De-risking Early Lateral Flow Assay Development: Antibody Selection

(8-10 minutes)

Antibody selection is one of the most influential decisions made during early Lateral Flow Assay (LFA) development. Whether antibodies are sourced commercially or developed as part of a broader LFA development service, the choices made at this stage directly affect assay sensitivity, specificity, reproducibility and long-term manufacturability. Applying a structured, risk based approach to antibody evaluation can significantly reduce technical and commercial challenges later in the development lifecycle.

Using Commercial Antibodies in Early LFA Development

Commercially available antibodies are frequently used during proof-of-concept and early feasibility studies. Their accessibility can accelerate development timelines and reduce initial costs. When carefully selected and appropriately qualified, commercial antibodies can perform effectively in lateral flow formats and support progression into assay optimisation.

However, commercial antibodies also introduce certain technical and commercial risks that should be considered early. Performance data is most commonly generated using ELISA formats rather than lateral flow systems and detailed information regarding immunogen design, epitope specificity or production processes may be limited. In addition, supply continuity, lot-to-lot consistency and long-term availability are not always fully under the developer’s control, particularly where antibodies are sold on a non-exclusive basis or may be discontinued. These limitations mean that careful consideration is required when selecting commercial antibodies for LFA development.

When is an antibody development service beneficial?

While commercially available antibodies can accelerate early feasibility studies, there are situations where a custom antibody development service may provide significant advantages. These services are particularly useful when commercial options are unavailable or limited, where greater control over supply, licensing, or intellectual property is desired or when long-term consistency and performance are critical.

Custom development allows antibodies to be designed with the intended assay and sample matrix in mind, ensuring that the final reagents recognise the correct form of the target antigen and are compatible with lateral flow formats. Partnering with experienced developers can also help anticipate potential issues with conjugation, immobilisation and assay performance before they become costly problems.

Other key considerations for using a development service include:

• Targeted epitope recognition: Antibodies can be raised against specific regions of the antigen that are most relevant for the assay, improving specificity and sensitivity.

  
  

• Enhanced reproducibility and supply control: Custom antibodies can offer more predictable lot-to-lot consistency and reliable long term availability, reducing manufacturing and regulatory risk.

  
  

• Intellectual property and clone ownership: Custom development can provide ownership or exclusive rights to antibody clones, offering greater commercial control, long term supply security and reduced dependency on third party suppliers. This can strengthen freedom to operate and minimise the risk of discontinuation or competitor access associated with non exclusive commercial antibodies.

  
  

• Clone sequencing and cell banking: Genetic sequencing of selected monoclonal antibody clones, combined with establishment of master and working cell banks, reduces the risk of genetic drift, hybridoma instability or cell line loss. This supports long-term supply continuity, scale-up and regulatory readiness, while preserving the option for future recombinant antibody production.

  
  

• Streamlined development workflow: Collaboration with experts enables early identification of potential technical challenges, supporting faster and more confident progression from candidate selection to functional lateral flow testing.

While engaging a development service adds time and cost compared with sourcing commercial antibodies, early investment can reduce technical risk, improve assay robustness and support long term manufacturability. Evaluating this option early ensures that the antibody strategy aligns with the broader development timelines.

What should be evaluated beyond the datasheet?

Selecting antibodies for LFA development requires evaluation beyond affinity and datasheet specifications. Key technical considerations include:

Immunogen relevance and antigen equivalence

Understanding how an antibody was raised is critical to predicting its performance in a real world test system. Developers should assess whether the immunogen used during antibody generation is genetically and structurally equivalent to the target antigen present in the sample matrix.

Where possible, antibodies raised against purified native antigen or against antigen regions that are exposed and present in the clinical matrix are preferred. Antibodies generated using recombinant fragments, truncated proteins or denatured antigens may fail to recognise the target in its native or matrix associated form.

A useful de-risking strategy is to evaluate antibody binding using antigen sourced from a different supplier than the antibody manufacturer. Where possible, testing with native or independently produced antigen helps break the closed loop between antibody and validation antigen and reduces the risk of overestimating performance, increasing confidence that results will translate to real samples.

Specificity and epitope recognition

Antibodies should recognise the target analyte in the form in which it appears in the test sample, whether intact, fragmented or structurally altered by the sample matrix. For sandwich assays, it is generally desirable to select antibodies that bind distinct, non-competing epitopes. While the exact spatial relationships of epitopes are typically unknown, functional antibody pairing assessments can determine whether antibodies are compatible and reduce the risk of signal suppression

Monoclonal antibodies are often preferred where high specificity is required due to their single-epitope recognition. However, in applications where broader detection is beneficial, such as bacterial targets, polyclonal antibodies may offer advantages by recognising multiple epitopes across a target organism.

Is the antibody biochemically suitable for LFA development?

High antibody purity is essential for reliable nanoparticle conjugation and consistent assay performance. Antibodies should ideally be supplied at high purity, free from residual host proteins, carrier proteins or stabilisers that may interfere with detector particle binding.

Purification strategies

Protein G affinity chromatography is commonly used to enrich IgG via Fc–ligand interactions; however, alternative approaches may be beneficial depending on downstream assay requirements. For example, Protein A can be considered when gentler elution conditions are preferred to help preserve antibody stability. Identifying the antibody subclass early can guide purification choices to ensure optimal recovery and functionality.

Antigen based affinity purification selectively isolates antibodies that bind a specific target, reducing weakly binding or non-specific IgGs. For monoclonal antibodies produced from hybridoma cell lines, whether harvested from ascites or culture supernatant, this step is generally unnecessary because the target antibody is effectively the only IgG present.

Polyclonal antibodies from serum, however, typically require antigen based purification to remove unrelated immunoglobulins. This approach can also be advantageous when high functional affinity or rapid binding kinetics are needed, such as in lateral flow assays, though it adds complexity to reagent preparation and process development. Purification strategy should therefore consider both subclass compatibility and the assay’s performance requirements.

For the purposes of LFA development it is also advantageous for antibodies to be supplied at a concentration of ≥1 mg/mL. This enables efficient early stage matrix assessment, allowing the same antibody candidate to be evaluated both as a capture reagent on the test line and as a detector antibody on the conjugate label (typically following dilution to working concentrations). Supplying antibodies at sufficient concentration reduces the need for reformulation or re-ordering and supports rapid, parallel evaluation of antibody function across assay components.

Buffer composition and formulation compatibility

The formulation in which an antibody is supplied can influence conjugation efficiency and assay performance. Antibodies should ideally avoid components that directly interfere with nanoparticle binding or surface chemistry, including:

• Carrier proteins such as bovine serum albumin (BSA)

• Sulfur containing preservatives (e.g. thiomersal) that can disrupt conjugation.

• Detergents or chelators (e.g. EDTA) that may affect surface chemistry or particle stability.

• Primary amine containing buffers (e.g. Tris) that can compete with chemical conjugation reactions.

Will the antibody perform under lateral flow conditions and how can early screening reduce risk?

Most commercially available antibodies are characterised using ELISA or other solution-based assays rather than lateral flow formats. ELISAs rely on long incubations, equilibrium binding, multiple wash steps and blocking reagents, so antibodies that appear strong in solution may underperform in lateral flow assays, which operate under non-equilibrium flow with very limited interaction time. Direct evaluation under LFA relevant conditions is therefore essential to identify candidates likely to perform reliably.

Testing should include antibody immobilisation on nitrocellulose and conjugation to detector particles, as drying and flow dynamics can significantly influence binding behaviour. Large-scale screening of multiple antibody pairs in LFA format is recommended to identify the strongest capture/detector combinations and confirm reliable signal generation under realistic assay conditions.

Parallel Screening and Performance Metrics

Rather than relying on a single antibody candidate or pair, early stage projects benefit from screening multiple antibodies in parallel. Combining high coverage early screening with focused lateral flow evaluation allows developers to efficiently identify robust antibody pairs while minimising reagent consumption and development risk.

Evaluating candidates based on conjugation efficiency, signal-to-noise ratio, background development and stability provides a realistic assessment of how antibodies will perform in the final device.

Additionally, assessing antibodies in the intended sample matrix early can reveal cross-reactivity and matrix interference that may not be apparent in buffer based assays. This is a common source of failure in later development stages, so early matrix assessment can significantly reduce risk and improve confidence in antibody choice.

Will the antibody remain consistent across lots and scale-up?

As assays move toward scale-up, consistency becomes critical. Monoclonal antibodies typically offer superior lot-to-lot reproducibility due to their single clone origin, reducing the need for repeated assay re-optimisation and simplifying validation and manufacturing transfer.

Monoclonals however are not always available for every target or epitope, and in some cases, polyclonal antibodies may be more suitable. Polyclonals can provide broader target recognition and enhanced detection of multiple epitopes, but they inherently have greater lot-to-lot variation. Strategies to manage this include:

• Careful sourcing and characterisation: Use suppliers with rigorous quality control and validated production processes.

• Pooling or blending batches: Combining multiple immunisations or batches can help reduce variability between lots.

• Early screening and benchmarking: Test new lots against a reference standard or previously qualified lot to confirm equivalent performance.

By considering both monoclonal and polyclonal options and implementing appropriate control strategies, developers can balance assay sensitivity and specificity with the practical realities of supply and reproducibility. Early planning for lot consistency helps mitigate risk, reduces downstream troubleshooting and supports consistent LFA performance.

Building a robust antibody strategy

Successful lateral flow assay development depends on making antibody choices that align with detection requirements, sample matrix and long-term manufacturing goals. Early decisions around immunogen relevance, antibody purity, formulation and conjugation behaviour all influence assay sensitivity, specificity, reproducibility and scalability.

To translate these principles into practice, many developers benefit from partnering with experienced lateral flow development services. At Fleet Bioprocessing Ltd., antibodies can be screened in parallel under LFA relevant conditions, helping identify the most promising capture and detector pairs while minimising reagent consumption and development risk. This approach ensures that antibody selection is informed by functional performance in real assays, not just datasheet specifications.

Long-term planning is also critical. Sequencing selected clones, creating master and working cell banks and considering recombinant production options can reduce the risk of genetic drift, ensure supply continuity and enable reproducible manufacturing. Intellectual property and clone ownership provide additional commercial control and freedom to operate. Collaboration with experienced developers helps anticipate potential challenges with conjugation, immobilisation or assay specific issues before they become costly problems.

By combining careful early evaluation with expert support, developers can build a resilient antibody strategy that supports both rapid proof-of-concept work and long-term manufacturing success.

Learn more about Fleet’s LFA development and antibody screening services here. Contact us today to discuss your project and explore how we can support your LFA development needs.

Get in Touch