Title: The lines of antigen-antibody interactions in vitro and their significance for sensitive and specific antigen and antibody assays, including hybrid ELISAs, and for the possibility of more efficacious vaccines
Abstract:
Basic studies of the reactions in conventional neutralization tests without interference from complement and in complement-enriched neutralization tests were published in 1978 and 1982 [1,2].
The reaction in the conventional neutralization test was found to be bi-factorial, consisting of 1) an early, short-lasting “over-neutralization” reaction and 2) a slowly progressing but enduring reaction following the formula . Here, kst is the binding or neutralization rate factor of the antigen-antibody system, Ag and AB are antigen and antibody titers, T is the reaction time, and q is a particular temperature-dependent co-determiner of the reaction rate, which was designated the log-antibody/log-virus equivalence factor of neutralization, i.e., a genuine measure of the antigen-antibody affinity. In the herpesvirus/IgG-antibody system investigated, q was found to be approximately 0.15 at 37 oC but 0.24 at 4 oC. The influence of temperature is shown indirectly by the varying values of factor q.
The action by the complement component C1q is central in the complement-enriched neutralization test. A significant neutralizing effect of otherwise non-neutralizing IgM antibodies could be demonstrated in serum as early as 4 days after experimental nasal infection and after 8 to 14 days in samples diluted 1:10.000 or more, illustrating an extraordinary potency of the non-neutralizing IgM antibodies in inactivating infectious agents.
The regular over-neutralization reaction in the conventional neutralization test was not understood but could later be explained by the simple aggregation of the test virus. Viruses can thus be inactivated in vitro by antibodies in three ways: 1) by binding to neutralizing antibodies, 2) by simple aggregation of the test virus particles synergistically by all antibodies, neutralizing as well as non-neutralizing, and 3) by aggregation, caused by the complement component C1q, of all complexes of virus bound to predominating non-neutralizing antibodies [3,4,5,6].
The lines for elaborating very sensitive assays demonstrating antigens and antibodies, including also rapid hybrid ELISAs incorporating prompt aggregation reactions, are outlined [1,2,3,4,5,6].
The reasons for the prompt aggregation reactions are recognized but so far unexplained specific forces attracting and binding 1) antigenic determinants on the infectious agent and antigen-binding sites on their antibodies, and 2) the Fc region of antibodies sensitized by being bound to their antigenic determinant on an infectious agent and binding sites on the C1q component of complement.
A primary adapted immune defense against infectious agents must take place on mucous membranes, where the secretory IgA antibodies constructed especially to promote aggregation of infectious agents predominate, and where the C1q component of complement must be anticipated to play an important role in aggregating antigen-antibody complexes.
For decades, vaccine producers have followed a trend making sub-unit vaccines giving rise to the formation of especially neutralizing antibodies, but without attention to the important role of non-neutralizing antibodies inactivating agents by aggregation as demonstrated in the in vitro studies. Further investigations are urgently needed to clarify the formation and the effector mechanisms of the fundamental attractive binding forces defined above and of the importance of aggregation reactions in vivo, which might give new information on how to produce better vaccines against infectious agents.
