This essay appears as the introduction to Section 3, Assay System Components, in our 2012-2013 Buyers Guide.
|Table 1. Capillary flow times for Hi-Flow Plus membranes.
*The range is for all measured values on a roll and represents ±3∂.
The acceptable range for the mean is ±10% of the target.
Selecting, evaluating, and sourcing critical raw materials is a strategic step in the development and production of diagnostic kits. This is also a strategic task for company management, because an IVD company always must optimize its return on investment (ROI). Developing outstanding IVD kits or reagent performance requires a combination of sensitivity, precision, robustness, value, ease of use, and stability. A collaborative approach is key: critical raw materials suppliers now routinely meet not only with the R&D team (test performance) but also with the production (batch size, scale-up), quality control (specifications, audits), purchasing (prices, supply agreement) and legal (nondisclosure agreements, license and patent restrictions) departments.
From development to production, each IVD assay requires critical raw materials that can be divided into two main categories: biological (i.e., monoclonal or polyclonal antibodies, bio-blockers) and chemicals (i.e., microspheres, nitrocellulose membranes).
Given the critical role they play in IVD products, raw materials should be sourced from qualified, certified, and validated suppliers.
In this article, we focus on biological components (antibodies and blockers) and chemical membranes and microspheres, which represent the current product offerings of our OEM Diagnostics group.
Immunoassays depend on the precise sensitivity, specificity, and robustness of antibodies to detect and monitor disease.
To manufacture polyclonal antibodies, the immunogen (antigen) of interest is injected into an animal, often in combination with an adjuvant to increase the immune response. The antibody response can be enhanced by subsequent booster injections of the antigen with or without adjuvant. Blood samples are obtained from the animal to assess the level of antibodies produced, and once a sufficiently high titre has been reached, the antiserum is prepared by blood collection followed by serum preparation, with subsequent purification of antibodies from the serum, if required.
The level and quality of the antibodies produced will vary from animal to animal and over time. Careful consideration should be given to the appropriateness of the chosen species and strain. Larger animals should be considered when larger quantities of antibody are required. For speed and efficiency, it is typical to contract with a company that specializes in polyclonal antibody production. These sources should offer complete project design including immunogen production, testing, purification, and conjugation. Projects may be conducted using your protocol or a standard procedure.
Monoclonal antibody (MAb) technology, first developed in 1975, has become an extraordinarily important resource for the IVD industry. These antibodies have a single, selected specificity and usually are secreted continuously by immortalized hybridoma cells. A hybridoma is a biologically constructed hybrid of an antibody-producing lymphoid cell and a malignant (immortal) myeloma cell. The first step in the production of MAbs is immunization of a mouse with an antigen. When the mouse begins to produce antibodies to the antigen, its spleen is removed. Antibody-producing cells from the spleen are then fused with a myeloma cell line that is not antibody producing and has been maintained in culture. The new fused cell line, which produces antibodies, is grown briefly in culture and then re-injected into another mouse’s peritoneum. Finally, the ascites fluid, which contains monoclonal antibodies, is harvested from the mouse.
Since mice are small and require little space, and because techniques for making monoclonals are well known, companies may find it tempting to try to create the monoclonals in their own facilities. However, specialized facilities that have developed and manufactured thousands of monoclonal antibodies can provide state-of-the-art production, project management, and cell banking services that ultimately save time and money.
One of the significant challenges in diagnostic product development is the presence of heterophilic antibodies in patient samples that can cause false positive or false negative results. Clinical laboratories and immunoassay manufacturers must be aware of the potential for erroneous results caused by interference from endogenous antibodies. These include anti-animal antibodies, rheumatoid factors (RF), other auto-antibodies and heterophilic antibodies. They can bind to antibodies from other species, as well as other cell fractions or assay components, and can cause false positive and false negative results by changing the binding ability of the target analyte or assay antibodies. Incorrect diagnosis and unnecessary or incorrect treatment may result. Interfering antibodies may persist in a patient for months to years and vary in affinity and concentration over time.
Immunoassay interference from human anti-animal and other antibodies represents a true antigen/antibody reaction. It is the specific reactivity of these antibodies with those in immunoassays that can cause false positive or false negative results. This
|Table 2. IVD applications using microspheres and nanospheres.
Sizes are given in nanometers (nm).
LF (lateral flow), IT (immunoturbidimetry), LAT (latex agglutination test), CLIA (chemiluminescent immunoassay), NAT (nucleic acid technology)
specific interaction is distinct from nonspecific binding (NSB), which generally causes high background signals. With NSB, proteins including antibodies and target analytes stick to the solid support or other components of the assay. Blocking by means of high-quality inert proteins such as bovine serum albumin (BSA) can usually overcome NSB. By contrast, interfering antibodies need to be blocked using specific antibodies and/or normal antibodies from the same species as the antibodies used in the assay.
For some assays, passive blocking is effective and can be accomplished by the addition of normal serum, purified normal immunoglobulins (IgGs), or subunits from the same species as the detector and capture antibodies. Passive blocking reagents use normal animal IgGs and other components to essentially overwhelm and reduce the concentration of interfering antibodies by providing alternate binding sites to those of the detector and capture antibodies. Passive blocking reagents may be combined and/or used with active blockers to optimize formulations for specific assays.
High-concentration or high-affinity interfering antibodies may overwhelm passive blocking systems and necessitate a more targeted approach. An active blocking strategy uses targeted antibodies that bind to and neutralize specific potential interfering components. Blocking formulations may need to incorporate a multitiered approach and include purified antibodies of the same subclass, subunits, polymers, specific antibodies, or a cocktail of several components that may neutralize such factors as HAMA and RF at the same time.
In some assays, low concentrations of blocking agents may be effective while other assays require higher concentrations of blocking reagents. Blocking formulations must be considered with respect to the specific assay and developed with positive and negative patient samples and those known to contain interfering components.
Before starting test development, it is important to establish a basic product design. Numerous product qualities should be determined at the outset, so that resources are not spent on an unmarketable design. Therefore, 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.
The membrane is probably the single most important raw material used in a lateral flow test strip. Physical and chemical attributes of the membrane affect its capillary flow properties. The capillary flow properties, in turn, affect reagent deposition, assay sensitivity, assay specificity, and test line consistency. By way of illustration, Table 1 shows the capillary flow times of a range of membranes specifically designed for use in this application by Merck Millipore.
Other physical properties of the membrane affect integration into finished test strips. The polymer from which the membrane is made determines most of its binding characteristics. If the membrane undergoes a secondary process that chemically alters the polymer or buries it under a second polymer, protein-binding properties may be dramatically altered. For the most part, a membrane’s protein-binding capacity is determined by the amount of polymer surface area available for
|Table 3. Size reproducibility: nanospheres used in IT reagents.
*Size obtained from 6 different lots.
immobilization. The membrane’s surface area is determined by pore size, porosity (amount of air in the 3-D structure), thickness, and, to a minor extent, structural characteristics unique to the polymer. All other parameters being equal, surface area decreases in a nonlinear fashion with pore size, increases linearly with thickness, and increases in a nonlinear manner with porosity. For example, the surface area of a 0.1-µm nitrocellulose membrane is about 10× greater than the surface area of a 10-µm membrane, not 100× greater. In a typical test strip, the test line is 1 mm wide. If the strip is 1 cm wide, the amount of capture reagent that can be bound is 5 – 20 µg (0.1 cm wide × 1 cm long = 0.1 cm2). This is 10 to 100× greater than required for most assays. Thus, protein-binding capacity is normally not an issue in test design.
Manufacturing schemes range from entirely manual to completely automated setups and can have a large impact on the performance of a lateral flow test strip. For reproducibility, certain steps require a high level of consistency. They are:
1. Application of reagents onto membranes, sample pads, conjugate pads, and other porous media.
2. Consistent lamination of membranes, sample pads, conjugate pads, and absorbent pads onto a support backing.
3. Precision cutting of sheets or rolls into strips of defined length and width.
4. Assembly of test strips in plastic housings.
In summary, developing a lateral flow assay is complex because of the large number of critical components brought together to produce a functional test strip. Changing one material or reagent typically affects the performance of others. Ultimately, it becomes necessary to understand how each component contributes to the overall performance of the test strip.
In 1956, Singer and Plotz first described the RF test, which is based on agglutination of white microspheres. Since then, tests or assays to identify, detect, or quantify serum proteins, autoimmune diseases, microbial and viral infections, hormones, antibiotics, or drugs have been developed, produced, and marketed by many IVD companies worldwide.
Using microspheres in lieu of a coated tube or ELISA plate offers many advantages including a large surface area (fast kinetics and low detection levels) and choice of surface chemistries (better protein coverage and antibody orientation). Microspheres can easily be used as the solid phase to enhance detection or separation of the targeted analyte. They may be composed of different materials (mainly styrene or styrene/acrylate) and they come in various sizes, surfaces, colors, magnetic contents, and densities.
White plain polystyrene microspheres are extremely uniform with excellent lot-to-lot reproducibility. These microspheres are mainly devoted to hydrophobic or passive immobilization of molecules (polyclonal or monoclonal antibodies, proteins, haptens) onto their surface.
With white functionalized polystyrene microspheres, the introduction of highly polar or ionizable chemical groups allows the covalent binding of biological ligands (antibodies, proteins, or peptides) and increases the colloidal stability of the suspension with less auto-aggregation.
Dyed microspheres are also extremely uniform and available in red, blue, green, black, yellow, or pink color. Dyed internally, their surface properties are unchanged, ensuring maximum color brilliance and preventing dye leaching in water or aqueous buffers.
Made in polystyrene (PS) or in polyvinyltoluene (PVT), fluorescent microspheres should be photostable, monodisperse, and uniform in size. To prevent dyes from leaching and to provide the maximum active surface, the fluorescent dyes are physically and irreversibly entrapped inside the microspheres. For long-term storage, fluorescent microspheres are protected from light.
Superparamagnetic microspheres are made with a combination of polymer and magnetic pigment. The magnetization of the microspheres increases with the applied magnetic field and falls back to zero when the field is removed. Neither hysteresis nor residual magnetization is observed, providing practical advantages to the end user.
When the magnetic field is removed, the microspheres demagnetize and redisperse easily. This property allows efficient washing steps, low background, and good reproducibility. The behavior of the microspheres is constant regardless of magnetization cycles. Such behavior is a key point for automated instruments.
Selection of microspheres (>0.1 µm) and nanospheres (<100 nm) can be confusing when trying to decide between hundreds of options; a critical raw material supplier should be able to help make the best choice for each application.
It is well known that for some, if not the majority of, IVD applications, specific sizes are recommended (see Table 2). A supplier offering the largest range of sizes is the best option in terms of R&D requirements, as this will allow test optimization.
The breadth of choice among microspheres of different compositions (polystyrene, styrene/acrylate, styrene/divinylbenzene, styrene/butadiene) and surfaces (plain and chemically or biologically modified) suggests that it is often, if not always, necessary to optimize immunoassay conditions for each type of microsphere in order to achieve optimal signal-to-noise ratio, stability, and margins.
Plain polymeric microspheres or microspheres with a low COOH content (<30 µEq/g for a size of 1 µm) are typically made of polystyrene and offer a hydrophobic surface dedicated for passive coupling. This is the easiest and cheapest approach to attach IgG or other large proteins onto the plain microspheres. As a direct consequence, the plain microspheres are particularly prone to interference with other blood or serum components.
For that reason, some assays need to be developed using surface-modified microspheres for covalent attachment. Many of them are modified with functionalized surface groups, including COOH, NH2, OH, or SH groups. Those chemical groups need to be activated (EDC, glutaraldehyde) to obtain the optimal covalent linking of antibody or other molecules.
More recently, interest has grown in pre-activated microspheres. They are modified with functionalized surface groups like chloromethyl, tosyl, or epoxy. These very reactive chemical groups don’t need to be activated to obtain the optimal covalent linking of antibody molecules or other ligands.
Some immunoassays are developed with bioactivated microspheres as solid phase. Many of them are biomodified with specific ligands, including streptavidine, anti-mouse or anti-human IgG, protein A or protein G, to name a few. The improved assay performance is attributable to the more favorable and specific binding orientation of the antibodies or their fragments on the chemically or bioactivated microspheres compared with the seemingly random orientation obtained with plain microspheres.
This bioconjugation approach is easy but more expensive than other options. As in every industry, IVD companies must focus on the best ROI, and quality must always correlate with cost.
Quantitative and qualitative immunoassays based on agglutination of polymeric microspheres have been used as diagnostic tools for many years. An immunoturbidimetric assay offering high precision and fast reaction kinetics needs extremely uniform microspheres from 50 to 300 nm. For assay optimization, it is crucial to evaluate bead sizes with slight differences. To fulfill R&D requirements, a microsphere supplier should be able to produce several lots of the same material at a high level of reproducibility (Table 3).
Polymer microsphere is a critical raw material for reagent and kit manufacturers, and new lots of materials need control and validation, which require time and money. For some IVD companies, a small amount of material on the order of a few grams will satisfy their annual production needs; for others, several kilograms of raw material may be needed.
An industrial facility allows the manufacture of industrial batch sizes (>10 kg for white microspheres and >4 kg for magnetic microspheres), ensuring that the supplier can satisfy microsphere or nanosphere demand.
Critical raw material validation is often an expensive weeks- to month-long process. Once validated, the
supplier must be able to keep the material for one year
or more. It is important to offer a product with a long shelf life (i.e., more than five years). Then, the validated material is secured and available at any time for immediate delivery.
We recommend that IVD developers and manufacturers work with critical raw materials suppliers who exclusively produce components and not finished products or complete diagnostic kits. Indeed, the main objective of IVD manufacturers is to develop, produce, and sell IVD kits and not to tie up in-house resources with the development and production of chemical components. For this reason only a handful of IVD manufacturers continue to produce chemicals and, in particular, microspheres in house.
An industrial microsphere supplier must be able to provide a large range of microspheres and nanospheres as regular products. In 90% of cases, standard products should be able to fulfill specific requests. If not, suppliers should be able to offer custom development services. Using state-of-the-art technology, the technical department should provide customized microspheres that will meet very specific needs and requirements.
To maintain fulfilling, long-term relationships, we recommend that IVD customers review supplier performance.
A raw materials supplier should undergo a customer review at least once a year. A formal service-level agreement will help in the evaluation of a supplier relationship. Companies that don’t use this approach to define the level of service required from suppliers should ask these questions:
• Are you always satisfied with the quality of products supplied? Does your supplier offer the possibility of conducting quality audits?
• Are you getting the best price for the requested product quality? Does your supplier offer bulk prices for mass production? Are you getting special offers for evaluation or validation?
• Does the material arrive in good condition? Is your supplier punctual? If you need material urgently, what is the lead time? Does your supplier offer safety stock?
• Does your supplier respond promptly to purchase orders or technical requests?
• Does your supplier offer custom or new products that might improve product and business performance? Does your supplier help you to achieve a better market position than your competitors? Does your supplier help to anticipate technology changes?
• Does your supplier offer regular technical support and face-to-face technical meetings?
Alan Doty, Senior Product Manager, Antibodies and Blockers, and Shawn Gaskell, Product Manager, Membrane Products, Merck Group; and Fabrice Sultan, PhD, Sales and Marketing Manager, Estapor Microspheres