FACTORS THAT DETERMINE PRECIPITATION 207 



in auti-egg albumin sera has recently been demonstrated by Heidelberger, Treffers 

 and Mayer (1940). 



With greater antigen excess, both the amount of antigen and the amount 

 of antibody precipitated fall off, until finally no precipitate forms. We might 

 expect the increase in antibody precipitated, with increase of antigen in the anti- 

 body excess zone ; the striking feature of the curves is the inhibition of precipitation, 

 partial and total, in the region of antigen excess. This feature is discussed below. 

 We must digress for a moment to consider factors, other than the presence of 

 antigen and antibody in optimal proportions, that determine the flocculation of 

 the antigen-antibody compound. 



The Factors that Determine the Flocculation of the Antigen-Antibody Compound. 



It has already been noted that, in the j^recipitin reaction, both antigen and 

 antibody are in a state of colloidal solution. Both antigen and antibody are 

 hydrophile colloids. That is, they carry at their surfaces groups with a considerable 

 attraction for water, so that each particle is surrounded by a more or less loosely 

 attached " atmosphere " of water, which maintains it in watery solution. If 

 some change occurs that makes these hydrophile groupings inaccessible to water, 

 or decreases their affinity for water, the antigen, or antibody, or both may become 

 hydrophobe colloids, and precipitate from solution. The many features that 

 antigen-antibody precipitation has in common with the non-specific precipitation 

 of colloids suggest that in the former we are dealing with a change from the hydro- 

 phile to the hydrophobe state. 



We have noted above that electrolytes play an essential part in the precipitin 

 reaction. One effect that they produce is a lowering of the electric charge on the 

 molecules of the antibody or of the antigen or of the antigen-antibody complex. 

 The action of electrolytes has, however, been studied in much more detail in relation 

 to agglutination, and will be more conveniently discussed when dealing with that 

 reaction (see pp. 211, 213). 



In most of the precipitating reactions that have been studied, by far the greater 

 part of the precipitate is derived from the antibody (see above). There has, there- 

 fore, been a natural tendency to regard the flocculation of the antigen-antibody 

 compound as due to some change in the antibody jirotein. It has been usual to 

 picture the particles of antigen as becoming covered with antibody globulin, this 

 globulin then imdergoing a change — perhaps becoming denatured — that alters 

 its condition from hydrophile to hydrophobe (see Dean 1917, Eagle 1930, 1932, 

 Boyd and Hooker 1934, Marrack 1938). 



Antibody protein, and some protein antigens, contain Hpoid substances, extract- 

 able by alcohol, ether and other fat solvents. The extraction of lipins in such a 

 way as to avoid denaturing the proteins has a marked effect on antibody proteins. 



Hartley (1925) found that ether extraction of rabbit antibody to horse serum proteins 

 greatly reduced its capacity to form specific precipitates. It seemed that thorough 

 extraction with ether prevented the antibody protein changing from the hydrophile 

 to the hydrophobe state as the result of antigen-antil)ody union. That union took place 

 was shown by the action of ether-extracted antitoxic serum, which, though unable to 

 precipitate with diphtheria toxin, nevertheless neutrahzed it. HorsfaU and Goodner 

 (1935, 1936a, h) confirmed and extended these observations. They showed that though 

 extracted horse and rabbit antipneumococcal sera retained their protective power, their 

 agglutinating and precipitating powers were reduced or lost. Small amounts of extracted 

 lipin restored the flocculating powers to the sera. Cephahn restored the activity of 



