The Importance of Using an Isotype Control

The Importance ofUsing an Isotype ControlWhite Paper

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What are Isotype Controls?Isotype control antibodies are essential negative controls for in vivo studies and can play an important role in standard immunoassays, such as flow cytometry and immunohistochemistry (IHC). They match the characteristics of the assay’s primary antibody (e.g. isotype, clonality), but having been raised against antigens known not to be present in common preclinical species (HEL, KLH, etc.), have no target cell/antigen specificity. This provides researchers with a suitable negative control to accurately discriminate between an antibody binding in an antigen-dependent specific manner from non-antigen dependent mAb binding due to Fc receptors (FcR) or other proteins. In the simplest terms, isotype controls help distinguish non-specific background signal from specific antibody signal.

Monoclonal, subclass specific (i.e. lambda vs. kappa immunoglobulin) isotype controls provide an especially reliable method that effectively differentiates between specificity versus background in a range of drug development assays.

Why is it Important to Use Isotype Controls?Structurally, all antibodies are very similar, with only a small hypervariable region determining antigen specific binding. The rest of the antibody molecule consists of the constant region, where sequences are often shared by antibodies of the same isotype.

The constant regions help determine antibody mechanism of action, by coordinating binding to the FcR present on many cell types. FcRs are expressed on a number of different cell types in the immune system, including (but not limited to):

• B lymphocytes • follicular dendritic cells • natural killer cells • macrophages • neutrophils • eosinophils • basophils • human platelets • mast cells.

The non-specific binding of antibodies to other proteins is affected by many factors, including:

• high concentration • charge • hydrophobicity.

Using an appropriate isotype control allows the true determination of specific vs non-antigen specific binding across a range of in vivo studies and Immunoassays

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Why PBS is not a Suitable Substitute for an Isotype ControlWhile the use of PBS instead of an isotype control antibody as a negative control is not ideal, it does occur in published research. Using this non-optimal control factor can result in a range of issues during efficacy studies and immunoassays.

1. Use of PBS in Efficacy Experiments Fails to Simulate the Effects Caused by Widespread FcR Engagement

Using PBS as a simplified negative control in a range of in vivo efficacy assays can result in inaccurate interpretation of results, due to performance differences observed between PBS and the isotype control. Figure 2 compares growth of the CT-26.WT syngeneic colon cancer model in the presence of PBS or rat IgG2a isotype control, with a clear difference observed between growth rates for the differing control agents.

Figure 1: Matching Primary Antibody Host, Isotype, Conjugation, Light Chain, and Concentration are Key Factors in Choosing an Isotype Control

ConcentrationConjugation

Host Isotype

Light Chain

By matching a range of primary antibody features, isotype controls provide an ideal negative control for precise data analysis

How to Choose an Appropriate Isotype ControlWhen choosing an appropriate isotype control, matching the characteristics of the primary antibody is key for accurate interpretation of study results. The main factors to consider when choosing the appropriate isotype control antibody for a study are host, isotype, conjugation, light chain, and concentration (Figure 1).

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Figure 2: Performance Difference between PBS and Isotype Negative Controls in Syngeneic Tumor Studies

Modest tumor inhibition can be observed with hIgG1 isotype control (Figure 3; shown for the H22 syngeneic liver cancer model). This helps to illustrate that the inhibitory effect on tumor growth of a corresponding target antibody must be compared with that of the hIgG1 isotype control, not PBS, for a correct calculation of tumor growth inhibition and a more accurate measurement of drug efficacy.

Using PBS in place of isotype controls isnot advised - performance differences can result in

incorrect data interpretation in in vivo studies

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Figure 3: Tumor Growth Inhibition Induced by hIgG1 compared with PBS

The importance of comparison with the correct control agent is also exemplified in the Collagen Antibody-Induced Arthritis (CAIA) model shown in Figure 4. In this model, rheumatoid arthritis in BALB/c mice is induced through the administration of an athrogenic antibody followed by LPS, which can then be treated with potential new agents.

In Figure 4, the model is treated with dexamethasone (with PBS control) or an anti-TNF mAb (with IgG1 isotype control). The PBS and isotype controls show a clear performance difference, which would lead to erroneous interpretation of anti-TNF mAb statistics if compared instead with a PBS control.


Figure 4: Performance Difference between PBS and Isotype Negative Controls in CAIA Model

Arthritis Index ScoringScoring occurs per paw suchthat the total score is 16.

0 = Normal paw

1 = One toe inflamed or swollen

2 = More than one toe but not entire paw

3 = Entire paw inflamed & swollen

4 = Very inflamed and swollen paw or ankylosed paw

2. PBS Use in Flow Cytometry Experiments Does Not Mimic FcR or Protein Staining

In flow cytometry experiments, antibody binding to FcRs or other proteins can cause non-specific staining in cell types, dependent upon FcR expression. This can appear deceptively specific due to the variation in FcR expression in different tissues. Therefore, it may be useful to have matched isotype controls which can mimic this staining, rather than use a non-binding PBS control.

As a result of this, in order to be useful in flow cytometry, isotype controls should ideally be the same species, heavy chain (IgA, IgG, IgD, IgE, or IgM), and light chain (kappa or lambda) class. For directly conjugated antibodies, isotype controls should be conjugated to the same fluorochrome at the same labelling ratio, such that an identical number of fluorescent molecules is conjugated to each antibody.

This is also referred to as the F:P ratio. Due to this level of complexity, researchers have different preferences regarding inclusion of isotype controls for flow cytometry. Differentially conjugated isotype controls can lead to misleading results.

3. Isotype Control Antibodies can Induce Differences in Tumor Infiltrating Lymphocyte Populations

Treatment with isotype control antibodies can result in differences in tumor infiltrating lymphocyte populations, which is not observed with PBS use. This is distinct from the staining controls in flow cytometry discussed above, as animals are dosed with the isotype control, and represents an actual change in immune cell representation within the tumor itself.

Matched isotype controls can mimic antibody staining in flow cytometry experiments; however, complete matching can prove complex

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Figure 5 displays this occurrence for the MC38 syngeneic colon cancer model. By Day 13, treatment with isotype control, at 10mg/kg twice per week, causes an increase in CD3+ T cells, which is not observed with PBS treatment. In contrast, this effect is not seen in CT-26.WT syngeneic tumors using the same isotype control, suggesting it is highly model dependent. Therefore, these data, utilizing the correct control are critical for understanding the full response of the anti-PD-1 treated tumors.

Using a secondary antibody alone as a control instead of an isotype control antibody is disadvantageous as the secondary antibody binds directly to the sample, rather than to the Fc fragment of a primary antibody or isotype control. The secondary could also have altered binding properties in isolation compared to binding with the appropriate isotype control. Instead, ideally, the negative control used for IHC should mimic the exact concentration and isotype of the primary antibody, which is why an isotype control is the most appropriate choice. Depending upon the tissue, the antibody isotype could cause a noticeable difference due to the potentially significant interaction with resident FcRs present in different tissue sections as demonstrated in Table 1.

Figure 5: Isotype Controls Induce TIL Differences for Specific Time Points and Models

Immunohistochemistry Experiments also Require Appropriate Isotype ControlsIsotype controls are also an ideal negative control for IHC assays. If isotype controls are not used, then they are usually swapped out for one of two options:

1. Directly staining with a secondary antibody

2. Staining with a polyclonal pool.

Isotype controls can induce TIL changes, making their use essential to understanding therapeutic antibody response data

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Table 1: Binding Preferences of Mouse, Rat, and Human IgG Isotypes(1)

Affinity (KA)

MouseIgG mFcγRI mFcγRII mFcγRIII mFcγRIV

IgG1 No bind ++ ++ No bind

IgG2a +++ + ++ +++

IgG2b ND ++ + +++

IgG3 ND No bind No bind No bind

Rat IgG2a No bind ++ + +

Human IgG FcγRI FcγRIIa FcγRIIb/c FcγRIIIa FcγRIIIB

IgG1 +++ ++ + ++ +

IgG2 No bind + + + No bind

IgG3 +++ + + ++ ++

IgG4 +++ + + + No bind

Figure 6 shows the differences observed between staining with mouse isotype controls IgG1 or IgG2 and a secondary antibody alone, when imaging CD138 and FGFR3 in the human tonsil. The isotype controls provide a more accurate control than using a secondary antibody alone and can provide confidence that the staining observed with the primary antibody is specific.

In IHC, isotype controls provide a more accurate negative control than staining with secondary only or a polyclonal pool due to

consistent binding properties with the primary antibody

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Figure 6: Isotype Controls Provide a More Accurate Negative Control in IHC Studies

CD138 staining: FFPE human tonsil stained with CD138 antibody, mouse IgG1, kappa isotype control, or secondary antibody only (20x magnification). FGFR3 staining: FFPE human tonsil stained with FGFR3 antibody, mouse IgG2a, kappa isotype control, or secondary antibody only (20x magnification)

CD138

Human tonsil

Human tonsil

FGFR3

Secondary Ab only

Secondary Ab only

Isotype control (Ms IgG1)

Isotype control (Ms IgG2a)

Similarly, using a polyclonal pool will also have altered binding properties compared to using a pure mAb. When using a monoclonal primary antibody, a monoclonal isotype control with matching isotype and light chain is ideal. Using a polyclonal mixture of immunoglobulins may lead to extra background or non-specific staining as the multiple isotypes contained in polyclonal mixtures may bind to additional targets. This is exemplified in Table 1, which details the binding preferences observed for different human, mouse, and rat isotypes. Polyclonal pool mixtures may contain variable amounts of any or all of these isotypes, dependent upon the donor.

Isotype control antibodies can also serve as a blocking or coating reagent in other immunoassays including IHC, which may help to minimize non-specific FcR mediated binding with a primary antibody. Other assays where an isotype control antibody could be used as a blocking agent include Western blots and ELISAs.

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SummaryIsotype controls are essential tools in a range of in vivo efficacy and immunoassays, providing appropriate negative controls for accurate measurement of antibody drug effects. The substitution of other agents such as PBS for isotype controls causes a range of issues which can hinder study performance and contribute to erroneous results. The use of high quality isotype controls provides a reliable method to accurately differentiate specificity versus background for precision drug development assays.

References1Vonderheide and Glennie. Agonistic CD40 antibodies and cancer therapy. Clinical Cancer Research 2013;19(5): 1035-43.

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CrownBio’s Isotype Controls are an Ideal Reagent Choice for your StudiesCrownBio provides a range of isotype controls, suitable for in vivo studies and immunoassays. Our controls have been carefully developed, providing a range of features, including:

• Primary antibody concentration (our isotype controls are supplied at 1-5mg/ml to match antibody drug concentrations)

• No carriers or additives - supplied in PBS only

• No cross reactivity with the species you are working with

• Very low endotoxin and high purity to ensure animal safety.

Specifically designed and well validated isotypecontrols provide a range of features and preferential

qualities over standard isotype controls

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The Importance of Using an Isotype Control