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Frequently Asked Questions about Antibody

What Is Recombinant Antibody?

Recombinant antibodies are antibody fragments produced in vitro by using synthetic genes which encode antibody heavy and light chains. These genes are introduced into expression systems (e.g. E. coli, yeast, insect cell line, mammalian cell or even transgenic plant). In order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns, almost therapeutic antibodies are still produced in mammalian cell lines. The recombinant antibody is high affinity, high consistent and can be used wherever you would normally use a traditional monoclonal antibody. For more information about our recombinant antibody, please see Recombinant Antibody Production Platform.

What Is the Difference Between Primary Antibody, Secondary Antibody and Isotype Control?

Primary antibody directly binds to a particular antigen of research interest and is useful for detecting protein expression, location and modification. Secondary antibody is raised against the host species of primary antibody and provide signal detection and amplification, which is usually labeled with fluorescent dyes or enzymes. Isotype control is an antibody that lacks specificity to the target, but match the primary antibody isotype. It is used as a negative control to help differentiate non-specific background signal from specific antibody signal.

How to Choose A Primary Antibody?

A right antibody is very important for experiment success. Some points should be noticed when choosing a primary antibody.

  • Specificity

Antibodies were firstly generated by immunizing host animals with an immunogenic substance, immunogen. These immunogens can be full-length proteins, fragments, peptides, whole organisms or cells. The immunogen of selected antibody should be identical to or contained the protein region that you want to detect.

  • Species Reactivity

Our antibody datasheets list the species reactivity except the non-species dependent antibodies. The species reactivity of selected antibody should be the same as your target protein species or share sufficient amino acid sequence homology.

  • Application

Our antibody datasheets list the applications we have tested or published in paper. If an application is not listed, it means that antibody is not appropriate for the application or has not been validated. Confirm your experiment and choose right antibody.

  • Host

It is better to choose a primary antibody of which species is different from your sample. This is to avoid cross-reactivity of the secondary antibody with endogenous immunoglobulins in the sample.

For more information, please see Primary Antibody.

How to Choose A Secondary Antibody?

Secondary antibody is directed against the host species and binds with primary antibody. if your primary antibody was raised in mouse, you will need an anti-mouse secondary antibody but the host of secondary antibody better not is mouse. Also, the secondary antibody has to be directed against the isotype of primary antibody. For example, if the primary monoclonal antibody is a mouse IgG2a, you will need an anti-mouse IgG2a secondary antibody. If the subclass of primary antibody is not known, the anti-mouse IgG Fab secondary antibody may be used because they recognize most mouse immunoglobulin subclasses.

Summary of immunoglobulin classes and subclasses:

  • Classes or Isotypes: IgG, IgM, IgA, IgE, IgD

Human IgG Subclasses: IgG1, IgG2, IgG3, IgG4
Mouse IgG Subclasses: IgG1, IgG2a, IgG2b, IgG3

  • Light Chain Classes: Kappa (κ), Lambda (λ)

Subclasses: λ1, λ2, λ3, λ4

  • Heavy Chain Classes: IgG (γ), IgM (μ), IgA (α), IgD (ε), IgE (δ)

For more information, please see Secondary Antibody.

How to Choose An Isotype Control?

Isotype control is used to confirm primary antibody specificity and non-specific binding. Therefore, the isotype control antibody should match host species, isotype, and conjugation of the primary antibody. For instance, if the primary antibody is an HRP-conjugated human IgG1, you will need an HRP-conjugated human IgG1 isotype control. Isotype control is not suitable for polyclonal primary antibody which contains more than one IgG subclass.

For more information, please see Isotype Control.

Our innovative antibody production team ensures emerging key specificities will be frequently added to our current catalog. Creative Biolabs owns comprehensive antibody products and related services. We are always ready to offer you the best products and services to help promote your projects. For more details about our antibodies, please feel free to contact us for assistance.

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