Agglutination of antibody coated human erythrocytes has been found to depend on the zeta‐poten‐tial at the surface of shear and the dimensions of the antibody molecules. The critical zeta‐potential above which agglutination cannot occur has been shown to be —23 millivolts and —13 millivolts for saline and albumin antibodies respectively. A zeta‐potential below —18 millivolts for 19S molecules and below —8 millivolts for 7S molecules has been found to be necessary to give optimum agglutination and titer. The value of the enzymes used in blood group serology has been shown to be related to their ability to reduce the net surface charge density with a consequent reduction in zeta. It is postulated that this effect is due to esterase rather than protease activity. Bovine albumin and three synthetic polymers were found to bring about agglutination by reducing the zeta‐potential as a result of raising the dielectric constant. No evidence was found to support the hypothesis that they reduce the surface charge of the erythrocytes. A satisfactory equation for calculating zeta, that can be used to predict the course of a serological reaction, has been computed from a determined value of —3190 esu/cm2 for the net surface charge density of human erythrocytes in NaCl‐buffer at 25 C. This has been extended to include electrolytes of other valences by taking into account the mean activity coefficient of the electrolyte. Evidence is presented to show that the minimum zeta associated with erythrocyte stability is about —7 millivolts. This was considered to be the minimum potential required to overcome the cohesive forces of interfacial tension. In reaction mixtures imparting a zeta‐potential of —7 millivolts, tests with ten anti‐K and ten anti‐Fya sera show them to react similarly to anti‐D. There was no evidence to support the claim that these antibodies are monovalent.
The underlying principles governing the second stage of hemagglutination, as described in 1965, have been reevaluated. This reappraisal reaffirms that red cell aggregation is dependent on the potential energy barrier (λ) between erythrocytes. This in turn is dependent on the ionic strength and dielectric constant of the bulk medium. Hemagglutination by IgG and IgM antibody is a function of the effective length of these immunoglobulins and the λ of the cells in the reaction mixture. Agglutination of cells occurs when the λ-potential is below a critical level. The enhancing action of natural and synthetic polymers (e. g., bovine albumin) is to lower zeta by raising the dielectric constant of the bulk medium. Several other recent concepts are discussed and the major differences discussed in light of data available.
Bovine Serum Albumin solutions (BSA) have been chemically polymerized to give solutions (PBSA) with superior serological properties. The new PBSA reagents are shown to contain covalently bonded dimers, trimers, tetramers, and larger polymers, the presence of which is directly related to hemagglutination by IgG antibody. Data are presented which show that reagents containing increased amounts of higher molecular weight polymers enhance hemagglutination, as demonstrated by serum albumin titrations of various antisera. Additionally, isolated polymers are shown to enhance hemagglutination as a function of their molecular weight with the high molecular weight tetramers being more effective than the trimeric form, which in turn is more effective than the dimeric and monomeric forms. The protein concentration does not play any significant role in these events. The data establish that the polymeric content of bovine serum albumin reagents is a major factor in the potentiating properties of such solutions. Additionally, it is shown that these reagents act not only on the second stage of hemagglutination as a function of the bovine albumin present, but also on the first stage of hemagglutination as a function of the ionic strength of these reagents. The polymeric content and ionic strength of albumin solutions are the major criteria in determining the effectiveness of these potentiating reagents.
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