The Laws of Virus-Antibody
Interactions in vitro


The Laws of Antigen-Antibody Interactions in vitro Viggo Bitsch viggo.bitsch@gmail.com

       The Characteristics of the in vitro Antigen-Antibody Interactions

1.  1.
Antibodies are regularly bound firmly and irreversibly to their antigenic determinants under physiological conditions.

2.  2.
The binding force is so far unknown, as is also the force binding the hexavalent complement component C1q to the Fc fragment of antibodies sensitized for that binding by being bound to their antigenic determinant (see below). The force binding antigens and antibodies is highly specific.

3.   3.
The function of the two distinct binding forces is magnetism-like, meaning that, within a certain range, related binding sites are attracted to each other and subsequently bound firmly.

4.   4.
The ntibodies are flexible molecules, and their attractive binding forces will draw and bend a reacting arm towards its related binding site.

5.    5.
Detailed knowledge of the involved binding mechanisms will be needed to better understand and possibly even control or influence them.

6.  6.
Virus-neutralizing antibodies constitute a minor part of all specific antibodies to antigenic determinants on the virion. It is unclear whether individual viruses have one or a few types of neutralizing antibodies.

7.    7.
The structure of antibody molecules, either in a divalent form (e.g., IgG with 2 binding sites) or a polymerized, polyvalent form (e.g., IgM with 10 binding sites), illustrates their capability of aggregating infectious agents. Polyvalent versions have the advantage of a wider span between their binding sites.
It is evident that the di- or polyvalent structure of antibodies serves one specific function: aggregating agents, thereby facilitating inactivation in various ways.

8. 8.
IgG antibodies persist regularly for life. For herpesviruses, the concentration of the non-neutralizing IgG antibodies at the highest concentration is approximately 8 times that of neutralizing antibodies, whereas for IgM antibodies, the difference is greater, ranging from approximately 100 to 10, decreasing over their reaction period.

9.   9.
For IgM antibodies, a significant virus-inactivating effect of the non-neutralizing antibodies was demonstrated in a complement-enriched neutralization test (see Point 15 below) in serum collected 4 days after experimental nasal infection and in serum diluted 1:10.000 after 8-15 days. IgM antibodies, of which a minor part is neutralizing: 1) are typically present from a very few days after infection, 2) are found in extremely high concentrations during the acute infection phase, and 3) disappear after some weeks, indicating that production stops as soon as stimulation by the presence of antigen has faded out. Their ability to aggregate is immense, especially due to their extremely high concentration, which greatly enhances their rapid synergistic effect during direct aggregation (see below).
This justifies classifying IgM antibodies as those to defeat acute infections.

10. 10.
Viruses are inactivated by antibodies in vitro in three ways: 1) via attachment to neutralizing antibodies, 2) via direct virus aggregation by the di- or polyvalent antibodies, or 3) via aggregation of virus-antibody complexes by C1q, the hexavalent aggregator of antigen-antibody complexes. The effect of C1q has so far been shown to be effective with IgG and IgM antibodies.
Although these reactions can be identified as separate, they will be integrated if circumstances allow.

11. 111.
The reaction in a conventional titration neutralization test, where complement in the antibody medium has been inactivated, is bifactorial. The initial prompt and short-lasting virus-inactivating reaction, originally called over-neutralization, is almost exclusively direct virus aggregation, synergistically mediated by the di- and/or polyvalent antibodies. The second reaction is the very slowly progressing, enduring, and first-order monovalent neutralization reaction induced by neutralizing antibodies, which, for herpesvirus IgG antibodies, can be observed as a separate first-order reaction with reaction times exceeding 2-3 hours at 37 oC.

12.  12.
The temperature-dependency of the neutralization reaction implies that the herpesvirus antibody titer, or the test sensitivity, at 37 oC for 24 hours in a titration neutralization test is not achieved at room temperature or 4 oC until after 4 and 16 days, respectively. Antigen-antibody tests should therefore not be performed at temperatures below 37 oC.

13.  13.
The reason for the monovalent, one-hit characteristic of the enduring neutralization process is that the reaction with increasing reaction time is determined exclusively by new hits arising from molecular movements of the reactants. A hit occurs when related binding sites come close enough to attract and bind.
The reaction remains enduring in a titration neutralization assay because the dilution of the antibody medium is compensated for by the increased reaction time.

14.  14.
The direct virus aggregation by di- or polyvalent antibodies will primarily be prompt
(the antibody concentration may be too low for this synergistic reaction, even with a measurable first-order neutralization), because all related binding sites within a certain range will react immediately. The subsequent aggregation reaction will depend on new hits arising from reactant movements, but will progress very slowly and be short-lasting. The antibodies react synergistically. The total effect is highly dependent on the antibody concentration and will be dramatically reduced at lower general antibody concentrations. It is readily diluted away in a titration neutralization test.

15.  15.
The in vitro aggregation of virus-antibody complexes by C1q is also prompt for the same reason. In a complement-enriched titration neutralization test with extended reaction times, the reaction immediately after complement addition is rapid (the C1q concentration can be adjusted to ensure immediate binding to the formed complexes). The inactivation by C1q will thereafter be first-order, reflecting the first-order hits from reactant molecular movements as the reaction time increases. An infectious complex formed by a single non-neutralizing antibody in the presence of C1q is promptly inactivated by its inclusion into an aggregate.

16. 16.
The antigen-antibody interactions, where aggregation is absent or eliminated, e.g., by extended reaction and inactivation of complement, are indicated in the standard, multivariable, antigen-antibody interaction formula:            
This formula can be used to evaluate the influence of the individual variables, to  compare the sensitivity of antigen-antibody assays, and to determine an appropriate sensitivity for practical use. See text for detailed information about symbols and relations.

17.  17.
In antibody assays, the sensitivity, or antibody titer, will be determined by the reacting antibodies present in the highest concentration and will be proportional to the reaction time.
It is worth noting that aggregation is not possible in conventional antibody ELISAs; therefore, the formula will account for the test reactions.

18.  18.
In antigen assays, the sensitivity will be raised exponentially with both increased reaction time and increased antibody concentration, illustrating the immense antigen-binding capability of antibodies. In the formula, this effect is influenced by the temperature-dependent factor q, which represents the actual strength of the attraction between the antibody and the antigenic determinant. The factor q will have values between 0 and 1: the lower q's value, the stronger the force. In the herpesvirus-IgG study, q was approximately 0.15 at 37 oC but 0.24 at 4 °C. More vivid molecular movements at higher temperatures may explain or contribute to the temperature dependence of the factor q.
It is worth noting that aggregations are eliminated in conventional antigen ELISAs.

19.  19.
On the basis of the demonstrated antigen-antibody reaction lines, 1) the ideal gold-standard assay for specifically virus-neutralizing IgG antibodies, and 2) the ideal gold-standard assay for reacting non-neutralizing IgG antibodies in the highest concentration, are concluded to be 37oC/24h modifications of 1) a conventional neutralization test and 2) a conventional antibody ELISA, respectively. Similarly, the ideal gold-standard assay for demonstrating antigens can be concluded to be a conventional 37oC/24h antigen ELISA.

20.20.
Additionally, reference standard assays or adequately sensitive tests for practical use can be elaborated from the reaction lines of the formula.

21.21.
The reaction recorded in an antibody blocking ELISA reflects the reactions in antigen-antibody mixtures indirectly. This reaction is essentially log-log linear in time, but with a slope that differs from 1, actually indicating it is decelerating.

  1. NB:  On false hypotheses

    1.  In immunology textbooks, it is stated that antigens and antibodies are bound by non-covalent weak forces, i.e., electrostatic interactions, hydrogen bonds, van der Waals forces, and hydrophobic interactions. However, the force mediating antigen-antibody binding, as well as the force binding C1q to sensitized antigen-antibody complexes, remains unknown.

    2.  The equilibrium and occupancy theories of neutralization have never been substantiated and are documented to be invalid.

    3.  The Law of Mass Action (the Law of Chemical Equilibrium) does not pertain to antigen-antibody interactions.

    4.  The Percentage Law, comprising two aspects: 1) the rate of virus neutralization (or of virus-antibody bindings) will be independent of the virus concentration, and 2) a persistent fraction of virus will be a regular feature of virus-neutralization and will constitute a certain percentage of the virus, is invalid.

    5.  Despite the invalidity of the Percentage Law, the original observation of an irregular course of neutralization under certain experimental conditions with hyperimmune serum definitely required further study.
     The reaction seen is likely explained by a partial-to-complete physical blockage of access for the neutralizing antibodies to bind very late to their antigenic determinants, i.e., a blockage created by non-neutralizing antibodies bound to the virion, due to an increasingly limited space over time. Under extreme experimental conditions, this circumstance cannot be considered a regular feature, nor linked to “a percentage law”.

    6.  The term “steric hindrance” is a complex concept in chemistry. Still, it has been used to describe what, so far, seems to be an increasing physical blockage of access of some antibodies to their antigenic determinants by other antibodies bound to the virion, due to an over time, increasingly limited space. Both terms, “a persistent fraction of virus” and “steric hindrance”, appear inappropriate.

    7.  The term “broadly neutralizing antibodies” seems to stem from an insufficient understanding of the in vitro lines of antigen-antibody interactions. Antigen-antibody interactions in vivo, found or thought to be irregular, shall be evaluated against regular, and even irregular, in vitro reactions. Regular in vitro interactions also include two aggregation reactions that result in immediate and irreversible virus inactivation.
     The term “broadly neutralizing antibodies” appears inappropriate.

      Introduction

    The elaboration of safe assays demonstrating either antigen or antibody is based on in vitro antigen-antibody interactions. Furthermore, observations on reactions in vivo should be evaluated against both the regular lines of virus-antibody interactions in vitro and the irregular in vitro observations. This underlines the significance of in vitro interactions.

    False hypotheses are frequently encountered when relations are complex, and problem-solving consequently requires systematic analysis to be understood. Antigen-antibody interactions are a key topic in biomedicine and are highly complex with many enigmatic aspects. It is therefore not

    surprising that attempts to understand have led to a considerable number of theories that have later been found invalid. To prevent continuous misunderstandings, false or unsubstantiated theories published must be identified and effectively eliminated.

    Investigations into antigen-antibody interactions have been performed almost exclusively with virus antigens. However, antibodies are the essential reactant and are likely to bind immutably to antigens, regardless of the antigen's character or origin, so this should not be considered a limitation.

    This overview of in vitro antigen-antibody interactions will briefly describe relevant investigations up to 1970, but will mainly focus on personal findings thereafter that clarified reaction lines in antigen-antibody assays, and, more generally, in mixtures of antigen and antibodies.

    Definitions
     Neutralizing and non-neutralizing antibodies: Neutralizing antibodies inactivate viruses by binding to their antigenic determinants on the virion. In contrast, non-neutralizing antibodies cannot inactivate the virus simply by binding to their antigenic determinants.

    Measurements of antigens or antibodies can be performed in screening or titration assays. A 37oC/24h test modification employs reaction at 37 oC for 24 hours.

    The reacting antibodies present in the highest concentration determine the titer recorded in an antibody titration test. This titer is also a measure of the test sensitivity.

    A reference standard antibody test is a modification of an antibody test recommended by authorities for practical use or to indicate the required level of sensitivity.

    Antigen and antibody assays demonstrate antigens and antibodies, respectively.

    The Basic and Regular Antigen-Antibody Interactions in vitro

    1. The Early Investigations

    The earliest in vitro studies of virus neutralization were performed using bacteriophages or animal viruses with artificial hyperimmune sera. Samples were taken at intervals from a mixture of virus and antibodies to measure the progress of neutralization and the influence of variables.  

    The neutralization rate in a virus-antibody mixture was found to be semi-logarithmically linear with the reaction time (Andrewes and Elford 1933) [1], proportional to the antibody concentration (Burnet et al. 1937) [2], temperature-dependent (Dulbecco et al., 1956) [3], and, erroneously, independent of the virus concentration [1].

    However, when the reaction in titration neutralization tests was investigated, it was found to be inconsistent. No substantial progression of neutralization was observed after reaction at 37 oC for approximately 1 hour or at 4 oC for 1 day, which led to the perception that after a particular reaction time, the reaction would reach a state of equilibrium in compliance with the Law of Mass Action, now most often called the Law of Chemical Equilibrium. Apparently, nobody realized that a neutralization test reaction might be bi- or even multi-factorial, implying that not one mode of virus inactivation, but several should be considered.

    2. Personal Investigations

    Around 1970, a BoHV-1 infection was diagnosed at an artificial insemination (AI) bull center in Denmark. The possibility of the spread of this infection with semen from AI centers to numerous herds was unacceptable. Early examinations indicated that the neutralization reaction in a neutralization test: 1) was dependent on the virus concentration (virus dose), 2) was highly dependent on the reaction time, and 3) allowed testing of serum undiluted. A 37oC/24h test version was therefore relatively soon introduced as the reference standard antibody assay for control of animals to be admitted to the AI bull centers.

    After the pioneering decision in 1970 to eradicate the BoHV-1 infection in the AI bull centers, all animals were controlled annually, and no BoHV-1-infected animals were ever found to have passed access control.

    To gain further insights into antigen-antibody interactions, systematic analyses of the reactions were performed, not in individual virus-antibody mixtures as earlier done, but primarily in titration neutralization tests; first in conventional neutralization tests with natural antibody samples (Bitsch 1978) [4], and then in complement-enriched neutralization tests with serum collected during the first 21 days after experimental nasal infection (Bitsch and Eskildsen 1982) [5].  

    2.1. Reactions in conventional titration neutralization tests
     All investigations [4] were planned and performed systematically to clarify the influence of the four independent variables: the concentrations of the virus and the reacting antibody, as well as the reaction time and temperature.

    Biology is, to a vast extent, logarithmic, so reaction times were planned and recorded on a logarithmic scale. This was crucially important.

    Figures 1, 2, and 3 illustrate the relationships between antibody titers and, respectively, short, extended, and excessive reaction times. Additionally, the influence of virus concentration is included in Figure 2, and the effect of reaction temperature in Figure 3.

     

    Figure 1. The progression of virus neutralization in titration neutralization tests at 37 oC with short reaction times. (From Bitsch,1978).
     VNA: virus-neutralizing antibody. Preincubation period: reaction time.
     Three sera with different late-infection antibody levels (IgG) were investigated. The reaction time was planned and plotted on a logarithmic scale. It should be noted that if the reaction time had been shown non-logarithmically, the neutralization lines would have been seen to “flatten out”, which, by many, would be interpreted as an indication of almost no improvement of the test sensitivity by increasing reaction time.

     

    Figure 2. The progression of virus neutralization in titration neutralization tests at 37 oC with extended reaction times, and with virus concentrations (virus doses) varying from 101.5 to 105.5 TCID50. (From Bitsch,1978).
     The neutralization lines can be considered the continuation of those shown in Figure 1. These log-log reaction lines approach a straight line with a slope of 1. From 2-3 hours after reaction onset, they can be considered linear, indicating a first-order relationship between the antibody titer and reaction time (see text). Please note that 1) if the reaction time for a fixed virus concentration with extended reactions is doubled, the antibody titer, or test sensitivity, will also be doubled, and 2) if for a fixed antibody concentration, the reaction time is doubled, or for a fixed reaction time, the antibody concentration is doubled, the amount of virus bound or neutralized will be increased by a factor of approximately 100, demonstrating a tremendous virus-neutralizing capability of antibodies, see text.


    Figure 3. Progression of virus neutralization in titration neutralization tests with excessive reaction times from 12 hours to 8 days, and with reaction temperatures of 4, 15, 26, and 37 oC. (From Bitsch, 1978).
     The log-log neutralization lines are linear with a slope of 1, indicating a first-order antibody reaction. It is worth noting that the sensitivity (titer) of an antibody test with a reaction at 37 oC for 24 hours is not achieved at room temperature or 4 oC until after reactions of, respectively, 4 and 16 days, i.e., another logarithmic relationship. The virus stock used was prepared to ensure that the active virus was only a few hours old at the time of storage, and controls were included in all experiments, demonstrating that no time-dependent virus inactivation other than neutralization by antibodies was involved.

    As reaction time increases, the log-log neutralization lines in these figures approach a straight line with slope 1. At 37 oC, the reaction in the virus-neutralization test is strictly first-order from 2-3 hours onward. This first-order antibody reaction will be understood from the following. In the simple first order equation, y = ax + b, the y intercept b shows where, in a coordinate system, the straight line will cross the y axis; the equation y = ax will, logarithmically transformed, be log y = log x + log a, which documents that a straight line with a log-log slope of 1 identifies a first order relationship for the variables y and x. For further details of the regular virus-antibody interactions in vitro, see Bitsch, 1978, 2017, 2024, 2024, 2025 [4,6,7,8,9] and Bitsch and Eskildsen, 1982 [5].

    The rapid neutralization, with short reaction times, that surpasses what should be expected for a first-order reaction, was concluded to be a regular phenomenon and was termed over-neutralization. This reaction could not be observed in the investigations by Andrewes and Elford because their antibody preparations had to be appropriately diluted.

    Brioen et al. [10] demonstrated in 1983 that virus was inactivated by being aggregated by antibodies, which logically explained that over-neutralization [4] was caused by this second mode of neutralization, i.e., simple aggregation of the virus by antibodies, here divalent IgG antibodies, without interference from complement [6,8,9].

    The neutralization reaction in a conventional titration neutralization test, where the antibody medium has been heated to inactivate complement, is therefore bifactorial, consisting of: 1) the prompt and early, short-lasting over-neutralization, i.e., virus inactivation by predominantly non-neutralizing antibodies, being, or becoming, within a certain range from their antigenic determinant, a range determined by the magnetism-like, attractive force also binding the reactants [8], and 2) the enduring, but slowly progressing, monovalent reaction of first order by specifically neutralizing antibodies observable separately only with extended reaction times [4].

    It should be noted that 1) all the various antibodies react synergistically in the early aggregation, and 2) the reason for the monovalent characteristic of the enduring neutralization process is the circumstance that the reaction is determined exclusively by “hits” arising from molecular movements of the reactants. A “hit” is when related binding sites come so close that they become attracted to each other and subsequently bound.

    The formula for the reaction in antigen-antibody media, not influenced by aggregation reactions, presented in 1978 and, with the present symbols, in 2017 [4,6,9], is:

    . In this formula, kst is the standard reaction rate factor, Ab and Ag are titers of the reacting antibody and antigen, T is the reaction time, and the factor q, a new co-determiner of the reaction rate unexpectedly observed, is a particular log-log antibody/antigen binding ratio independent of the reacting antibody and antigen concentrations but varying with the reaction temperature. In this way, q is a measure of the force that attracts and binds the reacting antibody to its antigenic determinant. The reaction temperature, the fourth independent variable, is indirectly indicated in the equation by the factor q, which varies with it. In the 1978 herpesvirus-IgG antibody neutralization study, q was approximately 0.15 at 37 oC but 0.24 at 4 oC. Detailed knowledge of the binding mechanism will be needed to understand better, and possibly even control or influence, these reactions.

    From the reaction lines expressed in this formula, tests demonstrating antigens and antibodies with previously unknown high sensitivity have been developed and used in the veterinary biomedical field for a long time [7,8,9]. With extended reaction times, not influenced by aggregations, the sensitivity of an antibody assay will be proportional to the reaction time, which is a considerable response. In contrast, the sensitivity of an antigen assay will rise exponentially with reaction time, to a degree depending on the value of the factor q.

    Regarding these relationships, the results in Figure 2, with an extended IgG antibody reaction with a herpesvirus not showing aggregations, illustrate:

    ·        For an antibody assay:
     A changed reaction time results in a directly proportional change in antibody titer or test sensitivity. An increase in the reaction time from 1-24 hours in a conventional neutralization test, used with late-infection antibody samples, will increase the sensitivity, not by a factor of 24, but by a factor of 16-18, because of a low residual aggregation recorded after 1 hour of reaction. This demonstrates the high sensitivity of the 37oC/24h recommended gold standard titration neutralization test for specifically neutralizing IgG antibodies [7]. Please note that antigen-antibody aggregation is impossible in a conventional antibody ELISA.

    ·        For an antigen assay:
     If either the antibody concentration or the reaction time is doubled, the number of virus particles neutralized or bound by the antibodies will be increased by approximately a factor of 100, illustrating the almost incredible neutralizing or antigen-binding potency of antibodies. These relations will appear from the formula when q is substituted with 0.15. If the reaction is performed at 4 oC (q = 0.24), the improvement in sensitivity for the antigen, corresponding to either a doubling of the antibody concentration or a doubling of the reaction time, will be approximately by a factor of 30. This illustrates the extreme sensitivity of a conventional 37oC/24h antigen ELISA [7,9]. Please note that antigen-antibody aggregation is impossible in a conventional antigen ELISA.

    The formula above expresses the regular reaction lines for the reacting antibody at the highest concentration and the antigen in an antigen-antibody mixture, under conditions that exclude aggregation. Therefore, the formula can be used for two purposes: 1) to understand, determine, and compare test sensitivities, and 2) to indicate the reaction characteristics in mixtures of antigen and antibody.

    2.2. Reactions in the complement-enriched neutralization test
     The investigations [5] were explicitly undertaken to better understand the inactivation of the virus by antibodies in combination with the C1q component of complement. Again, complement-enriched titration neutralization tests were anticipated to be more suited for investigation than individual antigen-antibody mixtures.

    The results of the investigations have been published in detail in various articles [5,7,8,9]. The C1q component is hexavalent and binds to the Fc region of antibodies sensitized for this binding by the action of being bound to its antigenic determinant on the antigen. This means that C1q will aggregate, not antigens directly as the di- or polyvalent antibodies do, but antigen-antibody complexes. The concentration of C1q can be adjusted to ensure immediate reaction with the virus-antibody complexes under in vitro conditions by using an appropriately low dilution of a stored complement source, e.g., guinea pig serum.

    Figure 4. The effect of complement in complement-enriched neutralization tests on the progress of reaction with an early convalescent-phase serum with IgM and IgG antibodies after nasal herpesvirus infection. (From Bitsch and Eskildsen, 1982)
     The SuHV-1 antigen and titration neutralization tests with extended reaction times at 37 oC were used. Preincubation period: reaction time. K0 and K1: no complement (K0) or heat-inactivated complement (K1) was added at the start of virus-serum incubation. For the neutralization test results 1-1, 2-2, 3-3, and 4, the complement was added at the start of the reaction or after 5, 11, and 23 hours, respectively. Due to the relatively high concentration of the reacting IgM antibodies, only IgM reactions are shown in titrations.

    Figure 4 illustrates the complement-mediated supplementary inactivation. First, after addition, the complement reacts readily with all immediately formed virus-non-neutralizing antibody complexes, inactivating them by aggregation. Thereafter, the inactivation elicited by complement is first-order, following the first-order binding of the non-neutralizing IgM antibodies in the highest concentration to their antigenic determinants. It is also evident from reactions 1-1, 2-2, 3-3, and 4 that, regardless of when the complement is added, the binding of the non-neutralizing antibodies is strictly first-order. If a considerable part had been reversible, a decelerating binding rate would have been seen.

     

    Figure 5. The consecutive appearance of non-neutralizing and neutralizing IgM and IgG antibodies during the first 21 days after experimental nasal herpesvirus infection, as shown in 37oC/24h conventional and complement-enriched titration neutralization tests. (From Bitsch and Eskildsen, 1982).
     Virus: SuHV-1. The sera were complement-inactivated at 56 oC for 30 min. and tested, either untreated (N) or treated with 2-mercaptoethanol (2-MC), which will inhibit the neutralizing effect of IgM antibodies, but leave IgG antibodies unchanged. In both cases, they were tested with and without the addition of complement (C´). The virus-serum mixtures were incubated at 37 oC for 24 hours, and where used, complement was added after 23 hours of reaction. Results from a complement fixation test are also shown (CF). The CF titers follow the titers measured for the dominant non-neutralizing IgM antibodies, albeit at much lower levels. Titers of non-neutralizing IgG antibodies were approximately 8 times (3 log base 2 units) higher than those of the neutralizing ones, as found earlier for other herpesvirus IgG antibodies.
     Titers measured for the antibodies are indicated by the symbols as follows:
       Symbols:  N+C':        non-neutralizing IgM antibody titers
               N:              neutralizing IgM antibody titers
               2MC+C':   non-neutralizing IgG antibody titers
                2MC:         neutralizing IgG antibody titers
                    CF:            titers from a conventional complement-fixation test

    Figure 5 shows the consecutive development of non-neutralizing and neutralizing IgM and IgG antibodies after experimental nasal infection with SuHV-1 virus, as demonstrated in a complement-enriched neutralization test.  A significant neutralizing effect of non-neutralizing IgM antibodies was demonstrated in serum collected 3-4 days after experimental nasal infection and in serum diluted 1:10,000 after 8-15 days. The binding of just one non-neutralizing antibody molecule to the virus resulted in prompt inactivation by inclusion of the virus-antibody complex into aggregates of such complexes formed by C1q [5,6,8,9].

    IgM antibodies are typically present only during the acute infection and shortly thereafter, and there seems to be no reason to doubt that their primary function is to combat infections. The in vitro investigations shown in Figure 5 even indicate that the non-neutralizing antibodies, present in concentrations determined primarily by the number of related antigenic determinants, have by far the highest virus-inactivating potency [5,8].

    The titers of reacting non-neutralizing IgG antibodies present in the highest concentration are approximately 8 times higher than those of neutralizing antibodies, a regular feature also for BoHV-1 herpesvirus IgG antibodies [5,9].

    In conclusion, there are two different aggregation modes of virus inactivation by antibodies: 1) the simple aggregation of virions by di- or polyvalent antibodies, involving predominantly non-neutralizing but to some extent also neutralizing antibodies, and 2) the C1q-dependent aggregation of virus-antibody complexes, again involving both groups, but predominantly non-neutralizing antibodies. The number of neutralizing antibody types to a virus is unknown, but presumably one or only a few, while the number of non-neutralizing antibody types is typically very high. The concentration of non-neutralizing antibodies will therefore be much higher than that of neutralizing antibodies, and just one non-neutralizing antibody bound to its antigenic determinant of the virus results, under in vitro conditions, in immediate virus inactivation. Furthermore, it should be noted that all the various antibodies react synergistically in aggregations. These characteristics, which imply that non-neutralizing antibodies are the most important virus inactivators in vitro, indicate that their in vivo virus-inactivating effect, in the extracellular space and/or on mucous membranes, may be extraordinary [8].

    References

    1. Andrewes CH, Elford WJ. Observations on anti-phage sera. I. "The percentage law". Brit J exp Path. 1933; 14: 367-376.
    2. Burnet FM, Keogh EV, Lush D. Immunological reactions of the filterable viruses. Austral J exp Biol med Sci. 1937; 15: 231-368.
    3. Dulbecco R, Vogt M, Strickland AGR.  A study of the basic aspects of neutralization of two animal viruses, western equine encephalitis virus and poliomyelitis virus. Virology. 1956; 2: 162-205.
    4. Bitsch V.  An investigation into the basic virus-antibody neutralization reaction, with special regard to the reaction in the constant-virus/varying-serum neutralization test. Acta vet scand. 1978; 19:110-128.  
    5. Bitsch V, Eskildsen M.  Complement-dependent neutralization of Aujeszky's disease virus by antibody. In: Aujeszky's Disease. Wittmann G, Hall SA, editors. Martinus Nijhoff Publishers, The Hague, Boston, London; 1982: 41-50. 

    6.  Bitsch V. The regular lines of antigen-antibody interactions in vitro. 2017.
    ISBN 978-87-994685-2-2. Available from: http://antigenantibodyinteractions.blogspot.com or ResearchGate

    1. Bitsch V. Antigen-Antibody Interactions in vitro: I. The Characteristics of Reactions in Tests for Antibodies to Viruses and Their Significance for Standard Assays and Adequate Routine Tests. Journal of Biotechnology and Biomedicine. 2024; 7: 241-255. DOI:10.26502/jbb.2642-91280148
    2. Bitsch V. Antigen-Antibody Interactions in vitro: II. The Non-Neutralizing Antibodies are by far the Most Potent Virus Inactivators. Journal of Biotechnology and Biomedicine, 2024; 7: 256-263. DOI:10.26502/jbb.2642-91280149
    3. Bitsch V. Systematic Analyses of Antigen-Antibody Interactions in vitro: Insights from Three Different Ways of Virus Inactivation and Their Specific Reaction Lines. Contemporary Research and Perspectives in Biological Science. 2025; 8: 64-92. DOI: 10.9734/bpi/crpbs/v8/3721
    4. Brioen PD, Dekegel, Boeyé A.  Neutralization of poliovirus by antibody-mediated polymerization. Virology. 1983; 127: 463-468.