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Immunochromatographic, Lateral Flow or Strip Tests Development Ideas

Provided by Bangs Laboratories, Inc. 9025 Technology Drive, Fishers, IN 46038-7034; e-mail: info@bangslabs.com; web site: bangslabs.com

About the Technology

Immunochromatographic assays, also called lateral flow tests or simply strip tests, have been around for some time. They are a logical extension of the technology used in latex agglutination tests, the first of which was developed in 1956 by Singer and Plotz.1 The benefits of immunochromatographic tests include:

1. User-friendly format.
2. Very short time to get test result.
3. Long-term stability over a wide range of climates.
4. Relatively inexpensive to make.

These features make strip tests ideal for applications such as home testing, rapid point of care testing, and testing in the field for various environmental and agricultural analytes. In addition, they provide reliable testing that might not otherwise be available to third world countries.

The principle behind the test is straightforward, and will be discussed in greater depth in a subsequent section. Basically, any ligand that can be bound to a visually detectable solid support, such as dyed microspheres, can be tested for qualitatively, and in many cases even semi-quantitatively. Some of the more common lateral flow tests currently on the market are tests for pregnancy, Strep throat, and Chlamydia. These are examples of conditions for which a quantitative assay is not necessary.

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Reaction Schemes

The two predominant approaches to the tests are the Non-Competitive (or direct) and Competitive (or competitive inhibition) reaction schemes. These can best be explained graphically, as shown in Figures 1 and 2:

Direct (Double Antibody Sandwich) Reaction Scheme


Figure 1


This format is used when testing for larger analytes with multiple antigenic sites, such as LH, hCG, and HIV. In this case, less than an excess of sample analyte is desired, so that some of the microspheres will not be captured at the capture line, and will continue to flow toward the second line of immobilized antibodies, the control line. This is species-specific anti-immunoglobulin antibodies, specific for the conjugate antibodies on the microspheres.

Competitive Reaction Scheme


Figure 2

This is used most often when testing for small molecules with single antigenic determinants, which cannot bind to two antibodies simultaneously. If this format is chosen, it is important to pay close attention to the amount of antibody bound to the microspheres, in relation to the amount of free antigen in the sample. If the sample does not contain an excess of free antigen, some of the microspheres will bind at the capture line, giving a weak signal, and making the test result ambiguous.

Typically, the membranes used to hold the antibodies in place are made up of primarily hydrophobic materials. Both the microspheres used as the solid phase supports and the conjugate antibodies are hydrophobic, and their interaction with the membrane allows them to be effectively dried onto the membrane. These hydrophobic interactions are very reliable, so much so, that getting the hydrophobically bound antibody/microsphere complexes to enter into the mobile phase upon sample introduction can be difficult. One variation to the above reaction schemes which has been proposed is the “Boulders in a Stream” approach2. This gets around the problem of protein-coated microspheres sticking to the membrane non-specifically by using a membrane that is inert, and does not bind antibodies. This makes migration of the mobile phase antibodies very efficient and reliable. The capture antibodies, rather than being physically bound by the membrane, are attached to large microspheres, which will be held in place physically, rather than chemically, as the sample passes by, much like boulders in a stream. This can be used for both of the above-mentioned reaction schemes, and is diagrammed below (Fig. 3).

Figure 3

These principles are well-documented in the literature, and appear very straightforward. However, in order to maximize efficiency and minimize development and production costs, there are some guidelines which, if followed, could possibly reduce some of the hurdles normally associated with the development of a new technology.

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Materials

Antibodies (three types):
  1. Stationary Phase
    1. Capture Line Antibodies
    2. Control Line Antibodies
  2. Mobile Phase
    1. Conjugate Antibodies (Antibodies on dyed microspheres, to which the sample analyte will bind initially.)
      (If you are testing for small molecules using the competitive binding format, you will also need purified antigen, or an antigen/carrier molecule (BSA) conjugate, for attachment to test lines.)

Membranes (dependent on the approach that you choose, as previously mentioned) Some options for this include:

  1. Nitrocellulose (High Protein Binding)
  2. Cellulose Acetate (Low Protein Binding)
  3. Glass Fiber Membranes (Non-Protein Binding)

Membrane manufacturers generally offer a wide variety of material types and pore sizes, so it is a good idea to investigate several options before deciding which specifications most closely match your test objectives.

Microspheres (several sizes and polymers to choose from) Conjugate antibody or antigen is attached, and microspheres migrate down the membrane upon introduction of your sample. Some hints to choosing an appropriate particle are as follows:

  1. Optimal flow rate is achieved by choosing microspheres 1/10 the pore size of the membrane through which they will migrate, or smaller.
  2. Optimal colors for visualization in various types of samples:
    1. Whole Blood: Black or Dark Blue
    2. Serum: Bright Red or Bright Blue
    3. Urine: Green, Blue, Red, or Black
    4. Saliva: Any Dark Color
    5. Cerebral Spinal Fluid: Any Dark Color
  3. To minimize hindered flow caused by the inherent hydrophobic interactions between membrane and particle (in the case of a hydrophobic membrane), pretreatment of the membrane with a substance that will maintain a small distance between the microspheres and the membrane, yet which can be easily rehydrated, is often helpful (Fig. 5). Examples of substances commonly used are sucrose, various water soluble inert polymers, and surfactants. The idea is to choose a substance that is stable in dry form, yet dissolves easily upon rewetting to allow the antibody bound microspheres to flow easily through the membrane upon addition of the sample. (A sample procedure for doing this is included later in this text.)
In addition to treating the membranes, the microspheres themselves can also be pre-treated with surfactants, synthetic or protein-based blockers. If done correctly, this can also help to reduce the problem of reagent mobility upon sample introduction. Much work has been done in developing optimum mixtures of these various polymers, detergents, and blockers.

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Reaction Kinetics

Now that we have looked at the principles behind these tests and some specifics regarding their manufacture, let’s consider some of the factors involved in choosing the appropriate raw materials.

Test developers are often concerned with reaction kinetics. A faster test will not only be more attractive commercially, but often will be more accurate. On the other hand, the test must proceed slowly enough that antibody/antigen reactions are able to occur. Some principles that govern the kinetics of immunochromatographic assays are as follows:

  • The reaction rate decreases with the square of the increase in flow rate.
  • Assay time decreases with increasing flow rate.
  • Sensitivity decreases with the square of the increase in flow rate.
  • Reagent usage increases with increasing flow rate.
  • Background (streaking on the membrane prior to the capture antibody line) decreases with increasing flow rate.
  • Flow rate decreases as distance from the origin increases.
  • The amount of protein bound decreases (for nitrocellulose membranes) as the pore size increases.
    Therefore, while increased flow rate is generally desirable, and it is known that one of the major influences affecting this is the relationship between microsphere and membrane pore size, there is a point of diminishing returns in trying to increase this variable. The above principles should be closely examined and weighed against each other in the research phase before deciding on the exact parameters for the final product.

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Calculations

Some of the variables to take into consideration when setting up a lateral flow test include:
  • Flow rate of membrane. This is determined empirically, and will vary according to the viscosity of the sample used. Data for the flow rates of specific membranes with specific sample types are supplied by the manufacturer.
  • Membrane porosity. This describes the fraction of the membrane that is air (e.g. a membrane with a porosity of 0.7 is 70% air.), and will have an impact on the flow rate of the membrane.
  • Membrane capacity. By definition this is the volume of sample that can pass through a given membrane per unit time, and is determined as a factor of the length (L), width (W), thickness (T), and porosity (P) of the membrane: L x W x T x P = Membrane Capacity
  • A second important calculation is the determination of the Amount of antibody that can be bound, per unit area of Membrane (pertaining to the capture and control lines). This calculation involves the following variables:
    1. Dimensions of representative capture antibody line: 0.1 cm x 0.8 cm= 0.08 cm2
    2. Binding capacity of membrane used for capture antibody (obtained from the membrane manufacturer). In this example, we will use 50 µg/cm2- a low end estimate for nitrocellulose membranes.
Therefore, the binding capacity of the membrane for the capture antibodies is simply a factor of these variables: 0.08 cm2/line x 50 µg/cm2 = 4.0 µg/line
This is a theoretical example, but from past experience we have learned that in practice, a tenth of this is normally sufficient. Therefore, as with all theoretical calculations, they can provide a baseline which is optimized for the specific conditions and reagents involved in each particular test.

There are other calculations involved in setting up this type of test, some of which are not within the scope of this text. However, the suppliers of the various raw materials are normally good sources for this information, and are generally happy to help ease the development process. For example, some important considerations involving the microspheres are the best type of binding, (covalent attachment or simple adsorption), as well as the proportion of antibody to microspheres for best sensitivity in the final product.

Useful information regarding this can be found in our TechNotes #13a “Adsorption Protocols” and #13c “Covalent Coupling Protocols”, both of which can be either downloaded from our web page or supplied in hardcopy form at no charge. Another good source for further information is a list of related references, which is supplied at the end of this note.

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Future Trends

The technology involved in these lateral flow tests is exciting in and of itself, in that it provides an accurate, easy to use, rapid diagnostic tool. Currently, the principles governing this test are being extended to allow for some exciting new possibilities for future tests. Some development possibilities that are currently being evaluated are:
  1. By using the same format for lateral flow tests and dyeing the solid support with a fluorescent dye, the possibility exists to create a truly quantitative test. If the spectral properties of the dyed microspheres to which the antibodies are conjugated are known, the amount of antibody bound at the capture line can be precisely quantified using a fluorimeter. The benefits to this would include those of all lateral flow tests that currently exist. In addition, the tests could, theoretically, become truly quantitative assays.
  2. By placing multiple lines of capture antibodies on the membrane, each for a different analyte, one can develop a single test for more than one analyte. An obvious application for this would be to create a drugs-of-abuse test panel. Biosite’s ‘Triage’ is based on this format.3 Diagnostically, this principle could be used for panels for which multiple analytes can be tested, such as immune diseases, allergies, or even Multiple Chemical Sensitivity Disorder. Also, as the technology involved in preparing these tests continues to develop, it should be possible to combine both of these ideas, 1) and 2), to make a low-cost, rapid quantitative diagnostic assay for multiple analytes.
  3. Another exciting application of this technology is in the environmental field. This format presents an opportunity to develop a rapid, reliable test that can be performed in the field for anything from water pollution to plant disease. Because these diagnostic tests must often be performed in harsh environments, the lateral flow format is ideal. With proper preparation and foil pouching, no refrigeration or special handling is required.
  4. As knowledge in the field of molecular genetics continues to expand rapidly, the interest in using a simple format for detecting various genetic markers, and DNA- or RNA- related infectious disease pathogens will increase. The guiding principle behind this type of test, the ability to bind a ligand from solution to a solid support, can be performed on genetic material as well as proteins, making the application of this technology in this field almost limitless.
  5. An idea that we think can be advantageous in terms of reduced development time, would be to use protein-coated microspheres, such as our ProActive® Streptavidin coated microspheres. By biotinylating the desired conjugate antibodies and then taking advantage of the strong affinity that biotin has for streptavidin, the antibodies are easily attached to the microspheres. Alternatively, Protein A coated microspheres will bind many IgG’s at the Fc region, allowing for optimized, directed antibody attachment. In this way, a series of tests could be developed rather quickly, using the same solid support, membrane, housing, etc. The only variable would be the conjugate and capture line antibodies used for each specific test. Additional information regarding these microspheres can be found in our TechNotes, #51-”ProActive® Protein Coated Microspheres”.

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References and Patents

  1. Tsuda, S., et al. Plant Disease 76, 466-469 (1992).
  2. Clausen, C.A. Nasa Tech Briefs 23a (1994).
  3. Brown, W.E.I., Safford, S.E. & Clemens, J.M. Solid-Phase Analytical Device and Method for Using Same, U.S. Patent: 5,160,701, Nov. 3,1992.
  4. Cole, F.X., MacDonnell, P.C. & Cicia, N.J., Porous Strip Form Assay Device Method , U.S. Patent: 5,141,850, August 25,1992.
  5. Fan, E., et al. Immunochromatographic Assay and Method of Using Same , International Patent: WO 91/12336, August 22,1991.
  6. Fitzpatrick, J. & Lenda, R. Method and Device for Detecting the Presence of Analyte in a Sample , U.S. Patent: 5,451,504, September 19, 1995.
  7. Imrich, M.R., Zeis, J.K., Miller, S.P. & Pronovost, A.D. Lateral flow medical diagnostic assay device U.S. Patent: 5,415,994, May 16,1995.
  8. Kang, J., Youn, B. & Oh, Y.H. Immunoasssay Devices and Materials U.S. Patent: 5,559,041, September 24, 1996.
  9. Koike, T. Immunochromatographic assay method , European Patent Appl.: 0 505 636 A1, August 7,1991.
  10. May, K., Prior, M.E. & Richards, I. Immunoassays and Devices Therefore, International Patent Number: WO 88/08534, November 3,1988.
  11. Rosenstein, R.W. Solid Phase Assay , U.S. Patent: 0 284 232 A1, July 3,1988.

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Literature Cited

  1. Singer J.M. and Plotz C.M. (1956) The latex fixation test. I. Application to the serologic diagnosis of rheumatoid arthritis. Am. J. Med. 21, 888.
  2. Bangs L.B. (1997) Immunological Applications of Microspheres. The Latex Course.
  3. Biosite Company, 11030 Roselle St., San Diego, Ca 92121.

    jd/11/98
    Copyright Bangs Laboratories, Inc., 1998
    All rights reserved.

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