Studies on the method to quantify PPD and its adsorption onto glass-surface

Abstract

The potency of PPD is very stable when kept as a lyophilized preparation or as a solution of high concentrations, but, as is well known, when diluted to the practical concentrations such as used in skin-testing for human, its potency diminishes rather rapidly. It has been considered that such decrease in potency is not due to denaturation of PPD, but due to its adsorption onto the surface of glass- or plastic-container. The adsorption of PPD had been studied by several authors with radiolabeled PPD or by the potency test in experimental animals or human. However, either method is not satisfactory because they are not quantitative and reproducible. Consequently, we developed a new method basing on ELISA to measure such low concentrations of PPD as used in skin-testing in human. Using this new method, we analyzed the adsorption of PPD onto glass-surface. The major characteristics of the PPD adsorption revealed by the present studies are as follows; (1) The adsorption of PPD takes place very rapidly immediately after the distribution of PPD solution to a new container and then proceeds in slower rates. (2). The rate of adsorption is influenced by both temperature and concentration of PPD solution. The rates of adsorption are almost the same at -80 degrees C and at 6 degrees C, much higher at 22 degrees C and further higher at 37 degrees C at any concentration tested. At -80 degrees C and 6 degrees C, there is no difference among the adsorption rates at three different concentrations, but at 22 degrees C and 37 degrees C, the rate of adsorption at the lowest concentration (0.25 microgram/ml) is much higher than those at higher concentrations. (3) The amounts of PPD adsorbed are determined by the surface area of glass in contact with PPD solution. (4) The larger the volume of PPD solution distributed, the bigger the absolute amount of PPD adsorbed, but the smaller the rate of adsorption. (5) So-called "volume effects" are evidenced. Commercially available Japanese PPD preparations have been produced and adjusted their potency taking such adsorption phenomena into consideration. But, if undue adsorption is induced by unappropriate handling and/or usage, the potency might be diminished. Especially, the single-dose preparation may be affected most strongly by so-called "volume-effects". Standard handling and usage recommended by the manufacturer are strictly required.