Affinity reagents that recognize the biological target are fundamental to lateral flow assay performance, both on the reporter particle and at the test line. While aptamers and other affinity reagents can be used, antibodies remain a common choice for sensitive and selective detection in lateral flow assays.
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Antibody selection can also be one of the more challenging parts of assay development. Each antibody has unique binding characteristics, specificity, kinetics, and formulation requirements that can affect conjugation, drying, rehydration, and performance on the strip. Selecting and screening antibodies early can reduce downstream optimization and improve assay reproducibility.
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Choosing Antibodies for Lateral Flow Assays
Selection of the optimal antibodies is a critical aspect of lateral flow assay design. For a sandwich assay, two antibodies that can simultaneously bind the target analyte with high sensitivity and specificity are required. For a competitive assay, the reagents will typically consist of one antibody and an analyte-protein competition reagent.
Antibodies can be sourced commercially or developed on a custom basis against a particular antigen. When commercially available antibodies exist, it is useful to screen as many unique candidates as economically feasible rather than investing heavily in the first promising antibody pair. Check clone numbers where available to make sure antibodies sold by different distributors are not the same material from the same original source.
Custom antibody development may be appropriate when suitable commercial antibodies are unavailable or when ownership and long-term control of the antibody clone are important. Although custom development requires additional time and resources, it can provide greater control over supply as an assay moves toward manufacturing.
Specificity should also be evaluated early. Antibodies that recognize related analytes or other components of the intended sample can generate false-positive results. Cross-reactivity testing should therefore be part of antibody down-selection rather than left until late-stage assay optimization.
Screen Antibodies in the Lateral Flow Format
Whether antibodies are commercially sourced or custom developed, they should be screened in the lateral flow format as early as practical. Antibodies can perform differently in lateral flow than in assays such as ELISA or Western blot because the kinetics, sample matrix, and assay conditions are substantially different.
In a lateral flow assay, the detector antibody must remain active after conjugation to the nanoparticle, tolerate drying and storage, and rapidly regain activity when rehydrated by the sample. Binding at the test line also occurs over a much shorter period than in many plate-based assays. ELISA can be useful for reducing a large pool of candidates, but promising antibodies should ultimately be evaluated under conditions that reflect the intended lateral flow assay.
Monoclonal vs. Polyclonal Antibodies
Monoclonal antibodies are homogeneous antibody preparations derived from a single clone and recognize a single epitope on an antigen. Polyclonal antibodies are heterogeneous mixtures produced by multiple B-cell clones and can recognize multiple epitopes on the same antigen.
Both formats can work well in lateral flow assays. Monoclonal antibodies are often selected as detector antibodies because their homogeneity can support consistent conjugation and specificity. Polyclonal antibodies may be useful as capture reagents because their recognition of multiple epitopes can increase opportunities for antigen capture. However, antibody performance is assay-specific, and the optimal detector-capture combination should be determined empirically.
| Polyclonal Antibodies | Monoclonal Antibodies | |
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Antibody Purification for Nanoparticle Conjugation
Before conjugation to a nanoparticle reporter, the antibody should be transferred into a buffer that is compatible with the selected conjugation method. Stabilizing proteins, salts, preservatives, and other formulation components can affect nanoparticle stability or interfere with coupling chemistry.
- Remove additional proteins such as BSA before nanoparticle conjugation.
- For passive adsorption, use a low-ionic-strength buffer and optimize pH for the antibody.
- For EDC/sulfo-NHS conjugation, avoid free primary amines from buffers such as Tris or glycine.
- Where possible, prepare antibody at a concentration of at least 1 mg/mL.
- Verify antibody concentration after purification or buffer exchange.
Antibodies can be buffer-exchanged using spin columns or dialysis tubing with an appropriate molecular weight cutoff. If an antibody formulation contains additional stabilizing proteins, an affinity purification method such as a Protein A or Protein G column may be required before final buffer exchange. Glycerol can also make antibody solutions more difficult to handle and is best avoided when possible.
After purification, verify the antibody concentration to ensure that the correct amount of protein is used during conjugation. Protein concentration can be measured using absorbance at 280 nm, a BCA assay, or a Bradford assay. At nanoComposix, we commonly use A280 when other proteins have been removed from solution.
For a detailed workflow, see our Antibody Purification Protocol. Our BioReady™ Conjugation Kits also provide reagents and materials for downstream passive or covalent nanoparticle conjugation.
Screening Antibody Pairs
For a sandwich assay, antibody pairs should be tested empirically to determine which detector-capture combination produces the best performance. Each antibody can be evaluated both as the detector antibody conjugated to the nanoparticle and as the capture antibody immobilized at the test line.
It is important to test both orientations of each pair. An antibody combination that works when antibody 1 is the detector and antibody 2 is the capture reagent may perform differently when those roles are reversed. The example below uses rows for detector antibodies and columns for capture antibodies.
| Antibody 1 | Antibody 2 | Antibody 3 | Antibody 4 | Antibody 5 | |
|---|---|---|---|---|---|
| Antibody 1 | — | 1 × 2 | 1 × 3 | 1 × 4 | 1 × 5 |
| Antibody 2 | 2 × 1 | — | 2 × 3 | 2 × 4 | 2 × 5 |
| Antibody 3 | 3 × 1 | 3 × 2 | — | 3 × 4 | 3 × 5 |
| Antibody 4 | 4 × 1 | 4 × 2 | 4 × 3 | — | 4 × 5 |
| Antibody 5 | 5 × 1 | 5 × 2 | 5 × 3 | 5 × 4 | — |
After preparing the conjugates, evaluate both conjugate stability and performance on the lateral flow strip. For each antibody pair, run a negative sample and a positive sample. Early screening is often easiest using analyte spiked into buffer rather than the final sample matrix, allowing the antibody interaction to be evaluated in a simpler system before matrix effects are introduced.
Promising antibody pairs should combine low non-specific signal in negative samples with strong test-line signal in positive samples. When several combinations perform well, additional factors become important:
- Cross-reactivity: Test structurally related analytes and other likely sample components early.
- Supply: Confirm long-term availability, bulk pricing, and commercial-use requirements with the antibody supplier.
- Conjugate stability: Confirm that the selected detector antibody remains stable after nanoparticle conjugation.
- Strip performance: Evaluate complete conjugate release, membrane clearance, background, and signal under the intended running conditions.
Control Line Antibodies
Both sandwich and competitive lateral flow assays typically include a control line that confirms that the sample and conjugate migrated through the strip correctly. The control line should generate a visible signal regardless of whether the target analyte is present.
A common approach is to use a secondary antibody that recognizes the species of the detector antibody. For example, if a mouse antibody is conjugated to the reporter nanoparticle, a goat anti-mouse antibody can be immobilized at the control line to capture the conjugate independently of the target analyte. If the detector antibody is derived from another species, the control reagent should be selected accordingly.
Next: Select the reporter nanoparticle