Choosing the Right Antibody for Immunohistochemistry

 

In immunohistochemistry (IHC), the choice of antibody can make or break an experiment and determine the outcome of the staining, either a false negative or a false positive result outcome.

Consequently, selecting the appropriate antibody is crucial for obtaining reliable and reproducible results.

However, navigating this terrain can be very complex, especially with the plethora of antibodies available on the market, ranging in specificity, sensitivity, and quality.

Monoclonal and Polyclonal Antibodies

Monoclonal Antibodies

In immunohistochemistry (IHC), researchers utilize two primary types of antibodies: monoclonal and polyclonal antibodies, each with distinct characteristics and applications.

Monoclonal antibodies are derived from a single clone of antibody-producing cells, typically generated by fusing antibody-producing B cells with immortalized myeloma cells to produce hybridoma cell lines.

These antibodies recognize a single epitope on the target antigen with high specificity. Due to their uniform epitope recognition, monoclonal antibodies offer batch-to-batch consistency and precise antigen detection, making them ideal for applications requiring high specificity, such as diagnostic assays and therapeutic targeting.

Polyclonal Antibodies

On the other hand, polyclonal antibodies are generated from multiple clones of antibody-producing cells, typically by immunizing animals (e.g., rabbits, goats) with the target antigen.

The immune response results in the production of a heterogeneous mixture of antibodies that recognize multiple epitopes on the antigen. This broad epitope recognition enhances the sensitivity of polyclonal antibodies, allowing for the detection of low-abundance antigens and providing versatility in recognizing different isoforms or post-translational modifications of the target antigen.

However, polyclonal antibodies may exhibit batch-to-batch variability and increased potential for non-specific binding, requiring careful validation and optimization in IHC experiments.

Understanding Antibody Specificity

One of the primary considerations when selecting an antibody for IHC is specificity. An ideal antibody should recognize the target antigen with high specificity, minimizing nonspecific binding to other proteins or structures within the tissue.

To ensure specificity, it’s essential to review the antibody’s validation data, including western blotting, immunoprecipitation, and previous IHC studies.

Assessing Sensitivity and Signal Intensity

In addition to specificity, the sensitivity of an antibody is crucial for detecting low-abundance antigens in tissue samples.

Antibodies with high sensitivity can reliably detect even weakly expressed antigens, providing robust staining signals.

Consider factors such as the antibody’s affinity for the target antigen and its signal amplification capabilities when evaluating sensitivity.

Validating Antibody Performance

Before committing to a specific antibody for your IHC experiments, it’s prudent to validate its performance in your specific experimental conditions.

Conducting pilot experiments using positive and negative control tissues can help assess the antibody’s staining pattern, signal-to-noise ratio, and background levels.

Collaborating with colleagues or consulting online resources for antibody reviews and recommendations can also aid in the validation process.

The Best Antibody for IHC Staining

The choice of antibody for IHC staining depends on various factors, including the specificity and sensitivity required for antigen detection, the availability of validated antibodies for the target antigen, and the experimental setup.

Researchers often conduct thorough validation experiments to assess antibody performance and optimize staining conditions for reliable and reproducible results in IHC experiments.

How Can Pathologists Select the Best Antibody?

Pathologists play a crucial role in diagnosing diseases and guiding treatment decisions based on histopathological examination of tissue samples. Selecting the best antibody for immunohistochemistry (IHC) staining is paramount for several reasons:

1. Accuracy of Diagnosis: The accuracy of the diagnostic assessment heavily relies on the specificity and sensitivity of the antibody used in IHC staining. Choosing the best antibody ensures precise identification and localization of target antigens within tissue specimens, leading to accurate disease classification and prognosis.
2. Differentiation of Disease Subtypes: Many diseases, especially cancers, exhibit heterogeneity at the molecular level, with distinct subtypes requiring tailored treatment approaches. The selection of appropriate antibodies allows pathologists to differentiate between different disease subtypes based on the expression patterns of specific biomarkers, facilitating personalized treatment strategies.
3. Detection of Prognostic and Predictive Markers: Certain biomarkers detected by IHC staining serve as prognostic indicators or predictors of treatment response. By selecting the best antibody for detecting these markers, pathologists can provide valuable prognostic information and guide therapeutic decisions, ultimately improving patient outcomes.
4. Avoidance of Misinterpretation: Inaccurate or nonspecific staining can lead to misinterpretation of IHC results, potentially resulting in erroneous diagnoses and inappropriate treatment decisions. By carefully selecting validated antibodies with proven performance in IHC, pathologists can minimize the risk of false-positive or false-negative results, ensuring the reliability of diagnostic assessments.
5. Optimization of Laboratory Resources: Limited resources, including time, tissue samples, and reagents, underscore the importance of efficient antibody selection in the laboratory setting. Choosing the best antibody streamlines workflow processes, minimizes experimental variability, and maximizes the utility of available resources, ultimately enhancing laboratory efficiency and productivity.
6. Quality Assurance and Regulatory Compliance: Pathology laboratories must adhere to stringent quality assurance standards and regulatory requirements to ensure the accuracy and reliability of diagnostic tests. Selecting antibodies from reputable suppliers with rigorous validation processes and regulatory compliance ensures adherence to quality standards and mitigates the risk of potential regulatory issues.

 

How Pathologist can determine they have a valid antibody?

Validating Antibodies in Immunohistochemistry: Standardizing the Staining Process

Pathologists must on standardized staining processes to detect when a wrong antibody or staining material has been applied. One effective tool for this purpose is the Process Record Slide (PRS) tool, accredited by ISO 13485, which aids in standardizing the staining process and detecting discrepancies in antibody performance.

The PRS tool consists of preformed slides embedded with surrogate antigens, each with a predetermined density or quantification.

These surrogate antigens mimic the target antigens of interest, allowing for the assessment of antibody performance in a controlled setting.

During the staining process, if a valid primary or secondary antibody is applied, the correct result is revealed, indicating successful binding to the surrogate antigen and appropriate visualization of the staining pattern.

However, if an invalid primary or secondary antibody is applied, which differs in antigen specificity or concentration from the surrogate antigen, the intended outcome will not be achieved.

This discrepancy in staining results serves as a red flag, signaling the potential use of a wrong antibody or staining material. By comparing the observed staining pattern with the expected outcome based on the PRS tool, pathologists can identify deviations and take corrective actions to ensure the validity and accuracy of their IHC assays.

Standardizing the staining process using the PRS tool offers several advantages in antibody validation:

1. Quantitative Assessment: The predetermined density or quantification of surrogate antigens allows for quantitative assessment of staining intensity, facilitating objective evaluation of antibody performance.
2. Quality Control: The PRS tool serves as a quality control measure, enabling pathologists to verify the reliability and consistency of antibody staining across different batches and experiments.
3. Detection of Errors: Discrepancies between the observed staining pattern and the expected outcome based on the PRS tool indicate potential errors in antibody selection or application, prompting further investigation and corrective measures.
4. Workflow Efficiency: Standardizing the staining process streamlines workflow procedures, reducing variability and optimizing laboratory efficiency in IHC assays.

 

By integrating the PRS tool into the staining process, pathologists can effectively validate antibodies, detect errors, and ensure the validity and reliability of IHC results. This standardized approach enhances the accuracy of diagnostic interpretations and contributes to the overall quality of patient care in pathology laboratories.

Conclusion

In conclusion, selecting a suitable antibody for immunohistochemistry (IHC) staining is crucial for achieving accurate and reliable results, as it can significantly impact the outcome of diagnostic assessments and research endeavours.

With a multitude of antibodies available on the market, each with its own specificity, sensitivity, and quality, navigating this landscape can be daunting.

However, by understanding the principles of antibody selection and validation, pathologists can ensure the validity and performance of antibodies used in IHC assays.

Standardizing the staining process through tools like the Process Record Slide (PRS), accredited by ISO 13485, offers a robust approach to validate antibodies, detect errors, and maintain quality control in the laboratory.

By integrating these standardized procedures into their workflow, pathologists can enhance the accuracy of diagnostic interpretations, differentiate disease subtypes, detect prognostic markers, and ultimately improve patient outcomes.