An isotype control antibody serves as a negative control in antibody-based detection methods, such as flow cytometry, immunohistochemistry or ELISA. It has the same type, host species, class and conjugation as the primary antibody but lacks specificity for the target antigen. This difference makes isotype controls ideal for differentiating between signals from non-specific background binding and specific antigen-antibody interaction.¹

Isotype controls are critical for validating antibody specificity and troubleshooting potential causes of background signals from primary antibodies, such as Fc receptor binding and autofluorescence. By comparing the signal from the specific antibody to that from the isotype control, researchers can improve the reliability of their antibody-based assays for biomedical applications.¹

What Is an Isotype Control Antibody?

Isotype control antibodies are non-specific immunoglobulins that share the same class, species origin and label (e.g., fluorochrome or enzyme) as the primary antibody used in experiments.²

Target antibodies are designed to recognize a specific antigen or epitope. In contrast, isotype controls are raised against antigens not present in the experimental sample or cell type, ensuring that they do not bind to the target. Their main role is to reveal the extent of background noise caused by artifacts, such as non-specific interactions, Fc receptor binding or autofluorescence.²

Why Use Isotype Controls?

An isotype control has the same properties possessed by the test antibody, except that it does not recognize the target antigen. Therefore, isotype controls:

• Detect non-specific binding and autofluorescence
• Validate antibody specificity
• Improve accuracy in assays like flow cytometry and IHC
• Prevent false positives and misinterpretation of data

Why Use an Isotype Control?

Background staining can significantly disrupt visualization, quantification and data analysis in flow cytometry and immunohistochemistry, especially when targeting rare cell populations or cells with low biomarker expression. Isotype controls provide an essential baseline for distinguishing true target staining from false positives and preventing the misinterpretation of non-specific signals as meaningful results.By comparing the isotype control signal with that of the target antibody, researchers can validate that observed staining or shifts in fluorescence are due to specific antigen recognition. Overall, using an isotype control in flow cytometry and immunohistochemistry improves the precision of and confidence in the collected data.^1,3

How Do Isotype Controls Work?

Isotype controls are engineered to possess the same qualities as the test antibody, except for target recognition.Researchers achieve this small but significant difference by raising the control antibodies against antigens not expressed in the sample or target cell type. Thus, they can simulate the background signal that might appear without true antigen binding. Therefore, differences in fluorescence or staining intensity between the isotype control and the target antibody reveal how much of the observed signal in the latter is specific or non-specific.¹

Researchers compare the control signal to that of the target antibody to determine whether the observed staining from the antigen-specific antibody exceeds the expected background level. This side-by-side approach helps set appropriate gates, thresholds and interpretation criteria.¹

For example, in flow cytometry, cells stained with an isotype control may show a low, diffuse fluorescence profile, representing non-specific binding. In contrast, cells stained with a target-specific antibody are expected to display a distinct fluorescence peak or a shift in intensity, indicating true antigen expression. If both profiles look similar, it suggests the signal from the target antibody is largely non-specific.⁴

Featured Product

Applications of Isotype Controls

Isotype control antibodies are widely used across immunoassays to help researchers discern between antigen-specific binding and non-specific background signals. They are especially common in flow cytometry, where precision and quantitative accuracy are essential.¹

In addition, isotype controls can be used as blocking agents or protein coating controls to eliminate the off-target interactions that generate a background noise.¹

The applications of isotype controls include:

• Flow cytometry
• Immunofluorescence (IF)
• Immunohistochemistry (IHC)
• Immunocytochemistry (ICC)
• Western blotting
• ELISA

Isotype Control in Flow Cytometry

In flow cytometry, isotype controls serve as a benchmark for researchers to set appropriate gates and thresholds, ensuring accurate identification of antigen-positive vs. antigen-negative cell populations. They are particularly useful in identifying markers with low expression.¹

Non-specific Antibody Control in Other Immunoassays

In assays such as ELISA and immunohistochemistry (IHC), as well as specialized drug-discovery assays, isotype controls help verify the specificity of antigen binding.

• For ELISA, an isotype control-coated well can reveal the background signal caused by off-target plate binding and separate it from the signals from the detection reagents.⁵
• In IHC or ICC, it helps differentiate target-driven tissue staining from non-specific adherence to cells or tissue sections.⁶
• In drug discovery assays, antibody detection shows how candidate compounds affect antibody-target binding. Isotype controls ensure accurate readouts, reflecting true therapeutic effects rather than detection artifacts. Using them, scientists avoid false positives (appearing active when they are not) and false negatives (missing target activity that is obscured).⁷

Other Research Applications

Beyond immunoassays and drug discovery, isotype controls are also used in diagnostic tests, quality control workflows and biopharmaceutical development. They help validate antibody specificity during early-phase antibody engineering, assess assay robustness and ensure that diagnostic antibodies bind only to their intended targets. ^8,9

Taken together, isotype control antibodies ensure that clinical assays and antibody-based biologics maintain high standards of specificity, reproducibility and reliability.

How to Select the Right Isotype Control Antibody

Choosing an appropriate isotype control is essential for accurately assessing background signal and ensuring meaningful interpretation of antibody-based assays. Essentially, the right control should closely mimic the characteristics of the test antibody. The key criteria can be summarized as follows:²

• The isotype control must come from the same host species as the test antibody
• The test antibody and the isotype control must be of the same class (e.g., IgG1, IgG2a, IgG2b or IgM) to match the affinity for Fc receptors
• If the primary is linked to a fluorophore, an enzyme conjugate or a metal tag, the isotype control must carry the same label
• The isotype control should not have a known reactivity with the Fab region of the target antigen in the sample

For example, a mouse IgG1 target antibody with FITC conjugation would require an FITC-linked mouse IgG1 isotype control for successful assay validation.

References

1. Spurgeon BE, Naseem KM. Platelet flow cytometry: instrument setup, controls, and panel performance. Cytom B-Clin Cytom 2020;98(1):19-27.
2. Schmit T, Klomp M, Khan MN. An overview of flow cytometry: its principles and applications in allergic disease research. Animal models of allergic disease: Methods and protocols 2020:169-182.
3. Kabir IM, Idris AT, Abubakar SD, Isah MM, Usman A, Yusuf L, et al. Immunohistochemistry as an indispensable tool in oncology. Indian J Gynecol Oncol 2024;22(3):106.
4. Park M, Lim J, Ahn A, Oh E-J, Song J, Kim K-H, et al. Current status of flow cytometric immunophenotyping of hematolymphoid neoplasms in Korea. Ann Lab Med 2024;44(3):222-234.
5. Oquendo E, Lin X, Ye S, Coble K, Grimaldi C. Using multiple platforms for critical reagents selection process to support pharmacokinetic ligand-binding assay development. Bioanalysis 2021;13(10):761-769.
6. Balaji S, Keswani S, Degala A, Li H, Goodman MD, Keswani SG. Considerations for immunohistochemistry. Success in Academic Surgery: Basic Science 2025:139-180.
7. Li H, Li H. Different antibody isotypes against tuberculosis: what we know and what we need to know. Front Immunol 2025;16:1682934.
8. Vicart A, Holland C, Fraser K, Gervais F, Aspinall-O’Dea M, Brown N, et al. Applications of cell-based protein array technology to preclinical safety assessment of biological products. Toxicol Pathol 2025;53(1):31-44.
9. Yang C, He B, Zhang H, Wang X, Zhang Q, Dai W. IgG Fc affinity ligands and their applications in antibody-involved drug delivery: a brief review. Pharmaceutics 2023;15(1):187.