Isopycnometric serology: a new technique based on buoyant density changes in latex beads.

WE wish to describe new serological techniques based on changes in buoyant density occurring when antibodies, or antigens, or both, are attached to latex beads. For the density change to be observed, the mass of the attached particles must be an appreciable fraction of the total mass. Using very small carrier beads less than one hundred antibody molecules or one virus particle per bead can be detected. The speed and sensitivity of the method make it directly applicable to the clinically important problem of rapid identification of infectious agents. If the volume and density of one member of a hybrid pair (termed here the carrier) are known, then the positive or negative buoyancy of the second member (or load) can be determined from the buoyant density of the hybrid. If the buoyant density of the load is known, then its volume, and hence its mass, can be calculated. For the application of this principle to subcellular particles, viruses, and macromolecules, a series of carrier particles of different size is required, with a narrow size distribution within each sample. The carrier particles should be either more or less dense than any of the load particles which may be encountered. Most particles of biological interest, excluding lipids and lipoproteins, fall in the range between 1.19 and 1.7 g/cc 2 • Carrier beads lighter than 1.18 glcc are preferable to those denser than 1.7 glcc because less viscous gradients and, therefore, lower speed centrifugation may be used. Fortunately, very uniform polystyrene latex beads are available 1 which cover the lower portion of the size range of interest here. Immunoglobulin (IgG) has been found to attach to these after mild heating in a slightly alkaline environment2, and flocculation of the resulting complex has been widely used to detect the human rheumatoid arthritis factor 3 • In all the experiments described here polystyrene latex with an average diameter of 0.109 11m (s.d. =0.0027 11m, Dow latex batch No. LSI044-E) was used. In sucrose gradients these particles band at a density of 1.049-1.050 g/cc 4 • Using a small linear gradient mixer .nd a six channel peristaltic pump, six identical sucrose grar lents were prepared and centrifuged in a Beckman SW50.1 swinging bucket rotor to study the banding behaviour of the particles. A band recovery apparatus 5 was used to observe and photograph bands and to recover samples. In some experiments plastic density-marker beads were used …