Major steps in LFA

3.1 Preparation of antibody against target analyte

Whether you can buy commercially available antibodies or relying on custom manufacturing, antibody selection is one of the most important steps in lateral flow assay design. Antibody cost, availability, sensitivity, kinetics, cross-reactivity and whether the antibody is monoclonal or polyclonal are all important factors when selecting an antibody. The first step is to research what antibodies are available for your specific application. The number of antibodies available for a specific analyte varies greatly. If an antibody is not available for the assay that you would like to develop, custom manufacturing of an antibody should be initiated as soon as possible. If antibodies are commercially available, the antibody selection may only include a few options, or there may be large selection of antibodies to choose from. As many antibodies as possible should be acquired to perform initial screening to determine which are most effective.

Typically a mixture of monoclonal and polyclonal antibodies will be selected for the first round of screening. Other considerations for down-selecting antibodies are the cross-reactivity characteristics, the immunogen used for antibody development, specificity, sensitivity, and any pairing information that the supplier might have regarding the specific antibody. Cost is also a factor as antibody costs can be a significant component of the bill of materials.

For conjugation of antibodies to nanoparticles, it is critical that the antibody is in the correct buffer. For best results, the antibody for conjugation should be purified and adjusted to a concentration of 1 mg/mL in a low ionic strength buffer. The concentration of antibody should be verified to ensure that the correct amount of antibody is being conjugated to nanoparticles. There are several ways to measure protein concentration including: absorbance at 280 nm, a BCA assay, or a Bradford assay.

3.2 Preparation of label   >> See more at technical note 3

3.3 Labeling of biorecognition molecules

Passive adsorption is the original method for attachment of proteins (antigens or antibodies) to lateral flow nanoparticle probes and is still widely used. The mechanism of passive adsorption is based on van der Waals and other attractions between the antibody and the surface of the particles. Small changes in pH can alter the association dynamics and affect the efficiency of conjugation. It is recommended that the pH of the adsorption buffer is slightly above the isoelectric point of the protein (which varies from antibody to antibody). A large excess of antibody with respect to nanoparticles surface area is typically used in order to ensure dense surface binding and high salt stability post conjugation. There are two main drawbacks to the passive adsorption. Firstly, every antibody requires slightly different conditions. Secondly, some antibodies may detach from the nanoparticle surface which can lead to a decrease in sensitivity and variable results.

Covalent coupling is more stable with less antibody desorption and requires fewer antibodies during conjugation. Amide bonds are typically utilized to connect a carboxylic acid functionalized nanoparticles to free amines on the antibody. This covalent bond is achieved through an EDC/Sulfo-NHS intermediary generated from a carboxylic acid surface particle.

Figure 4-1 EDC-NHS reaction scheme

3.4 Assembling

An overview of a typical card based FLA manufacturing process is shown in Figure 4-2. The basic processing steps for a cassette-based LFIA involve dispensing of reagents, drying of components, lamination of materials, cutting into strips, cassette assembly and packaging. Small scale production runs use backing cards that are 30 cm long and can be cut into strips with a 3 mm, 4 mm or 5 mm width. For highly reproducible striping of test and control lines, a dispenser with a flexible hollow glass fiber connected to a syringe pump is used. The conjugation is applied to the glass fiber conjugate pad using a non-contact spray head on the dispenser. Once the solutions are applied to both the nitrocellulose membrane and the conjugate pad, these components are dried and cured in the oven at 30 °C. After drying, the nitrocellulose, conjugate pads, sample pads, and wick are transferred to a dry room where they are attached to self-adhesive backing cards using a clam laminator. To assemble the 30cm master cards, the nitrocellulose is applied first, followed by the conjugated pad, wick pad and then sample pad. The laminator ensures an accurate placement of master card components with the correct overlaps and controlled pressure. The assembled cards are then cut into individual strips with a guillotine and assembled into a plastic cassette which is sealed in a foil lined bag with desiccant.

Figure 4-2 General procedure for the card-based assembly of a lateral flow device

3.5 Assay Optimization

There are many components that need to be meticulously optimized to develop a high sensitivity lateral flow assay. The optimization process includes choosing the appropriate antibody pair, conjugation conditions, sample pad and conjugate pad material and treatment, nitrocellulose membrane, test line concentration, wick pad material, running buffer, cassette, and sample volume. There is a relationship among all of these components that needs to be carefully balanced to produce an effective and functional assay. Accordingly, the development process is not linear. After each stage of optimization, the preceding stages often need to be revisited and reoptimized, resulting in an iterative and recursive process.

Usually a first step in the development of the assay is the optimisation of the concentration of the label and the recognition element. The second step is the optimal position of the test line. Both are aimed at optimal sensitivity of the assay. Strip material, the label and the detection strategy are usually the personal choice of the research group developing the assay. However, often the development of a new test is aimed at circumventing existing patents.

To develop a LFA, various factors, such as the test reaction rate, should be taken into consideration. For example, a rapid reaction rate affects the accuracy of the test and low reaction rate elongates the test time. The decrease in the reaction time causes insufficient time for antibody-antigen reaction. Because of this, a test should be slow enough for a sufficient antibody-antigen reaction. In brief, an increase in flow rate causes a decrease in the raction rate, in assay time, in the sensitivity of test, in the background signal, and an increase in the reagent usage. An increase in the distance from origin decreases the flow rate. The increase in the pore size of a nitrocellulose membrane decreases the amount of bound protein.

As it has been mentioned before, the optimization of the experimental conditions is important to develop a LFA with excellent performance and high sensitivity. Good sensitivity, low immunoreagent consumption, and good color intensity are important criterias for developing a LFA. When these points are considered together, the flow rate is the main factor for conducting LFA, which is affected by label and membrane pore size. The immunoreaction time depends on the migration time of buffer and the property of the nitrocellulose membrane. Another investigated optimization parameter was the amount of anti-detection antibody on the test line. The high concentration of anti-detection antibody on the test line causes nonspecific adsorption of the conjugates in the test line, resulting in a high background signal, and thus low S/N ratio is obtained. Apart from this, the excess amount of antibody on the test line increases the stereo-hindrance effect which may decrease the efficiency of immunoreactions. Another factor, which affects the sensitivity of LFA, is the amount of labeled-reaction antibody conjugates on the conjugate pad. The color intensities of the test and control lines are affected by the amount of labeled-reaction antibody. Because the captured labeled-antibody amount on the test and control lines is proportional to the amount of labeled reaction, antibody conjugates on the conjugate pad. The running buffer, which is used in the study, affects the response of LFA. The accurate selected buffer minimizes the nonspecific adsorption, increases the sensitivity and reproducibility of the LFA.

When optimizing an assay, the metrics to evaluate are increased signal intensity and elimination of non-specific signal. Because each antibody and conjugate is different, it is important to re-screen all parameters individually for each antibody. For covalent conjugations we generally recommend starting with a 2 hour incubation time. During optimization, shorter and longer incubations should be tested. In circumstances where you are limiting the number of antibodies per particle rather than saturating the surface (i.e. competirive assays) we generally recommend a shorter incubation time (as short as 5 minutes) before quenching to reduce the chances of antibodies folding and binding to several available acid groups on the surface, in turn decreasing antibody functionality. Covalent conjugation is a great choice if you’d like to control the amount of antibody per particle. When decreasing the number of antibodies on the surface, it may be desirable to use a short incubation time, as mentioned above. Always be sure to quench (stop the reaction by adding a solution containing primary amines such as tris, glycine, hydroxylamine etc.) any remaining NHS-ester prior to processing to avoid crosslinking of particles. Conjugate diluent components will vary significantly depending on sample media. These will need to be investigated and optimized when moving from a clean system (analyte spiked into buffer) to a clinically relevant sample. We recommend using real samples as early as possible during development. A suitable starting point for the conjugate diluent includes salt buffer, blocking agent and surfactant. Tween is typically reduced if non-specific binding is observed, and increased if there is reduced signal intensity at the test and control lines. Each component will likely need to be optimized for each individual assay. If high concentrations of detergents are required, it is recommended to use the more robust, covalent attachment for your conjugate so that the detergent doesn’t negatively interfere with colloidal stability.

It is important to introduce your analyte in its matrix early in assay development. Obstacles generally arise when switching from a “clean” analyte + buffer system to using actual clinical samples (saliva/plasma/stool etc.). Most sample matrices are very complex and will require investigating blocking agents and detergents in an effort to minimize sample variation.

Colored detector reagents, such as gold nanoparticles (AuNPs), colored latex beads, magnetic particles (MPs), carbon nanotubes (CNTs), quantum dots (QDs), and enzymes are used as labels for the construction of LFA. They provide sensitive analysis and maintain the conjugation between analyte and biorecognition elements. The key factors for chosing label are that the label does not change the properties of biorecognition element and the label must provide stable conjugation with biorecognition element. The use of label is important for sensitive analysis. The optical properties of label enhance the sensitivity of LFA. Gold nanoparticles have been mostly used in literature due to their good optical properties. They have high affinity to biomolecules and the functionalization of AuNPs is easy. An alternative biorecognition molecule of antibody is immunogen, which is a cheaper biomolecule than an antibody, since the production cost of antibody is high. The binding of antibody to colloidal gold occurs by means of hydrophobic residues in the antigen-binding site of antibody. This way, the binding sites of an antibody are decreased. This problem can be solved by using immunogen instead of antibody.