Originally Published IVD Technology October 2004
Biochemicals and chemical reagents
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| Choices of colloidal gold, antibodies, antigens, and other reagents in diagnostic applications must be made carefully. |
The basic performance characteristics of immunoassays are defined by the interaction
of a variety of material, chemical, and biological components. The proper function
of even the simplest devices (e.g., lateral-flow assays) during their entire
shelf life may require the appropriate interaction and performance of dozens
of component materials and reagents. Carefully selecting reagents and reagent
suppliers is critical to ensure the proper function of the device and the long-term
manufacturability and performance of the final product.
The biochemicals and chemical reagents category encompasses a broad range of
assay components. This article will examine the major systems in a lateral-flow
assay. Some of the obvious as well as less-obvious places and ways in which
reagents are used will also be discussed. In addition, the issues associated
with sourcing reagents and choosing suppliers in light of recent developments
in the lateral-flow marketplace will be presented. Most of what is discussed
in this article regarding lateral-flow assays applies to the broader field of
diagnostics, including other immunoassay formats, molecular diagnostics, and
other areas.
Lateral-Flow Assay Systems and Components
A lateral-flow assay comprises several defined systems and components. Such
systems require treatment with biochemicals and chemical reagents to make sure
the assay performs correctly.
The sample-application pad is made of cellulosic paper, glass fiber, or polyester
material. In this pad, IVD manufacturers are able to perform chemical and biological
treatments on a sample before it reaches the conjugate pad. Such treatments
might include removing red blood cells without lysis, lysing red blood cells,
filtering out particulates, changing a sample’s pH, or removing mucins
or other interfering components from a sample before it reaches the conjugate
pad. In one particular treatment method, manufacturers can use surfactants to
make the pad hydrophilic, particularly if it is a glass fiber or polyester pad.
IVD manufacturers can add polymers to harden the pad and control the flow rate.
Manufacturers can also add antibodies or other biochemical reagents to capture
red blood cells or mucins. In this case, IVD manufacturers include buffering
components to ensure that a sample will be at an appropriate pH when it reaches
the conjugate pad.
The conjugate release pad is also made of glass fiber or polyester material.
With this pad too, IVD manufacturers can add surfactants and polymers to control
flow and make the material hydrophilic. Moreover, manufacturers can add other
critical reagents to the conjugate release pad. The most important reagent is
the conjugate itself. The conjugate may be a biological component (e.g., antibody,
antigen, hapten) that is bonded to a visible label (e.g., colloidal gold, colored
latex particle), or an invisible label (e.g., paramagnetic particle). IVD manufacturers
add the conjugate to the pad by immersion or dispensing with a quantitative
dispenser. The dispensing is done in a sugar solution that stabilizes the conjugate
and assists in its release from the conjugate pad. Manufacturers place the conjugate
in a buffering solution to make sure that reactions take place at a proper pH.
The membrane substrate system usually consists of a nitrocellulose membrane,
while other membranes are less commonly used. IVD manufacturers dispense the
test and control lines on the membrane with antibodies or antigens that are
sometimes coupled with anchor proteins (e.g., avidin, streptavidin, biotin).
The membrane is blocked using blocking buffers that contain bulk proteins, (e.g.,
casein, bovine serum albumin) and treated with surfactants for its long-term
stability and flow characteristics. Manufacturers add certain reagents to the
striping solutions to ensure more-consistent dispensing and binding, and prevent
hydrophilicity at the test and control lines (Tween 20 is used at very low concentrations).
IVD manufacturers can also use low concentrations of alcohols to precipitate
proteins onto the membranes to assist in binding.
Depending on the assay, a sample may require pretreatment. Such pretreatment
ensures that the correct antigenic sites are available for the assay, or removes
interfering components before adding a sample to it. Samples will often be run
in a running buffer or with a chase buffer so that the test functions correctly
and the assay goes to completion. Using these buffers also reduces nonspecific
binding and general background noise in the test. To accomplish such functions,
IVD manufacturers may use buffering salts that are coupled with polymers, surfactants,
and biological reagents.
IVD manufacturers should also realize that, in addition to the chemicals that
may be included in a lateral-flow assay, material suppliers have already added
other chemicals into the systems and components they provide. Such chemicals
may include surfactants in membranes, binders in glass fibers or polyesters,
and adhesives in backing materials. All of these chemicals can affect an assay,
sometimes positively and sometimes not, and should be considered when designing
assays and choosing materials.
Selecting Reagents and Suppliers
Choosing the right reagents for lateral-flow assays and the right suppliers
of such reagents is critical. Consequently, certain important questions should
be addressed: What factors should IVD manufacturers take into account when selecting
the biochemicals and chemical reagents they use in lateral-flow assays? What
should manufacturers look for in the suppliers that provide such component materials?
Purity, Quality, and Reproducibility. All of these terms are relative, depending
on the reagent. With antibody supplies in particular, purity is a crucial issue.
IVD manufacturers choose between monoclonals and polyclonals based on availability
and performance issues. While manufacturers often prefer monoclonals, they may
not be available. Performance or cost concerns may also force the use of polyclonals
in at least half of a lateral-flow assay. When using monoclonals, IVD manufacturers
should consider the following: Is the antibody in ascites or culture? How is
it being purified? Is it necessary to worry about anti-id or interfering anti-species
antibodies? Is the clone securely backed, or are there risks involved due to
how the clone is being handled? Alternatively, when using polyclonals, manufacturers
should consider the species, cross-reactivity issues, and the lot-to-lot reproducibility
of the antibodies.
Stability. In this area, IVD manufacturers need to address certain issues: Are
the supplies and suppliers stable? Will the suppliers provide a pricing agreement
that insulates manufacturers against severe market fluctuations?
Price. Cost is always a crucial element, particularly with lateral-flow assays
since they use more antibodies per assay than, for example, enzyme linked immunosorbent
assays (ELISA). In a standard lateral-flow assay, 0.1–1.0 micrograms of
antibody are used per assay on a test line. Using expensive monoclonal antibodies
that cost $500 per mg is clearly not economically feasible, especially if they
are used for the capture line.
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| Diagnostic Consulting Network LLC (Irvine, CA) is a provider of antibodies, antigens, colloidal gold, and streptavidin (shown above). |
In addition to capture-line antibodies or antigens, the other major reagent
costs are the conjugates. An important decision for IVD manufacturers is whether
to produce the labels, (e.g., colloidal gold) and the conjugates in-house. From
a cost perspective, manufacturing colloids and conjugates in-house could result
in huge cost savings. However, if implemented without considering technical
capacity and quality assurance, the results can be disastrous, with the potential
for final product rejection or even product failure in the field.
When choosing a supplier of labels (e.g., latex, gold colloid, paramagnetic
particles), and conjugates or custom conjugation services, IVD manufacturers
should make sure that technical and quality issues can be resolved, and that
pricing in the long term will be acceptable. Some conjugate manufacturers try
to bundle conjugate pricing into other services, such as contract manufacturing.
Manufacturers should be cautious with this approach since few companies have
the ability to provide excellent service in areas that require diverse skill
sets. Good technical companies do not always make good contract manufacturers,
and vice versa.
On Demand and Long-Term Availability. IVD manufacturers should consider whether
a supplier has the materials in stock on a regular basis, and whether they will
experience long lead times each time an order is placed.
Technical Support. Does a supplier have the ability and willingness to work
with IVD manufacturers when technical difficulties that may be related to the
reagents arise? This is a crucial decision point. The suppliers’ capabilities
must be known and evaluated. Like any good equipment or materials supplier,
a good reagent supplier should want to build a relationship with the manufacturer,
and act as a partner in targeting product and company growth, and assisting
in problem solving whenever possible.
Supplier Focus and Ultimate Source. IVD manufacturers should know about their
reagent suppliers and the products they are providing. Is the supplier a primary
manufacturer, or is it sourcing the reagents and reselling them? If the supplier
is sourcing the reagents, does it have a supply agreement with its supplier?
Will the supplier’s prices fluctuate, or will its supply disappear?
Having suppliers that are focused on the IVD industry is ideal, although not
always possible. Large chemical companies, from which ultimately a portion of
surfactants, salts, and polymers are sourced, sometimes cannot control their
products to the degree that the IVD industry would like. This is not a reflection
on the quality of the supplier or the product per se, but rather a difference
in focus. As a result, IVD manufacturers should be careful when switching bulk
chemical suppliers and when changing bulk chemical lots. Lot-to-lot performance
matching can be crucial to ensure proper performance of the final assay product.
Market Trends and the Effects on Reagent Requirements and Suppliers
The developmental focus of the lateral-flow assay market has been shifting.
The market used to focus almost solely on qualitative assays in the clinical
market for infectious diseases and a few biochemical markers. Today, there is
a trend toward semiquantitative and fully quantitative assays for biochemical
markers, as well as in markets not related to clinical healthcare, including
veterinary, environmental, and food-related applications. As a result of the
shift toward quantitative assays in particular, issues of reproducibility and
process CVs are coming to the forefront of IVD manufacturers’ development
processes in ways not before seen. This in turn places requirements on the biochemical
and chemical reagent suppliers to improve their products’ reproducibility
and performance. The value of lateral-flow assays in areas other than clinical
diagnostics has been gaining recognition. In addition to the increased globalization
of the IVD marketplace and a limited pool of experienced developers of lateral-flow
products, the demand for technology transfer of developed products to third
party manufacturers worldwide has been growing during the past few years. Under
a technology transfer scenario, a company wishing to enter the lateral-flow
market or expand its existing product lines without the expense of doing in-house
development will approach service companies that offer developed assays for
sale and transfer.
Technology transfer consists of transferring the manufacturing standard operating
procedures (SOPs) to the client company, installing the necessary equipment,
establishing the manufacturing facility and processes, training manufacturing
personnel, and providing ongoing consulting services. By using such service
companies, manufacturers can enter the marketplace quickly and economically
with a functional product. In this scenario, manufacturers must be aware that
they are purchasing a set of SOPs that recommend using a particular set of biochemicals
and chemical reagents. Manufacturers should perform due diligence to ensure
that the reagent supply is secure, the quality is assured, and pricing is acceptable
and guaranteed for a period. Such reagents are the lifeblood of the product
that the manufacturer has purchased. Manufacturers should make sure that the
technology transfer service companies can guarantee an ongoing reagent supply.
Conclusion
In a constantly shifting and growing marketplace, as technology races ahead
and IVD manufacturers race along with it, a few things still remain constant.
A diagnostics product is only as good as the reagents used in its production,
and the reagents are only as good as the supplier of such reagents. Manufacturers
should take time to perform due diligence early in the development cycle while
forming partnerships with reagent suppliers. Doing so will pay off in the long
run.
Michael Surmanian,
Diagnostic Consulting Network LLC (Irvine, CA)
Copyright ©2004 IVD Technology
