Superior stability, sensitivity, and precision and reproducibility of manufacture make gold suitable for use in membrane-based tests.
The demand for rapid membrane-based tests encompasses applications in a wide variety of fields (see box, below). The number of such potential applications will likely increase as these low-cost alternatives to expensive instrumented methods of testing become more sensitive and more specific.
Applications for rapid tests
(Areas duplicate most human clinical applications)
This article focuses on the importance of a critical component of such tests—the detection label—and how its manufacture can affect the performance and reliability of the whole test system. Specifically, the article will address the advantages of using gold conjugates in this capacity.
What Is a Rapid Test?
A rapid test is an inexpensive, disposable, membrane-based assay that provides visual evidence of the presence of an analyte in a liquid sample. Such tests can be formatted either as freestanding dipsticks or as devices enclosed within plastic housings. Typically, as little as 200 µl of liquid sample is required to perform the test, which is usually complete within 2–5 minutes. In clinical assays, the sample may be urine, blood, serum, saliva, or other body fluids. In nonclinical tests, the sample may be a small volume of solution prepared from soil, dust, plants, or food, and similarly applied directly to the membrane test strip. No instrumentation is required to perform such tests, which can be used in clinics, laboratories, field locations, and the home—often by inexperienced personnel.
A large flask of gold conjugate. Photo by Hugh Burden, courtesy BB International
Rapid tests come in two forms—lateral flow and flow-through. The lateral-flow format is by far the most common because it is easier to manufacture and use. Although the same principles apply to both formats, the lateral-flow test is discussed here.
The base substrate of a rapid test is typically a nitrocellulose strip onto which is immobilized a capture binding protein, usually an antibody or antigen (see Figure 1). A pad (often glass fiber) containing dried conjugate is attached to the membrane strip. For the majority of currently available tests, this conjugate pad contains gold particles adsorbed with antibodies or antigens specific to the analyte being detected. A sample pad, usually paper, is attached to the conjugate pad. When applied to the sample pad, the liquid sample migrates by capillary diffusion through the conjugate pad, rehydrating the gold conjugate and allowing the interaction of the sample analyte with the conjugate. The complex of gold conjugate and analyte then moves onto the membrane strip and migrates towards the capture binding protein, where it becomes immobilized and produces a distinct signal in the form of a sharp red line. A second line, a control, may also be formed on the membrane by excess gold conjugate, indicating the test is complete.
Figure 1. Construction of a lateral-flow rapid test.
By definition, rapid tests should provide results in a short time, preferably minutes. Such tests must be convenient, accurate, reliable, inexpensive, disposable, and foolproof. They must also be easily and unambiguously interpreted, even by users without experience. From the manufacturers' point of view they should carry a large added value and be easily marketed worldwide to users who may be either experienced or inexperienced in the use of such tests. (see sidebar, below).
Rapid tests are versatile. By switching the antibodies and making small adjustments to the chemistry of the strip format, the same test design can be used for many applications.
Why Use Gold?
Early rapid tests used colored latex to form the visual signal, and some current versions continue to use this method. Latex was originally, and still is, the prime labeling method used in agglutination tests. This is because of its predisposition to agglutinate in the presence of binding components. For rapid tests, in which stability of the conjugate is critical for avoiding false positives, this predisposition to agglutinate can become a major problem.
Because of their greater potential stability, gold labels were introduced into membrane-based rapid tests in the late 1980s. Gold particles of any accurately defined size can be manufactured reproducibly under the appropriate manufacturing conditions. Different sizes may be used for different applications. Its superior stability, sensitivity, and precision and reproducibility of manufacture make gold suitable for use in rapid tests. Gold is essentially inert and forms almost perfectly spherical particles when properly manufactured. Proteins bind to the surfaces of these gold particles with enormous strength when correctly coupled, thus providing a high degree of long-term stability in both liquid and dried forms. Also, when accurately stabilized during manufacture, nonspecific interaction of gold conjugates can be reduced to zero.
A comparison of the benefits of different labels used to mark antibodies and antigens is shown in Table I.
|Ease of preparation||***||***||**||***||**||**|
|Ease of use||***||***||**||***||**||*|
|* Limited application|
|**Acceptable for some applications|
|***Outstanding and applicable to most tests|
Table I. Comparison of the characteristics of labels commonly used in rapid tests.