Antigen-Antibody Interactions in vitro: Analyses Demonstrate a Dominating Role of the Non-Neutralizing Antibodies as the Most Potent Inactivators of Viruses | Chapter 9 | Contemporary Research and Perspectives in Biological Science Vol. 7

The understanding of the regular lines of reactions between virus and antibodies has been hampered by the common perceptions: 1) that neutralization of virus in neutralization tests would lead to a state of equilibrium and 2) that it was generally thought that non-neutralizing antibodies did not participate in the neutralization in vitro. The regular lines of neutralization by antibodies in vitro have been presented earlier, and in a recent article, the reaction characteristics in five different assays demonstrating antibodies to viruses were analyzed and recommendations for gold and reference standard tests and routine adequately sensitive tests were given. Inactivation of the virus in a conventional neutralization test was found to be a bifactorial process, consisting of: 1) a prompt and short-lasting reaction originally termed the over-neutralization phenomenon and 2) an enduring but slowly progressing reaction of first order. The over-neutralization could later be concluded to be neutralization due to the aggregation of viruses. The reaction of first order followed the lines of a demonstrated formula for the regular antigen-antibody interactions in vitro, not including aggregation. Tests for demonstration of viruses and antibodies of very high sensitivity and specificity have been elaborated on the basis of these reaction lines and used in diagnosing, controlling and eradicating viral infections in the veterinary medical field for a long time. 

In the present article, the role of the non-neutralizing antibodies in the neutralization process in vitro is analyzed. The non-neutralizing antibodies inactivate viruses by aggregating the agents. An extremely high virus-inactivating potency of these antibodies was demonstrated. The principal characteristics of virus aggregation were concluded to be the following: 1) IgG and IgM antibodies to various antigenic determinants aggregate virions synergistically and rapidly, 2) the virus-inactivating aggregation by the polymerized isotypes of antibodies is greatly enlarged by their polyvalency, and 3) the complement component C1q will promptly attach to antibodies sensitized by being bound to their antigenic determinant on a virus and inactivate such virus-antibody complexes by including them in aggregates. In complement-enriched neutralization tests, C1q will 1) promptly aggregate antigen-antibody complexes formed and 2) with increasing reaction times aggregate the virus following the first-order binding of non-neutralizing antibodies to their antigenic determinants, thereby inactivating viruses with the same rate as neutralizing antibodies. 

In a herpesvirus complement-enriched neutralization test, the titers of reacting non-neutralizing IgG antibodies in the highest concentration were found to be approximately 8 times higher than that of the neutralizing antibodies, indicating that also the concentration of the reacting non-neutralizing antibodies was 8 times higher than that of the neutralizing ones. The total concentration in blood of non-neutralizing antibodies in blood by far exceeds that of the neutralizing ones, theoretically being largely proportional to the number of antigenic determinants on a virion. The capability of the non-neutralizing antibodies to neutralize by aggregation, most pronounced in cooperation with the C1q complement component, and their high concentration, imply that the non-neutralizing antibodies have a much greater neutralizing potency than neutralizing antibodies. One non-neutralizing antibody bound to an antigenic determinant will result in almost immediate inactivation of the virus due to the incorporation of the formed antigen-antibody complex in aggregates by the C1q component. 

The formation of the pentameric and decavalent IgM antibodies is the first humoral immune response after infection and the virus-inactivating potential of these acute-infection phase antibodies is enormous. A significant neutralizing IgM antibody response could be detected in blood by a complement-enriched neutralization test 4 days after nasal infection and samples taken after 8 to 15 days were positive in dilutions 1:10.000 or higher. The IgM antibodies, non-neutralizing as well as neutralizing, must in cooperation with the C1q component of complement be considered the ultimate virus inactivators and of almost unimaginable importance in the defeat of infections. 

Recognized but unidentified specific forces attracting: 1) the binding sites of antibodies and their specific determinants on viruses, and 2) the binding sites of the C1q component and the Fc fragment of sensitized antibodies bound to their antigenic determinants, are concluded to be one reason for the prompt reaction, which is characteristic of the virus aggregation process. 

Further investigations into the effector mechanisms determining: 1) the specificity and attractive binding of the antibodies to their antigenic determinants and 2) the reaction between the complement component C1q and sensitized antibodies are urgently needed.

 

Author (s) Details

Viggo Bitsch
The State Veterinary Serum Laboratory, Copenhagen and the Department of Cattle Diseases, The Danish, Dairy Board, Aarhus, Denmark.

 

Please see the book here:- https://doi.org/10.9734/bpi/crpbs/v7/2582

Systematic Analyses of Antigen-Antibody Interactions in vitro: Insights from Three Different Ways of Virus Inactivation and Their Specific Reaction Lines | Chapter 5 | Contemporary Research and Perspectives in Biological Science Vol. 8

Antibodies neutralize viruses in three different ways. In contrast to what is commonly perceived, very sensitive and specific tests demonstrating antigens and antibodies can be elaborated on basis of the regular reaction lines demonstrated. The action of the non-neutralizing antibodies is of the greatest importance in combatting and preventing infections.

 This analytical review aims to clarify the complex lines of antigen-antibody interactions in vitro, to achieve a better understanding of the impact of various antibodies and the reaction lines on the basis of which sensitive and standardized tests for demonstrating antigen and antibody can be elaborated. A variety of aspects of the antigen-antibody binding reactions are evaluated, including 1) the reactions in a conventional neutralization test, 2) the first-order antigen-antibody reaction demonstrated, 3) the formula for the regular reaction without aggregations presenting the log antibody/log virus equivalence factor of neutralization, 4) the lines of two different aggregation reactions inactivating viruses, 5) irreversibility of antigen-antibody bindings, 6) lines for elaborating gold and reference standard assays and highly sensitive routine tests, 7) a “persistent fraction of virus”, and 8) the “Percentage Law”. A series of important conclusions are presented.

 Antibodies are bound firmly and irreversibly to their antigenic determinants on a virus. The binding reaction does not lead to an equilibrium state as commonly perceived. The sensitivity of antigen-antibody tests may consequently be both variable and adjustable to high levels. Antibodies neutralize viruses in three clearly distinct ways. Neutralization of virus by antibody in a virus-antibody mixture without presence of the complement component C1q proceeds as two separate reactions, i.e., 1) a first-order reaction caused by particularly neutralizing antibodies being bound monovalently to their antigenic neutralization determinant and 2) an early “over-neutralization” reaction that can be attributed to the formation of virus aggregates created by the di- and polyvalent antibodies which to a wide extent are non-neutralizing. This aggregation is caused by the various antibodies, in a synergistic action. It is highly dependent on a sufficient antibody concentration and is prompt and short lasting. In the serial dilutions of a conventional neutralization test, an early aggregation is readily diluted away with increasing reaction periods. In contrast, the first-order neutralization reaction is slowly progressing but enduring. The test sensitivity, cf. Definitions will be directly proportional to the reaction time but temperature-dependent. The third neutralization reaction is dependent on the presence of a complement. The hexa-valent C1q component will enhance the neutralizing effect of antibodies by aggregating virions that have been bound to a non-neutralizing antibody. Such an antibody is sensitized for binding to the C1q component by being bound to its antigenic determinant on the virion. In a complement-enriched neutralization test, two different reaction rates will be observed. Practically instantly, C1q will react with all pre-formed virus-antibody complexes and, because of the multi-valency of that molecule, include them in aggregates but thereafter, with increasing reaction time, the reaction will be of first-order following the continuous first-order binding of non-neutralizing- antibodies to their antigenic determinants. In a herpesviruses complement-enriched neutralization test, the reaction with non-neutralizing IgG antibodies increased titers recorded for neutralizing IgG antibodies in a conventional neutralization test by a factor of approx. 8, but for IgM antibodies the neutralizing effect by complement was enormous. A significant neutralizing effect could be demonstrated in blood as early as 4 days after experimental nasal infection and after 8 to 15 days, the neutralizing effect was demonstrated in serum diluted 1:10.000 or more. The huge neutralizing potential of the non-neutralizing antibodies in a joint action with complement indicates a fundamental role of these antibodies in combatting infections. The so-called Percentage Law. according to which the rate of virus neutralization will be independent of the virus concentration was found invalid. The formula for the regular lines of antigen-antibody interactions in vitro not comprising aggregation has been presented earlier. Highly sensitive antigen-antibody tests, elaborated on basis of the reaction lines documented, have been in use for a long time in the veterinary medical field.

 

Author (s) Details

 

Viggo Bitsch
The State Veterinary Serum Laboratory, Copenhagen and The Department of Cattle Diseases, The Danish Dairy Board, Aarhus, Denmark.

 

Please see the book here:- https://doi.org/10.9734/bpi/crpbs/v8/3721