IVD Lateral Flow Assay Development

Making IVD Lateral Flow Test Strips for Analytes

Lateral flow test strips based on the principles of immunochromatography exist for a wide array of target analytes. The first tests were made for the detection of human chorionic gonadotropin (hCG). Today, commercially available tests monitor ovulation, detect infectious disease organisms, analyze drugs of abuse, and measure other analytes important to human physiology. Products have also been introduced for veterinary testing, agricultural applications, environmental testing, and product quality evaluation. While the first tests presented qualitative results based on the presence or absence of a signal line, test design has progressed toward semi-quantitative and quantitative assays and the integration of hand-held readers.

Most lateral flow test strips are modeled after existing immunoassay formats. Thus, test strips for hCG are typically sandwich assays; and drugs of abuse are analyzed by competitive or inhibition assays. In serum assays, antibodies are detected as indicators of various disease states. Many variations are possible, but they all have in common the formation of a complex between a detector particle that is free in the sample stream and a capture reagent that is bound to the membrane at the test line (Figure 1).

Figure 1. Lateral flow diagram

Taking a lateral flow test strip from the design stage through product development to final manufacturing is a process that employs principles from biology, chemistry, physics, and engineering. A test design that works well in the R&D lab is of little use if it is difficult to manufacture reliably in high yield. Similarly, even though a product may work well during in-house testing, if it fails to work reliably in the field, it is unlikely to be successful. Uncharacterized variations in a single material, reagent, or process can be sufficient to ruin consistency of performance.

With these considerations in mind, the purpose of this guide is to provide information on the key aspects of immunochromatographic test strip design, particularly the materials used and their integration with reagents and manufacturing processes. Understanding these principles will help to provide a framework for more rapid test development.

Getting Started

Product Description

Before starting test development, it is important to have a basic product design established. There are numerous product qualities that should be determined at the outset so that resources are not spent on a design that isn’t marketable. Necessarily, market information will be required to establish certain performance criteria. Key test parameters include: detection limits (sensitivity), required specificity, assay speed, detection format, detection system, test line format, results interpretation, sample matrices, housing design, packaging, labeling, stability requirements, and target cost. It is also critical to understand how much time is available for the test development program.

Antibodies and Other Capture Reagents

Antibodies used in immunochromatographic tests must have sufficient sensitivity, specificity, purity, and stability to accomplish the performance requirements of the finished product. Depending on the assay design, antibodies may be used as a capture reagent at the test line, as a conjugate on the detector particle, or both. As a manufacturing consideration, the antibodies should be available in sufficient supply to meet production forecasts. Purification and consistency of supply are also important. Since the antibodies may be bound to a membrane and detector particle, contaminating proteins will compete for binding sites. There is also the decision to be made as to whether polyclonal or monoclonal antibodies will be used. Minimally, the antibody preparation should be affinity-purified.

A key consideration in antibody selection is the chemical extremes to which the antibodies will be subjected during test strip manufacture and storage. Minimally, the antibody must remain reactive after being adsorbed to a solid surface, retain its structural integrity when completely dried, and then be instantly reactive when rehydrated by the sample. Antibody systems derived from other assays such as ELISA or western blots may not meet these requirements, necessitating screening for alternatives. Also, strategies used to stabilize antibodies in solution are often incompatible with immunochromatography tests.

While formation of an immunocomplex at the test line is most commonly used as the result indicator, it is theoretically possible to achieve a result using any ligand recognition system where a detector particle becomes bridged to a capture reagent on the membrane. In such systems the reagents employed, whether purified from a natural source or prepared as a synthetic construct, are subject to the same requirements for sensitivity and stability as antibodies. It is important to know how these reagents are prepared, what contaminants may be present in the ‘purified’ product, and what lot-to-lot variations can be anticipated. Purity should not be assumed, but rather demonstrated by the supplier using relevant analytical methods.

Detector Reagents

Various types of detector reagents can be used for the visualization of a signal. The most commonly used materials in commercially available tests are latex beads and colloidal gold particles. Other possibilities include enzyme conjugates, other colloidal metals, fluorescent particles, and magnetic particles. One of the most important features of the particles is that the population is monodisperse with consistency of size and spherical shape. When a test is run, the particles are required to move through the torturous pore structure of the membrane.

Smaller particles move faster than larger particles. Particle preparations with different size and shape distributions will move through the membrane differently. This can lead to differences in apparent sensitivity and specificity, even when all other components of the test are identical.

Methods for preparing various particles and conjugating antibodies to them can be found in the literature.

Commercial sources are also available. Since the conjugated detector particles are one of the key reagents in the finished test strip, methods for their preparation and handling should be fully validated. Similarly, relevant quality control methods need to be established. If they are purchased from a vendor, they should come with specifications on the attributes relevant to performance in lateral flow tests.

Licensing Agreements

There are many patents covering technologies, formats, reagents, and materials that may be of great value in the development of immunochromatographic test devices. Test developers may need to consider licensing one or more of these patents prior to the commercialization of final product. We do not provide legal counsel, nor can we assign rights for any of the patents that have been issued. A list of pertinent patents can be found in Table 1.

Some patents (e.g., US4855240, US4703017, US4376110 and EP0810436A1) are broadly based and may apply to many different test formats. Other patents are more restricted and may not apply based on reagent selection and test device formatting. Finally, there may be additional patents that pertain to immunochromatographic tests and immunodiagnostic assays in general. To comply with international trade and licensing agreements and to prevent possible legal problems after product launch, it is prudent for test developers and manufacturers to review the patent literature prior to commercialization.

Table 1. Partial List of Patents Relevant to the Development and Commercialization of Immunochromatographic Assays

Patent No.	Assignee & First Inventor	Priority Date	Brief Description of the Invention/Application (Abbreviated Primary Claim)
(US) 4313734	Akzonn, Inc. JHW Leuvering	01 FEB 82	A method, test kit, and labeled component for the detection and/or determination  of one or more analytes. The labeled component is comprised of colloidal metal (e.g., gold), > 5 nm in diameter to which a detector reagent (antibody or antigen) is coated.
(US) 4376110	Hybritech GS Davis	08 MAR 83	Immunometric analysis for detection and determination of antigens that uses two monoclonal antibodies to form a sandwich ternary complex. This is a reagent patent that applies to all immunometric (sandwich-type) assays.
(US) 4435504	Syva Company R Zuk	06 MAR 84	Chromatographic immunoassay employing a specific pair member and a label conjugate which delineates a border whose distance from one end of the chromatography relates to the amount of analyte present
(US) 4703017	Becton Dickinson RL Campbell	27 OCT 87	Solid phase assay for an analyte wherein binder is supported on a solid support (e.g., nitrocellulose membrane) and the tracer is comprised of ligand labeled with a colored particulate.
(US) 4855240	Becton Dickinson RW Rosenstein	08 AUG 89	Test device and assay for determining analyte wherein tracer and sample may be simultaneously applied to different absorbent material portions both in capillary flow communication with a solid phase (e.g., nitrocellulose membrane) with binder for analyte.
(US) 4954452	Abbott Laboratories DA Yost	04 SEP 90	A method of performing a diagnostic immunoassay utilizing colloidal non-metal (e.g., selenium, tellurium, sulfur, etc.) particles having conjugated thereto a binding component capable of specifically recognizing an analyte to be determined.
(US) 5028535	Biosite KF Buechler	02 JUL 91	Ligand assay with controllable thresholds for signal production by competitive reaction with labeled conjugate and receptor (e.g., antibody), both present in predetermined and controlled amounts.
(US) 5075078	Abbott  Laboratories EW Osikowicz	24 DEC 91	Self-performing immunochromatographic device: Chromatographic test strip containing immobilized reagents that produces distinctly different read-outs (e.g., –, +) depending on the medical diagnosis.
(WO) 95/16207	SmithKline Diagnostics, Inc. HM Chandler	07 DEC 93	An assay device for detection and determination of an analyte in a test sample that uses a barrier containing an aperture to control the application and flow (including in the reverse direction) of reagents, including the sample.
(US) 5654162	Bio-Metric Systems PE Guire	05 AUG 97	A device capable of generating a signal indicative of the presence of an analyte, the device comprising a liquid permeable solid medium to which a reactant that can bind the analyte is immobilized and a detector reagent (labeled antibody) that can also bind to the analyte.
(EP) 0810436A1	Unilever PLC PJ Davis	03 DEC 97	An analytical test device incorporating a dry porous carrier to which a liquid sample suspected of containing an analyte can be applied indirectly, the device containing a mobile detector reagent and an immobilized capture reagent, and the means to control and detect analyte and detector reagent binding.

Have a Manufacturing Plan in Each Phase of Development

Manufacturing schemes range from entirely manual to completely automated. For reproducibility, there are certain steps that require a high level of consistency.

Choose the Right Manufacturing Process

The choice of process depends primarily on the amount of product to be manufactured and the funds available to staff and equip the manufacturing facility.

• Manual – process materials in a batch mode.
• Automated – process materials in a continuous mode.
• Combination of manual and automated.

Key Elements for Manufacturing Consistency

Consider the following elements of the manufacturing process to ensure consistency from R&D through manufacturing.

• Consistent lamination of membranes, sample pads, conjugate pads, and absorbent pads onto a support backing.
• Precision cutting of sheets or rolls into strips of defined length and width.
• Control for consistency of reagent application onto membranes, sample pads, conjugate pads, and other porous media.
• Placement of test strips in plastic housings.

Minimize Waste, Maximize Yield

And finally, when designing the assay and planning for progression to a manufactured product, use the following steps to minimize waste and maximize yield.

• Match lot sizes of materials and reagents during planning.
• Continuously train personnel to ensure proper execution of manufacturing processes.
• Continuously conduct in-process inspections to ensure sub-assemblies are being manufactured to specification.
• Ensure that materials chosen are compatible with manufacturing machinery so they perform predictably in the finished device.