Neutralization of virus by antibody in vitro

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Introduction

The first studies of the lines of the virus-antibody neutralization reaction in vitro were undertaken around 1930, and over the following decades more comprehensive studies have revealed regular lines of antigen-antibody interactions, which are of importance for the performance of a variety of in vitro assays for demonstration and quantification of specific antigens and antibodies. Specific antibodies to viruses tested in vitro will usually be of either the divalent IgG or the decavalent IgM isotypes directed specifically against the various antigenic determinants of the virus.

Virus-neutralizing antibodies, in contrast to non-neutralizing antibodies, are defined as antibodies abolishing the infectivity of the virus for tissue culture cells by binding to their specific antigenic determinants on the virus.

The neutralization reaction in vitro is bifactorial

In 1933 it was reported that the rate of neutralization in a mixture of virus and antibody proceeded linearly with time, but also erroneously, as indicated below, that this neutralization rate was independent of the virus concentration. A fraction of the virus was found to be resistant to neutralization... In 1937 it was found that this rate was proportional to the antibody concentration . These relationships were confirmed by others over the following years, also in a publication from 1956, where it was further concluded that the rate of neutralization was temperature-dependent . In 1978 it was documented that two different, regular features of neutralization were involved in the neutralization process, i.e., (1) a basic and enduring first order (i.e., linearly progressing) reaction corresponding to the reaction demonstrated earlier and (2) a time-limited reaction, which was termed over-neutralization, because it was a neutralization reaction additional to the first one. The persistent fraction of virus was found not to be a regular feature of the neutralization process (investigations were performed with herpesviruses), see also review from 2017 . In 1983 it was demonstrated that also the aggregation of virus particles by antibody might result in neutralization , , see also review from 2001. Over-neutralization was concluded to be neutralization by aggregation of virus particles. The regular reactions involved in virus neutralization in vitro are, therefore, (1) neutralization by specifically neutralizing antibodies and (2) neutralization by the formation of virus aggregates created predominantly by the non-neutralizing antibodies.

Neutralization by specifically neutralizing antibodies

The neutralization by neutralizing antibodies is associated with exclusively monovalent binding, as di- or polyvalent binding will be included in the aggregation process. Furthermore, it is progressing as a first-order reaction. The rate of neutralization in a virus-antibody mixture can be expressed by the formula (Eq. 1) $k={\frac {D}{T}}\,log{\frac {V_{0}}{V_{T}}}$ , if the dependence on antibody concentration is included , and by the formula (Eq. 2) $k_{s}={\frac {D}{T}}\,V_{0}^{q}$ , if the dependence on virus concentration is included as well. In these formulas k and k_s are neutralization rate factors (s for standard), D is (relative) antibody concentration, T is the reaction time, V₀ and V_T are the concentrations of infective or non-neutralized virus (virus titer) after 0 or T hours of reaction, while q expresses a certain antibody-virus equivalence factor of binding, which is a characteristic of the reaction.

From Eq. 2 follows that the antibody concentration D as determined in a neutralization test will be directly proportional to the reaction period T, if neutralization by aggregation can be disregarded. In neutralization tests with herpesviruses at a reaction temperature of 37 C^o, for example, the sensitivity by increasing the reaction period from 3 to 24 hours was correspondingly raised by a factor of 8, whereas the sensitivity by increasing the reaction period from 1 to 24 hours was raised, not by a factor of 24, but only by a factor of approx. 18 because of a still remaining low degree of neutralization by aggregate formation after 1 hour of reaction.

It was furthermore documented that reaction temperatures below 37 C^o would lower the test sensitivity to such a large extent that reaction temperatures lower than 37 C^o should regularly not be used. The sensitivity of a 37 C^o/3hrs. neutralization test, for example, was not reached at a reaction temperature of 4 C^o until after 2 days of reaction.

Neutralization by formation of aggregates

Aggregates of virus created by antibodies (natural antibodies)

This neutralization due to the formation of virus aggregates, in the 1978 investigation called over-neutralization, is caused predominantly by non-neutralizing antibodies, but also aggregates created by neutralizing antibodies bound to one or more virions will be included. The rate of the aggregation reaction is initially tremendously high, because the whole variety of non-neutralizing antibodies is involved. In the herpesvirus investigations, it was rapidly decelerating after a few minutes and practically absent after 2 hours of reaction at 37 C^o. The reaction is dependent on the antibody concentration and can be diluted away. The reason why this reaction was not observed in the investigations of neutralization rates in virus-antibody mixtures is that the antibody samples had to be diluted appropriately to facilitate the observation of the neutralization reaction over time.

Aggregates of complexes of virus bound to non-neutralizing antibodies created by complement

Complement-dependent neutralization is also caused by aggregation. Complement has a certain ability to bind to antibodies that have been "opened" for attachment by binding to their antigenic determinants. The aggregates accordingly consist of complexes of virus bound to non-neutralizing antibodies, where these complexes are held together by complement. As the progression of binding of non-neutralizing antibodies to their antigenic determinants will follow the lines of a first-order reaction, the complement-dependent reaction will also be of first order. With a herpesvirus, use of complement raised the sensitivity of a neutralization test used on serum with mainly IgM antibody by factors varying from approx. 100 to 500, while with mainly IgG antibody samples it was increased by a factor of 8

Aggregates of complexes of virus bound to antibodies created by anti-isotype antibodies (artificial antibodies)

Also, anti-isotype antibodies (antibodies to immunoglobulins produced in a different animal species), can, again because of their di- or polyvalency, aggregate complexes of virus bound to non-neutralizing antibodies. The neutralization reaction in a virus-antibody mixture will, therefore, be increased considerably by the addition of anti-isotype antibodies

Antigen-antibody reactions in laboratory tests

This section is included only to exemplify the involvement of the two different basic antigen-antibody interactions in laboratory tests. Details of the various tests must be sought elsewhere.

The two reactions, (1) the reaction between an antigenic determinant and its specific antibody, which is enduring and of first order and (2) the aggregation reaction which is tremendously fast, practically instantaneous, are used in various tests for documenting presence of, but also for quantifying, antigens and antibodies. For example are agglutination tests, also haemagglutination tests, and complement fixation tests based primarily on the rapid aggregation reaction. As shown above, are both reactions involved in tests for demonstration of neutralizing antibodies in ordinary polyclonal serum samples, but it is the enduring, time-dependent reaction by specifically neutralizing antibodies after cease of the aggregation reaction that gives the high sensitivity associated with the use of extended reaction periods. Samples tested for the presence of neutralizing antibodies are regularly heat-inactivated to prevent interference from complement being present. If complement then is added under controlled conditions, complement-dependent aggregation can further increase the neutralization reaction. An ELISA for demonstration of antigen using capture antibody is based on the binding reaction between an antigen and its specific antibody or antibodies, so the test sensitivity will be time-dependent. In a traditional indirect ELISA for demonstration of antibody, the test sensitivity will similarly be time-dependent, implying that high sensitivity will require extended reaction periods.

References

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