TESTS OF THE LATTICE HYPOTHESIS 221 



Eagle (1938) subjected diphtheria antitoxin and antipneumococcal serum to pro- 

 gressively intensive treatment with formaldehyde. At one stage the antibodies lost 

 their precipitating power, but were still protective, a separation of activities that suggests 

 a non-specific element in aggregation. Kleczkowski (19416) and Bawden and Kleczkowski 

 (19426) made complexes of antibody globulin with albumin, in which the antibody retained 

 its specific combining power, but was incapable of in vitro flocculation. Phenomena of 

 this kind do not necessarily invaUdate the lattice hypothesis, but they give an indication 

 of the degree to which non-specific factors may be operative in aggregation. 



Topley, Wilson and Duncan (1935) devised a test of the multivalence of antibody, 

 using mixed agglutination systems. Two antigens are chosen, of approximately equal 

 particle size, and in similar concentration. The amount of antibody which in each case 

 will flocculate the antigen in a given time is determined, and the reaction of the two antigens 

 and antibodies allowed to take place in a mixtm-e. If the lattice hypothesis holds, the 

 aggregates that form wiU consist of one antigen or the other, since they are held together 

 by specific antibody. If flocculation is non-specific, the aggregates wiU be mixed. More- 

 over, in the first case, the speed of flocculation of each antigen will be independent of 

 the presence of the other ; but if antibody merely sensitizes the antigens to the action 

 of electrolyte, then the concentration of flocculable material in the mixtures will be doubled, 

 and the speed of the mixed reaction much greater than that of either reaction separately. 

 Topley, Wilson and Duncan found that pneumococci, and the microscopically distinguish- 

 able coliform bacilli, formed separate aggregates. With mixtures of two distinguishable 

 types of red blood cell, Abramson (1935) and Hooker and Boyd (1937) found mixed aggre- 

 gates. Wiener and Herman (1939), on the other hand, found separate aggregates with 

 mixtures containing red cells and a precipitating antigen, red ceUs and pneumococci, 

 and two types of red cells. Mixed aggregates were formed with the latter when antibody 

 was in excess. Non-specific flocculants also gave mixed aggregates. With precipitating 

 systems, Hooker and Boyd (1937) found an increased speed of flocculation in mixtures, 

 indicating non-specific aggregation. Duncan (1938) confirmed this but, with agglutinating 

 systems, found no increase in speed of agglutination. He suggested that both mechanisms 

 were operative, but that one or the other might predominate according to the system and 

 the circumstances of its reaction. Heidelberger and Kendall (1937) saturated pneumo- 

 cocci with horse antibody. The addition of more pneumococci to the over-sensitized 

 cell§ resulted in prompt agglutination of aU the pneumococci, a result strongly suggestmg 

 that, in addition to the valencies employed in binding antibody to the original pneumo- 

 cocci, there were on the bound antibody free valencies available for combination with 

 the added cocci. Boyd and Hooker (1938) suggest that in mixed systems specific combina- 

 tion may operate in the second stage, and a lattice may form in antigen excess ; in antibody 

 excess, the " two-stage " reaction appears to take place, for they found that red ceUs 

 saturated with a very marked excess of antibody were still susceptible to the flocculating 

 action of salts. Hershey's (19436) recent experiments, made on somewhat similar lines 

 with bacteriophage, also support the lattice hypothesis. A living Bad. coli bacteriophage 

 adhered firmly to a precipitate consisting of dead coli bacteriophage and homologous 

 antibody. Other specific aggregating systems, with e.g. staphylococcal phage, serum 

 proteins, pneumococcal polysaccharide, absorbed only a small amount of the phage. He 

 obtained no evidence that the particles of antigen were rendered non-specificaUy " sticky " 

 by their coating of antibody, a condition to be expected if aggregation was non-specific. 



Another experimental approach to the problem of antibody valency is possible. 

 No safe assumption can be made of the precise valency of natural antigens. But, 

 as we shall see later (p. 258), certain fractions of natural antigens will react with 

 antibody, provided they possess the chemical groupings that characterize the 

 full antigen. These reacting fractions are called haptens, and in some cases can 

 be prepared synthetically. The reaction of monovalent and divalent synthetic 

 haptens with antibody should provide a clue to the valency of antibody. Mono- 



