Secondary antibodies bind to primary antibodies, facilitating the detection, sorting, and purification of target antigens. By recognizing the species and isotype of the primary antibody, they enhance protein detection. Various labels, such as dyes or enzyme conjugates, can be attached to accommodate specific applications.
Secondary antibodies play a vital role in immunoassays, such as western blotting, immunohistochemistry (IHC), immunofluorescence, and enzyme-linked immunoassays (ELISA). Validation experimentally confirms an antibody’s suitability for its intended use. In immunoassays, it ensures selective binding to the target on a membrane, even within complex samples like cell or tissue lysates.
However, the most well-characterized secondary antibody can produce suboptimal results if not appropriately diluted or used in combination with an effective blocking strategy. Assay conditions, like blocking reagents, can significantly affect antibody performance.
Factors determining the performance of secondary antibodies
- Specificity: The ability of a secondary antibody to detect its target antigen while avoiding structurally dissimilar antigens.
- Sensitivity: The amount of antigen a secondary antibody can detect, influenced by binding affinity, labeling, and degree of labeling.
- Consistency: The ability of the antibody to perform reliably across different batches, affected by its source and clonality.
The role of blocking in improving antibody performance
Blocking involves using a blocker or blocking buffer to reduce non-specific antibody binding to off-target proteins and the membrane. It stabilizes blotted proteins while preserving their retention. This also improves antibody specificity and enhances the signal-to-noise ratio for target detection. Blocking buffers should not interfere with antibody-antigen interaction or target detection.
Common blocking agents like BSA, nonfat dry milk, and casein vary in effectiveness. Their blocking strength can sometimes affect target protein detection in specific assays. For example, in immunohistochemistry protocol, protein blocking solution prevents non-specific binding and can include 2–10% normal serum from the same species as the secondary antibody.
The choice of blocker should match the assay type and target detection method. For example:
- Nonfat dry milk is a common, inexpensive blocker but may mask antigens. However, endogenous biotin and immunoglobulin G (IgGs) in milk may cross-react with sheep or goat secondary antibodies.
- Casein, a phosphoprotein in nonfat dry milk, can interfere with phospho-specific antibodies and increase background signals.
- Bovine serum albumin (BSA) is an alternative suitable for biotin or alkaline phosphatase detection but can increase nonspecific banding and background in fluorescent assays.
- Nonmammalian blockers may reduce background noise and cross-reactivity compared to mammalian-based reagents.
Key considerations for blocking
- Compatibility with detection system: Always check if the buffer contains any components like casein or biotin that can interfere with the detection reagents.
- Effect on antibody-antigen interaction: Extended blocking times or high concentrations can mask interactions, reducing signal intensity.
- Protein loss from membrane: Excessive blocking, especially with nonfat dry milk, may cause progressive elution of blotted proteins.
- Optimization for low-abundance proteins: The strength and incubation time of the blocking agent should be carefully adjusted for sensitive detections. For example, for western blotting, incubation is done with gentle rocking overnight at 4°C or for 1 hour at room temperature.
- Buffer selection: Tris-buffered saline (TBS) and phosphate-buffered saline (PBS) are commonly used for blocking, incubation, and washes. However, TBS is preferred for phosphoprotein detection and alkaline phosphatase-conjugated antibodies.
Optimizing secondary antibody dilution
The concentration of the secondary antibody influences the selectivity of the primary antibody. Excess secondary antibodies can cause off-target binding, while too little may result in weak signals. Moreover, the antibody-antigen binding rate is influenced by temperature, pH, buffer components, and relative concentrations. Since antigen concentration is usually fixed, the optimal antibody concentration must be determined through dilution experiments. Optimal dilution of secondary antibody varies based on the antibody conjugate type and detection method used. For example, if a datasheet recommends a 1:200 dilution, it is best to test a range of dilutions, such as 1:50, 1:100, 1:200, 1:400, and 1:500.
A dilution curve helps determine the optimal concentration for the target proteins. Secondary antibody solutions are usually prepared in the same buffer and blocking solutions as that for primary antibody. If a high background appears, further optimization may be needed.
The concentration of whole antisera, culture supernatants, or ascites fluid is often unknown, and unpurified antibody preparations can vary significantly. When specific antibody concentrations are unknown, estimated values can serve as a rough guideline. However, these dilutions are just a starting point and may require adjustments based on experimental results.
Here is a table showing the dilution needed for each immunoassay for the respective secondary antibodies.
| Secondary antibody | Western blot | ELISA | IHC | ICC | Flow cytometry |
| Goat Anti-Rabbit IgG H&L (HRP) | 1/2000-1/20000 | 1/120000 | 1/1000 | 1/1000-1/5000 | NA |
| Goat Anti-Rabbit IgG H&L (HRP) | 1/2000-1/50000 | 1/5000-1/20000 | 1/2000-1/50000 | NA | NA |
| Rabbit monoclonal [H169-1-5] Anti-Human IgG Fc | NA | 1/1000 | NA | NA | NA |
| Goat Anti-Mouse IgG H&L (Biotin) | 1/2000-1/20000 | 1/350000 | NA | 1/1000-1/5000 | NA |
Factors influencing optimal dilution
The optimal dilution of the secondary antibody depends on the target protein’s expression level and the primary antibody used. Other factors include:
- Sample abundance: Highly expressed proteins, like glyceraldehyde-3-phosphate dehydrogenase (GAPDH), require higher dilutions to avoid signal saturation.
- Antibody format and conjugate: Secondary antibodies are conjugated with labeled compounds like fluorophores or enzymes for detection that have varying signal intensities. Horse-radish peroxidase (HRP) and alkaline phosphatase (AP) are the most commonly used enzymes. HRP should not be used with sodium azide, as it inhibits enzyme activity and requires the proper washing of membranes before detection.
- Detection method: Fluorescent secondary antibodies have a lower detection range like chemiluminescent methods. They are more suitable for detecting proteins present in higher abundance. Thus, it needs more precise dilution.
- Time and place of incubation: Secondary antibody incubations are usually done at room temperature for about an hour. Longer incubations may increase nonspecific binding and background noise. Adjusting antibody concentration while keeping incubation times consistent is often more effective.
Conclusion
Optimizing secondary antibody performance requires careful consideration of blocking strategies, dilution factors, and assay-specific conditions to achieve accurate and reproducible results. Proper blocking minimizes nonspecific binding, while selecting the right dilution prevents weak signals or excessive background noise.
By systematically adjusting antibody concentrations, incubation times, and detection methods, researchers can enhance the specificity, sensitivity, and consistency of immunoassays for reliable experimental outcomes.
