What are conjugated primary antibodies?
Conjugated primary antibodies are chemically modified antibodies that allow for the direct attachment of a label, such as a fluorescent dye or enzyme. These conjugated antibodies streamline detection by directly binding to their target, enabling the visualization or detection of specific target molecules in biological samples. There are several benefits to using conjugated primary antibodies including reduced assay time, improved specificity, less background noise, ease of use, high multi-plex labeling, and convenient for use across different samples. Conjugated primary antibodies can be used on multiple platforms and in various applications such as flow cytometry, immunohistochemistry, western blot, and immunocytochemistry.
Direct versus indirect detection methods
There are two types of detection methods that can be used with primary antibodies—direct and indirect. Direct detection utilizes a primary antibody conjugated to a label (i.e. fluorescent label) for “direct” detection of a target, without the use of a secondary antibody. This method offers speed and simplicity. Direct detection is a quick, one-step process excellent for high-throughput and multiplex assays. Indirect detection utilizes a secondary antibody conjugated to a label that binds to an unlabeled primary antibody, resulting in the “indirect” detection of a target (Figure 1). Learn more about direct versus indirect detection methods in the table below.
Figure 1. Schematic of direct and indirect detection methods. Direct detection involves a fluorescent label conjugated to a primary antibody that binds to a target of interest. Indirect detection involves detection of a target using an unconjugated primary antibody binding to a target of interest followed by binding of a secondary antibody with a fluorescent label.
Comparison of direct versus indirect detection
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| Direct detection | Indirect detection |
| Description | Primary antibody is directly conjugated to a fluorescent dye, without the need for a secondary antibody | Secondary antibody with a conjugated fluorescent dye is used to bind to an unlabeled primary antibody |
| Multiplex | High multicolor staining is based on the number of fluorophores your detection system can identify | Limited by the availability of primary antibodies for various species, often restricted to one species |
| Background | Significantly less background noise by eliminating the use of a secondary antibody | Non-specific binding may increase background noise caused by secondary antibody cross-reactivity |
| Convenience | Reduced assay time by eliminating the need for additional secondary antibody incubation steps | Additional time and steps needed for secondary antibody binding |
Conjugated primary antibodies for IHC
Fluorescence labeling is a highly versatile approach to immunohistochemistry (IHC), offering the ability to detect numerous targets simultaneously with increased sensitivity and specificity. Conjugated primary antibodies for IHC offer innovative multiplex staining techniques with a streamlined workflow and less time-consuming process for tissue labeling.
Conjugated primary antibodies have helped transform the world of spatial biology by offering researchers the ability to understand the spatial organization, function, and behavior of individual cells within tissues. This analysis of single cells allows for deep insights into:
- Cell types, cell states and cell functions
- Cell-cell interactions
- Cellular communication
- Cellular environment
- Tissue microenvironments and architecture
Thermo Fisher Scientific offers high-quality, easy-to-choose, and easy-to-use conjugated primary antibodies for IHC and spatial biology designed to enable detailed cellular profiling and tissue imaging. Our conjugated primary antibodies have been validated across various tissue types and confirmed against secondary antibody IHC labeling. These conjugated primary antibodies are offered in a variety of fluorophores, including Alexa Fluor and Alexa Fluor PLUS, for a convenient single-step labeling method. Directly conjugated primary antibodies allow researchers to achieve high-plex labeling with 8–10 biological targets on one staining mix (Figure 2).
Figure 2. FFPE normal human tonsil (A) and tonsil lymphoma (B) labeled with primary antibody conjugates. With this advanced 9-plex technology, the enhanced expression of CD138 (tumor marker in myelomas, lymphomas), FoxP3 (immunosuppressive T-regulatory cells), and other biologically relevant markers in the lymphoma tissue can be visualized, allowing mapping of the tumor microenvironment.
Find publications, protocols, and technical guides to start or evolve your multiplex imaging experiments and research.
Related spatial biology protocols:
- IHC FFPE Tissue Low pH Antigen Retrieval—Direct Method
- IHC FFPE Tissue High pH Antigen Retrieval—Direct Method
- Download imaging protocol handbook
Spatial biology posters:
Antibodies for immunocytochemistry
Our range of immunocytochemistry (ICC) antibodies are conjugated to many fluorophore types, including Alexa Fluor and eFluor dyes. These conjugates enable visualization of the subcellular localization of a target protein.
Antibodies for flow cytometry
Our range of flow cytometry antibodies are conjugated to many fluorophore types, including Alexa Fluor and eFluor dyes. These conjugates are designed to help answer complex cell biology questions with our wide range of fluorophores for traditional or spectral analysis.
Antibodies for western blotting
Our range of western blot antibodies are offered as polyclonal, monoclonal, and recombinant antibodies. They can be used for direct or indirect detection. These conjugates offer clean, definitive, and reproducible western blot results.
Advanced Verification
Thermo Fisher Scientific is committed to adopting higher validation standards for the Invitrogen antibody portfolio. We have implemented additional specificity tests to help ensure high confidence levels in our products. You can identify the products that have already undergone this testing with the Advanced Verification badge, shown above. This badge can be found in antibody search results and at the top of product webpages. The data supporting the Advanced Verification status can be found in the product specific data galleries. To learn more about our testing standards, please visit Invitrogen Antibody Verification.
For Research Use Only. Not for use in diagnostic procedures.