

An Introduction to HuCAL Technology

Order your printed copy of An Introduction to HuCAL Technology, which combines the first two sections of the Technical Manual (Introduction to Recombinant Antibodies and HuCAL Technology).

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