The binding of human serum albumin by monodisperse latex particles was studied applying the radioisotope labelling technique. The radiochemically active substance was 1311-labelled albumin and the latices were monodisperse samples ranging in particle size from 200 to 940 nm. All experiments were performed in buffered solutions at pH 8.0 and the ionic strength 0.05. I n one type of binding experiments human serum albumin was added in great excess leading to a saturation of latex particle surface. The excess albumin was removed by centrifugation and washing of the sediments by pure buffer. For two latex samples the binding isotherms were determined. The results of the experiments with excess albumin suggest that a nearly constant amount is bound per unit particle surface irrespective of the latex sample and particle size. One washing was shown t o take away approximately some loo/, of bound albumin from the particle surface demonstrating fairly strong binding. From the binding isotherms the binding constants K and the standard free energies of binding AGO were estimated. Values of K about 6 pM-l and AGO about -9 kcal/mol indicate high affinity of human serum albumin to the latex particle surface. The results of the present experiments are discussed from the point of view of recent findings on protein interactions with detergent molecules. The results indicate a possible expansion of the tertiary structure of the human serum albumin molecule in its part attached to the particle surface whereas the part of the molecule oriented to the solution remains in its native conformation as is seen from the preserved immunochemical activity against specific antibodies.
The stability of monodisperse polystyrene latex particles in the immunochemical system human serum albumin-rabbit anti-human albumin serum was studied by using a photometric method for the detection of latex particle agglutination. The latex particles were coated with human serum albumin and mixed with dilutions of both a hyperimmune and a normal rabbit serum. The experiments were carried out with latex particles of different size at varying pH, ionic strength and temperature. From the agglutination curves it was possible to derive conclusions on the specific and nonspecific immunochemical interactions with latex particles as well as on the effects of electrolyte coagulation. Maximum sensitivity ofthe latex reagent was found a t pH values between 8 and 9. At pH < 6 the latex reagent becomes unstable, a t pH < 4 the immunochemical interactions disappear, and at pH > 11 the immunochemical reaction becomes indistinct. Thermal inactivation could eliminate some of the nonspecific interactions, but the most efficient method was in experimenting at high ionic strengths ( I = 0.5).Latex particle agglutination methods, having been in use in serologic laboratories for some fifteen years, belong to very sensitive methods for the detection of immunochemical interactions. Since the first report of Singer and Plotz [l] on the use of synthetic polymer latex particles for serologic purposes in the detection of rheumatoid factors, an appreciable number of papers have appeared describing various latex agglutination tests. The predominant part of this work has been connected with the problems of the application of latex tests in serology, as can be concluded from extensive reviews [2,3]. Far less work, however, has been devoted to the study of latex agglutination in well defined immunochemical systems containing only specific antibodies against a given antigen.If a systematic study of latex particle agglutination in immunochemical systems is to be undertaken, one has to choose a model system consisting of a single well characterized antigen and a homologous specific antibody. Renoux [4] was the first, as far as we know, to describe latex agglutination in a model immunochemical system. His work on the system ovalbumin-antiovalbumin, aimed at comparing the sensitivity of the latex agglutination method with the precipitation and passive hemagglutination methods. Renoux only sketched the idea and described a few experiments but did not investigate the influence of a number of important factors which may influence the immunochemical interaction, such as pH, ionic strength, temperature, etc. An extensive work in studying the influence of the variation of several physicochemical parameters on latex particle stability and agglutination was done by Singer and his collaborators [5-101. They experimented, however, with human gamma-globulin coated latex particles and rheumatoid sera, a system much more complex than model systems. For that reason their results are not fully comparable with data obtained on model systems and could be obscured t...
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