The Labeling Component

In order to visualize the binding of specific antibodies to drug molecules in DOA testing, the antibody component is often conjugated (labeled) to a color medium. For this purpose, a number of particulate conjugates have been used, with varying degrees of success. Latex was among the earliest used. Latex particles offer high visibility (in a variety of colors), low cost, and ease of preparation and conjugation. Its natural tendency to aggregate after binding to the ligands makes latex the ideal choice in agglutination assays, but not in rapid tests, in which stability of the antibody conjugates for an extended period of time is essential. Other media for color detection such as enzyme-linked immunosorbent assays (ELISAs) are commonly incorporated into clinical test kits. These assays offer high sensitivity and visibility, but the requirement for multiple color-development steps makes this approach not suitable for rapid tests. Inert particulates such as gold, silver, and carbon have been used in many applications, and offer considerable advantages in rapid tests. All three are stable, low-cost conjugates that are easy to prepare in large scale. Colloidal gold probably offers the highest visibility, sensitivity, and reproducibility, and has thus become the most commonly used conjugate for rapid tests (11).

2.2.1. Colloidal Gold Conjugates

Gold colloids are formed by the reduction of gold tetrachloric acid (HAuCl4) through a nucleation process, in which central icosahedral gold nuclei of eleven atoms are first formed. Further reduction reaction causes the remaining gold atoms to bind to the nuclei to eventually form the colloid state (12). Common reducing agents include sodium thiocyanate, sodium citrate, and sodium borohydride. The size and shape of the colloids depend on the type and amount of reducer used (13). A larger amount of reducer will allow formation of a larger number of nuclei and hence a smaller size of the particles.

To make a high-quality and reproducible label, it is essential that a good-quality gold colloid and high-purity antibody be used (12). Although it is considered easy to make a gold conjugate, it is not easy to manufacture a good gold conjugate with consistent reproducibility. To start with, the gold colloid should always be uniform in shape with an even distribution of single particles (as shown in Fig. 1). Poor-quality gold will contain particles with irregular morphologies. These particles will be prone to aggregation even before the addition of the detector reagent. In addition, irregular sizes and shapes will affect the amount of antibody that can be loaded onto each particle, and this will directly affect the performance of each batch of finished conjugate.

Once the ligand has been conjugated, the quality of the gold conjugate must be assessed before incorporation into the rapid-test assay. Usually, electron microscopy is employed as a quality-control measure. Figures 1 and 2 compare the appearance of a good-quality gold sample with that of a poor-quality gold sample. Note the size and shape of the particles. The presence of clusters in a gold conjugate is indicative of a poorly optimized product. With time, the frequency of these clusters and the number of particles per cluster

Fig. 1. Good quality gold particles. Note the regular size and shape of the particles.
Fig. 2. Poor quality gold particles. Note the irregular shape and size distribution of the particles. Also notice the aggregation of particles.

can increase. In inhibition assays, this results in false negatives (14) and poor stability of the final product. Any excess antibody present will compete with labeled antibody for binding sites with the capture reagents, and this will lead to reduced sensitivity and potentially false-positives.

Gold particles can be produced that range in size from 5 to 100 nm in diameter. In a rapid test, the colloidal gold particle must be large enough to be seen. The most common size used is 40 nm. This size gives maximum visibility with the least steric hindrance in the case of IgG conjugation. Other sizes from 5 nm to 100 nm have been conjugated with success. However, 5-nm particles do not have the bright red color of the large-size particles and hence give only a very weak to no signal at the detection line, no matter how many particles are used. Only particles larger than 20 nm give a meaningful signal. On the other hand, 100-nm particles are too large compared with the antibody molecule. An IgG antibody has a molecular weight of 160 kDa and a length of approx 8 nm, of which only 4 nm extends out from the surface of the colloidal gold particle. This size differential creates steric hindrance and makes it difficult for the short surface antibody to interact with other molecules.

It is accepted that three amino acid residues play an important role in the conjugation of proteins to gold particles (15). These are: lysine, which ishighly positively charged, and is attracted to the negatively charged gold particle; tryptophan, which binds through hydrophobic interactions; and cysteine, which forms the strongest attachments via dative bonds through the formation of sulfur bridges with the gold surface, such that the antibody and gold particles share electrons.

The success of the conjugation in terms of performance in the assay depends on the location of these amino acid residues in the protein to be conjugated. If they should be located in a region of the antibody near the antigen combining site (the Fab region) (1), then the gold label can interfere with the binding capacity of the antibody. This is termed steric hindrance, and is almost impossible to overcome without compromising the integrity of the molecule supplied for conjugation. For optimal sensitivity of the assay, the three amino acids—lysine, tryptophan and cysteine—should be located in the Fc region (the constant portion) of the antibodies, and for antigens should be topologically isolated from the working reactive epitopes.

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