Antibody Validation for ELISA: Ensuring Accurate and Reliable Results

Written by Nerea Gómez de San José, PhD in protein biomarker research.

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Despite being one of the most widely used tools in life science research, antibodies face a persistent challenge – cross-reactivity. Several studies highlight the lack of specificity among commercially available antibodies (1-4) emphasizing the importance of proper antibody validation. Enzyme-linked immunosorbent assays (ELISAs) are a widely used immunoassay technique for quantifying proteins in a crude biological sample. The success of an ELISA depends largely on the specificity and affinity of the antibody pairs used. Therefore, validating antibodies is a critical step in ensuring accurate and reproducible ELISA results.

Here, we will delve into the essential aspects of ELISA antibody validation, providing a comprehensive guide for researchers aiming to optimize their assays.

Antibody Validation

Antibody validation is critical for several reasons:

• Specificity: To ensure that the antibody binds only to the target antigen and not to other proteins.
• Sensitivity: To detect even low concentrations of the antigen.
• Reproducibility: To obtain consistent results across different experiments and laboratories.
• Accuracy: To correctly quantify the target antigen.

Several initiatives have established guidelines to ensure antibody reproducibility in an application-specific manner. In 2016, Uhlen et al. proposed five pillars for antibody validation to address the reproducibility crisis. This guide encompasses the following strategies:

Figure 1: Antibody Validation Strategies

Assay Specific Antibody Validation

It is crucial to note samples are prepared differently for various antibody-based assays. For flow cytometry and ELISA, proteins are generally in a native form, whereas they are partially or completely denatured for western blotting, immunohistochemistry, and immunocytochemistry. Due to differences in protein conformation and target accessibility, an antibody that works well in one assay may perform poorly in another. This complexity makes it challenging to establish a single benchmark for assessing antibody performance across all possible applications. Thus, methods for antibody validation must be tailored to the specific application and context.

Validation of antibodies for sandwich ELISA

ELISA experiments detect soluble substances such as peptides, proteins, antibodies, and hormones. They are usually performed in a "sandwich" format (Figure 1). A sandwich ELISA identifies analytes by using a capture antibody bound to the ELISA plates to secure the target protein, while a detector antibody generates a read-out signal. The use of antibody pairs significantly increases the specificity and sensitivity of the sandwich ELISA compared to indirect formats, and antibody validation is a critical step in ensuring accurate and reproducible results.

Figure 2. Sandwich ELISA workflow

Finding and Validating Antibody Pairs for ELISA

1. Understand the Target Protein

• Protein structure: Antibody pairs bind to complementary epitopes on the antigen surface. The native confirmation of the target protein must be understood to explore the suitability of individual epitopes as antibody targets.
• Biological relevance: Consider the role and expression of the target protein in the biological context you are researching.
• Post-translational modifications (PTMs), isoforms, and cleavage products: Be aware of any modifications that may affect antibody binding.

2. Include appropriate controls

• Expression confirmation: Ensure the target protein is expressed in the chosen samples.
• Controls: Use positive and negative controls to validate antibody specificity.

3. Antibody validation

• Ensure company validation data has been performed in the same sample type as you require, as matrix effects may interfere with antibody binding. You may test suitability of antibody pairs in your sample type using spike and recovery and linearity of dilution.
• If needed, perform further validation following the five pillars for antibody validation.

4. Identify optimum antibody concentrations

• Perform a checkerboard titration to determine optimum concentration for both capture and detection antibody.

ELISA assay validation

To validate an ELISA method for measuring analyte concentrations in biofluids, the following steps are recommended based on the guidelines described in the paper "A Practical Guide to Immunoassay Method Validation" by Andreasson et al.:

• Precision: Measure of closeness of repeated measurements
  • Within-Run Precision: Analyze multiple replicates of at least two samples (one high and one low concentration) within a single run.
  • Between-Run Precision: Analyze multiple replicates of the same samples across different runs and days.
• Sensitivity: Establish the limit of quantification (LOQ) by analyzing samples with known low concentrations. The LOQ is the lowest concentration at which the analyte can be reliably quantified with acceptable precision and accuracy.
• Matrix effects refer to the interference from substances present in the matrix. They can be reduced by diluting the sample. Matrix effects can be checked by spiking the analyte of interest in the sample and analyzing the recovery.
• Protein stability: Assess the stability of the analyte in the sample under various storage conditions (e.g., room temperature, 4°C, -20°C) and after multiple freeze-thaw cycles. This is important to avoid systematic errors related to sample collection and storage.
• Cross-Reactivity Testing: Test the antibody against a panel of related proteins to assess cross-reactivity. This step ensures the antibody is specific to the target antigen and does not bind to similar epitopes on other proteins. Tools such as Blast® can be valuable in this process.

ELISA Troubleshooting

Despite rigorous validation, some issues may still arise during ELISA. Here are common problems and their potential solutions:

• Non-Specific Binding
  • Problem: High background signal or non-specific binding.
  • Solution: Optimize blocking conditions and washing steps.
• Low Sensitivity
  • Problem: Poor detection of the target antigen.
  • Solution: Bring all reagents to room temperature 30 minutes before assay, pipette at an angle, and do not touch the bottom of the well, perform serial dilution of samples so they fall within detectable range
• Inconsistent Results
  • Problem: Variability between replicates or assays.
  • Solution: Standardize protocols, ensure consistent reagent quality (e.g., lot-to-lot variability), and maintain strict environmental controls (e.g., temperature).

Conclusion

Antibody validation is a critical component of ELISA development, providing confidence in the assay results. By following application-specific validation strategies and adhering to established guidelines, researchers can significantly improve the reliability of their experimental outcomes.

References

1. Herrera M, Sparks MA, Alfonso-Pecchio AR, Harrison-Bernard LM, Coffman TM. Lack of specificity of commercial antibodies leads to misidentification of angiotensin type 1 receptor protein. Hypertens Dallas Tex 1979. 2013 Jan;61(1):253–8.
2. Schonbrunn A. Editorial: Antibody Can Get It Right: Confronting Problems of Antibody Specificity and Irreproducibility. Mol Endocrinol. 2014 Sep;28(9):1403.
3. Andersson, S., Sundberg, M., Pristovsek, N. et al. Insufficient antibody validation challenges oestrogen receptor beta research. Nat Commun 8, 15840 (2017).
4. Kahn RA, Virk HS, McPherson PS. Heed a decade of calls for antibody validation. Nature. 2023 Aug;620(7974):492.
5. Uhlen, M., Bandrowski, A., Carr, S. et al. A proposal for validation of antibodies. Nat Methods 13, 823–827 (2016).
6. Andreasson U et al., E. A Practical Guide to Immunoassay Method Validation. Front Neurol. 2015 Aug 19;6:179

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