Isotype Specific Monoclonal Secondary Antibodies

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Overview

Secondary antibodies are key components of the detection system; the selection of an optimum secondary antibody can improve the positive signal in addition to reducing false positive or negative staining.

Recombinant Monoclonal Antibodies 
- the Solution to Cross-Reactivity in Multiplexing

Recombinant monoclonal antibodies directed against the three main mouse isotypes, IgG1, IgG2a, and IgG2b are an alternative to cross-adsorbed secondary antibodies. They are capable of detecting individual isotypes without any species or isotype cross-reactivity, enabling multiple unlabeled mouse monoclonal antibodies to be used simultaneously. Multiplexing without species issues is therefore straightforward and an alternative species does not need to be sourced.

Click here to learn more about multiplexing using secondary antibodies.

Read our overview on multiplex fluorescent western blotting to discover how it can benefit you.

Isotype Specific Secondary Antibodies Video

Watch our new video which demonstrates practically how using Bio-Rad’s mouse IgG isotype specific secondary antibodies can deliver results you can trust in imaging multiplexing, without the risk of false positives, and in only two simple steps.

Benefits of isotype specific secondary antibodies:

1. Advantages of recombinant monoclonal secondary antibodies over polyclonals:

• Polyclonal isotype specific antisera are produced by depletion of reactivity to an undesired target which is not 100% efficient. Whereas recombinant human monoclonal antibodies are selected against epitopes unique to the isotype
• Polyclonal antibodies can show variation between batches; recombinant monoclonal antibodies do not

2. High specificity: the signal detected by each secondary is specific to each primary.
3. Minimum nonspecific background: isotype specific secondary antibodies do not bind to non-target IgG.
4. Suitable for multiplexing: no binding to non-target primary antibodies in a multiplex experiment.
5. Easy-to-use: directly substitute for any secondary, no special protocol or extra steps required
6. Versatile: can be used in IHC, IF, flow cytometry, and western blotting.

Isotype specific secondary antibodies in different applications:

• Western Blotting
• Imaging – IHC and IF
• Flow Cytometry
• ELISA

Western Blotting

In a western blot experiment, detection of multiple targets can be achieved using isotype specific secondary antibodies, as directly labeled primary antibodies may not be bright enough.

The western blot image below shows the specific detection of the target antibody by the three isotype specific antibodies in lanes 4, 6 and 8 compared to lane 2 showing the presence of all three antibodies:

• IgG1 isotype detection
  • Human Anti-Mouse DyLight 488 (HCA309D488), clone AbD24121 recognizes the IgG1 isotype of Mouse Anti-Actin Beta (VMA00048), 42 kDA molecular weight, as seen in lane 4. AbD24121 does not recognize Mouse Anti-PCNA IgG2a or Mouse Anti-Ezrin IgG2b
• IgG2a isotype detection
  • Human Anti-Mouse DyLight 650 (HCA310D650), clone AbD24124 recognizes the IgG2a Isotype of Mouse Anti-PCNA (VMA00018), 29 kDA molecular weight as seen in lane 6. AbD24124 does not recognize Mouse Anti-Actin Beta IgG1 or Mouse Anti-Ezrin IgG2b
• IgG2b isotype detection
  • Human Anti-Mouse DyLight 550 (HCA309D488), clone AbD24127 recognizes the IgG2b isotype of Mouse Anti-Ezrin (VMA00344), 80 kDA molecular weight as seen in lane 8. AbD24127 does not recognize Mouse Anti-Actin Beta IgG1 or Mouse Anti-PCNA IgG2a

Imaging – IHC and IF

The three isotype specific secondary antibodies are designed for use in multiplex imaging:

• Highly specific: detect monoclonal antibodies from the same species and detect three different antigens/antibodies with no cross-reactivity
• Versatile: further increase your multiplexing capabilities by combining isotype specific and species specific secondary antibodies. Remove the need to use different species for subsequent antibody identification

In the five images below you can see the highly specific fluorescent staining of all three isotype specific secondary antibodies.

Flow Cytometry

The use of secondary antibodies in flow cytometry is not recommended; it is preferable to use directly conjugated antibodies when multiplexing. However this may not be possible if:

• A conjugate is not available
• Or,  if the antibody cannot be conjugated because:
  • It is impure or only partially pure e.g. tissue culture supernatant
  • It contains carrier protein
  • It is too dilute
  • It is in insufficient quantity

Indirect staining involves careful optimization of your experiment, to avoid nonspecific binding. To help with this, Bio-Rad has created a how-to guide so you can achieve the best specific flow staining using secondary detection reagents.

The images below show specificity of the secondary antibodies in both surface and intracellular staining.

ELISA

The isotype specific secondary antibodies are ideal for use in ELISA as illustrated below.

Isotype Specific Secondary Antibody Range