Lateral flow strip test

The immunochromatographic lateral flow strip test is a one-step test that facilitates low-cost, rapid identification of various analytes at the point of care. We have developed lateral flow strip tests for the specific qualitative or semiquantitative detection of antigens, antibodies, and haptens, such as drug residues. Here, we describe in detail the preparation of three examples of the strip tests for detection of (a) the infectious bursal disease virus (b) Tricbinella specific antibodies, and (c) Clenbuterol residues in urine samples. 

Key words Immunochromatography, Lateral flow strip test, Infectious bursal disease virus, Tricbinella, Clenbuterol. 

Principles and characteristics of lateral flow strip assays are reviewed. Recent technology developments permit the use of inexpensive electronic readers for interrogating lateral flow strip test results, thus avoiding the inevitable variation and subjectivity of visual inspection to assess the capture of reporter-labeled analyte on test lines of the strip. Protocols for developing lateral flow assays are described, including two specific case studies for assaying cotinine (a small-molecule metabolite of nicotine) in a competitive format, and assaying HIV antibodies in a sandwich-type assay format. 

Fig. 1. The lateral flow strip structure (A) Schematic representation of the lateral flow strip. A lateral flow strip typically consists of four zones of a sample pad, a conjugate pad, a membrane containing the test and control lines, and an absorbent pad on a backing plate. (B) Lateral (toft and over (bottorft views of a lateral flow strip. The outmost layers of the ends of the strip are plastic cover films.

Lateral flow strip structures and methods of operation should be designed and implemented in view of desired performance requirements and other constraints. These design criteria inputs reflect specific parameters along with performance objectives with respect to sensitivity, specificity, reproducibility, reliability, ease-of-use, and cost. These parameters and performance metrics should be quantified, even for qualitative tests. 

As with all assays, each reagent must be adjusted to optimize test performance. A lateral flow test that is visually read is complicated by the fact that the interpretation of test results is subjective, dependent on ambient conditions and the experience of the tester. For this reason, it is preferable to use an instrumented reader to interrogate lateral flow test results. Recently, new inexpensive readers have become available. One such device (Avagotech, Menlo Park, CA) utilizes an inexpensive CCD camera, such as those found in mobile phones along with simple electronics to capture images of the lateral flow strip and its capture lines. Other more sophisticated reader devices are also available. The costs for any reader are dependant upon the capabilities of the reader hardware and software. Newer systems are now under development for fluorescence and chemilumenescent analysis. This new generation of readers is expected to increase lateral flow test analytical capabilities considerably. 

Dyed particles also are commonly used in diagnostic lateral flow tests (like the common home pregnancy test), as the colors can be seen with the eye without the need for special detectors. In this type of assay, antibodies or antigens are coupled to the dyed particles and a sample solution applied to the test strip carries them along within a membrane. The particles then are captured at points in the membrane that represent either a control or a positive sample result. Large numbers of color particles docking at these points within the membrane create the visual lines associated with these disposable tests. 

Analytical tools to detect this particular protein rely on the use of antibodies, which specifically recognize the newly introduced protein. Two major applications are available lateral flow devices (also known as strip test or dipstick), and ELISA. An overview and an in-depth discussion of protein-based methods for the detection of grain derived from modern biotechnology can be found in the literature e.g., [3,4],... 

Wang, X., K. Li, D. Shi, et al. 2007. Development of an Immunochromatographic lateral-flow test strip for rapid detection of sulfonamides in eggs and chicken muscles. J. Agric. Food Chem. 55 2072-2078. 

A growing application of the strip type of technology for immunoassay is for infectious disease testing and the rapid detection of various infectious antigens and antibodies including Chlamydia,group A streptococci, " Helicobacter pylori, ° infectious mononucleosis, and human immunodeficiency virus. These tests commonly use a lateral-flow format for urine, serum, plasma, or whole blood samples in conjunction with analytical techniques, such as enzyme immunoassay and immunochromatography. An alternative... 

The technology of the immunochromatographic test, also called lateral flow test or strip test, has been used for many years. However, its application to food safety, especially in mycotoxin testing is quite recent. The typical immunochromatographic test strip consists of a sample pad conjugate pad, membrane absorbent pad and adhesive backing. 

S. J., and Guo, J. Q. (2006) Development of an immunochromatographic lateral flow test strip for detection of -adrenergic agonist... 

Rapid lateral flow test strips considerations for product development. (Bedford, MA Millipore Corp., 2002)... 

Fig. 1. Lateral flow tests are often constructed from a series of materials that sequentially overlap. The goal is to imbed all reagents so that a flowing sample rehydrates and moves all materials up a test strip. Analytes and reagents then interact in zones placed on the strip. The result is a rapid test that provides information easily visible to the eye.

Finally, before beginning work on a lateral flow test, some specialized equipment is needed to ensure precise delivery of reagents onto the lateral flow test materials. Specific procedures for using some of this equipment are described below in the examples. Note that the goal here is to describe equipment appropriate for the research bench. There are also companies that offer equipment capable of manufacturing large numbers of test strips in highly automated fashion. 

Our laboratory has been using an Avagotech reader for evaluation of reagents for HIV and cotinine lateral flow assays. Figure 2. shows an example of results for a sample diluted in our prototype HIV antibody detection lateral flow test strips. The Avagotech reader allows the data to be plotted and coefficients of variation to be determined, making the screening more quantitative and less subjective. 

Commercial lateral flow assays are assembled in plastic housings to simplify sample handling and to protect users from the contact with assay components and potentially dangerous samples. Samples are usually mixed with a diluent buffer and loaded onto the strips. After absorption of the sample, strips can be washed with a second chase buffer to decrease background and to increase sensitivity of detection. In a research environment, these tests can be run in 96-well plates with nitrocellulose strips dipped into wells containing samples. 

The reliability of the test strip immunoassay was determined by carrying out the test with the uncontaminated samples spiked with detected target at three levels and analyzed by strip test and instrumental analysis. The results obtained by strip test should be consistent with the results obtained by instrument. The correlation between the two methods should be good. An example of validation of gold-based lateral flow immunoassay and analysis of food samples for the detection of carbaryl was shown in the following. 

At the same time, much simpler yet very successfiil microfluidic analysis systems based on wettable fleeces emerged First very simple dipsticks for e.g. pH measurement based on a single fleece paved the way for more complex test strips that have been sold as lateral-flow tests in the late 1980s [14]. Examples that are still on the market today are test strips for pregnancy [15], drug abuse [16-18], cardiac markers [19] and also upcoming bio-warfare protection [20]. 

In lateral flow tests, also known as test strips (e.g. pregnancy test strip), the liquids are driven by capillary forces. Liquid movement is controlled by the wettability and feature size of the porous or microstructured substrate. All required chemicals are pre-stored within the strip. The presence of an analyte is typically visualized by a colored line. 

The first immunoassay performed in a capillary driven system was reported in 1978 [67]. Based on this technique, the commonly known over-the-counter pregnancy test was introduced into the market in the middle of the 80 s. Today, this microfluidic platform is commonly designated as a lateral flow test (LAT) [14]. Other terms are test strip , immunochromatographic strip , immunocapillary tests or sol particle immunoassay (SPIA) [68]. Astonishingly, hardly any publications from a microfluidic point of view or in terns of material classification exist, and apparently many company secrets are kept unpublished [69]. 

Lateral flow tests were among the first successfully commercialized micro-fluidic products. A huge amount of assays has been developed on the capillary test strip platform during the past 30 years [76]. Today, they serve a wide... 

Lateral-flow immunoassays (LFTAs), also known as immunochromatographic test strips or dipsticks, are a form of rapid and portable immunoassay in which the test sample extract flows along a solid substrate via capillary action. The dipstick system is simple operationally, whereas the technology behind it is advanced and subtle. 

The lateral flow home pregnancy immunoassay shown schematically in the figure is a qualitative test for hcG. Urine is applied to the sample pad at the left end of a horizontal test strip made of nitrocellulose that serves as a wick. Liquid flows from left to right by capillary action. Liquid first encounters detection reagent on the conjugate pad. The reagent is called a conjugate because it consists of hcG monoclonal antibody... 

Dry tests or test strips (also called lateral flow devices or immunomigration strips) are popular as... 

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