Optical Study of the Reaction between Transferred Monolayers of Lancefield's “M” Substance and Various Antisera

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Abstract
Summary: The Blodgett-Langmuir method has been adapted to the approximate determination of the extent to which reactive sites on a plane surface become occupied by molecules of a dissolved reactant, certain simplifying assumptions, based on experimental observation, being made as to the thickness of the adsorbed layer. The method in this modified form has been used in a study of the reaction between transferred monolayers of Lancefield's “M” substance and various antisera. It is pointed out that the experimental conditions are characterized by the absence of the complicating secondary effects essential to most immunological observations. By varying the method of preparation, monolayers of “M” of varying thickness can be obtained. Even the thinnest of these, in which the surface-degradation of the molecules has been most complete, are capable of reacting specifically with the homologous serum. Certain cross-reactions with other antistreptococcal sera have been observed, and also the reaction of mixed monolayers of “M” and egg-albumin. The rate of deposition of antibody has been studied semiquantitatively. The time required for half the active surface of “M” to become occupied by antibody-molecules varies from 5 minutes for whole serum to 30 minutes for 1:300 serum at room-temperature. The initial state of the process has been described approximately by an equation based on the assumption that the rate of reaction is proportional to the number of unoccupied reactive sites. The value of the velocity-constant thus obtained increases with the dilution of antiserum used. Accompanying this increase in velocity constant is a considerable decrease in the thickness of the deposited layer of antibody, which varies from about 150 Å for whole serum to 55 Å for 1/300 serum. A limiting value somewhat less than 50 Å is probably approached at greater dilutions of serum. Variation in velocity-constant and thickness of the deposited antibody-layer with dilution may be attributed either to the dissociation of molecular aggregates of antibody upon dilution, or to differences in the orientation of antibody-molecules at the active surface. The available evidence appears to favor the latter hypothesis. The sensitivity of the optical method is such as to permit the detection of antibody-concentrations of about 4 × 10−7 g/ml in the system under discussion.

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