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.

Cross-Adsorbed Antibodies

Issues with species and isotype cross-reactivity are often overcome by the use of cross-adsorbed secondary antibodies. Here, unwanted cross-reactivity is removed by pre-adsorption of the secondary antibody with the cross-reacting antigen, to yield a more specific secondary and therefore reducing nonspecific background staining. Read our overview on cross-adsorbed secondary antibodies to discover why and how you should use them, how they are generated, and a list of the cross-adsorbed secondary antibodies available.


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.

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

Benefits of multiplexing with isotype specific secondary antibodies illustrated by imaging

Benefits of multiplexing with isotype specific secondary antibodies illustrated by imaging



Isotype specific secondary antibodies in different applications:


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

Fluorescent western blotting using isotype specific secondary antibodies.

Fluorescent western blotting using isotype specific secondary antibodies.

HeLa lysate (25 µg) was run in lanes 2, 4, 6, and 8. Detection was carried out as follows:

Mouse IgG1 Anti-Actin Beta (VMA00048) at 0.5 µg/ml was used in lanes 2 and 4, followed by IgG1 specific Human Anti-Mouse Dylight 488 (HCA309D488) at 0.3 µg/ml (green).

Mouse IgG2a Anti-PCNA (VMA00018) at 0.5 µg/ml was used in lanes 2 and 6, followed by IgG2a specific Human Anti-Mouse Dylight 650 (HCA310D650) 0.3 µg/ml (blue).

Mouse IgG2b Anti-Ezrin (VMA00344) at 0.5 µg/ml was used in lanes 2 and 8, followed by IgG2b specific Human Anti-Mouse Dylight 550 (HCA311D550) 0.3 µg/ml (red).

Visualization with the ChemiDocMP Imaging System.
 

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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.

Chromogenic staining of human colon using isotype specific secondary antibodies.

Chromogenic staining of human colon using isotype specific secondary antibodies.

FFPE human colon adenocarcinoma stained with Mouse Anti-Cytokeratin 18 (MCA1864) and Human Anti-Mouse IgG1 (HCA309HRP) (brown) and Mouse Anti-PCNA (MCA1558) and Human Anti-Mouse IgG2a HCA310AP (red).
 


Fluorescent staining of human colon using isotype specific secondary antibodies.

Fluorescent staining of human colon using isotype specific secondary antibodies.

FFPE human colon adenocarcinoma stained with Mouse Anti-Cytokeratin 18 (MCA1864) and Human Anti-Mouse IgG1 (HCA309D488) (green) and Mouse Anti-PCNA (MCA1558) and Human Anti-Mouse IgG2a (HCA310D550) (red).
 


Fluorescent staining of human colon using isotype specific secondary antibodies.

Fluorescent staining of human tonsil using isotype specific secondary antibodies.

FFPE human tonsil stained with Mouse Anti-Macrophages/Histiocytes (MCA1478) and Human Anti-Mouse IgG2b (HCA311D550) (red) and Mouse Anti-PCNA (MCA1558) and Human Anti-Mouse IgG2a (HCA310D650) (green).
 


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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

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

Surface staining using fluorescently conjugated secondary antibodies.

Surface staining using fluorescently conjugated secondary antibodies.

Mouse Anti-Human CD3 (MCA463) labeled with Human Anti-Mouse DyLight 488 (HCA309D488) specific for mouse primary IgG1 and Mouse Anti-Human CD20 (MCA1710) labeled with HuCAL® Anti-Mouse DyLight 650 (HCA311D650) specific for mouse primary IgG2b.

All experiments performed on red cell lysed human blood gated on lymphocytes in the presence of 10% human serum. Data acquired on the ZE5 Cell Analyzer.
 


Intracellular staining using fluorescently conjugated secondary antibodies.

Intracellular staining using fluorescently conjugated secondary antibodies.

Mouse Anti-Human IFN Gamma (MCA1581) labeled with Human Anti-Mouse DyLight 488 (HCA309D488) specific for mouse primary IgG1 and Mouse Anti-Human CD3 (MCA2184) labeled with HuCAL Anti-Mouse PE (HCA310PE) specific for mouse primary IgG2a.

All experiments performed on red cell lysed human blood, fixed and permeabilized with Leucoperm (BUF09), gated on lymphocytes in the presence of 10% human serum. Data acquired on the ZE5 Cell Analyzer.

 

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ELISA

The isotype specific secondary antibodies are ideal for use in ELISA as illustrated in the images below, which clearly show the isotype specificity of all three antibodies.

ELISA detection of IgG1 only using 22isotype specific secondary antibodies

ELISA detection of IgG1 using isotype specific secondary antibodies.

Mouse IgG1, IgG2a, IgG2b and BSA were used to coat duplicated rows on an ELISA plate which was then probed with Human Anti-Mouse IgG1 AP, clone AbD24121, (HCA309A) at doubling serial dilutions. HCA309A only recognizes mouse IgG1, not IgG2a or IgG2b.
 


ELISA detection of IgG2a only using isotype specific secondary antibodies.

ELISA detection of IgG2a using isotype specific secondary antibodies.

Mouse IgG1, IgG2a, IgG2b and BSA were used to coat duplicated rows on an ELISA plate which was then probed with Human Anti-Mouse IgG2a AP, clone AbD24124, (HCA310A) at doubling serial dilutions. HCA310A only recognizes mouse IgG2a, not IgG1 or IgG2b.
 


ELISA detection of IgG2b only using isotype specific secondary antibodies.

ELISA detection of IgG2b using isotype specific secondary antibodies.

Mouse IgG1, IgG2a, IgG2b, and BSA were used to coat duplicated rows on an ELISA plate which was then probed with Human Anti-Mouse IgG2b AP, clone AbD24127, (HCA311A) at doubling serial dilutions. HCA311A only recognizes mouse IgG2b, not IgG1 or IgG2a.
 


Isotype Specific Secondary Antibody Range
 

Human Anti-Mouse IgG1

 

Human Anti-Mouse IgG2a

 

Human Anti-Mouse IgG2b

 
Format Catalog Number Format Catalog Number Format Catalog Number
DyLight 488 HCA309D488 DyLight 650 HCA310D650 DyLight 550 HCA311D550
DyLight 550 HCA309D550 PE HCA310PE AP HCA311A
DyLight 650 HCA309D650 AP HCA310A HRP HCA311P
PE HCA309PE HRP HCA310P Dylight 650 HCA311D650
HRP HCA309P DyLight 550 HCA310D550 PE HCA311PE
Alkaline phosphatase HCA309A        

Optimize your Secondary Antibody Detection System Protocols and Resources

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