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

Author(s): Viggo Bitsch

Interactions between antibodies and viruses are diverse and function in different ways in tests demonstrating virus or antibody. Three antibody tests based on three different neutralization reactions by antibodies and another two ELISA modifications, one demonstrating a binding reaction and the other a blocking reaction by antibodies, represent five different antigen-antibody interactions used for demonstrating antibodies to viruses.

These five tests are the first-order virus neutralization test, the virus aggregation neutralization test, the complement-enriched virus neutralization test, the conventional antibody ELISA, and the blocking antibody ELISA. Basic versions of these tests are evaluated. The reaction in each test is described. The reacting antibodies in the highest concentration measuring the antibody titer and the test sensitivity are, where relevant, defined as either neutralizing or non-neutralizing, and modifications adjusted to possess an appropriately high sensitivity are evaluated for routine use and for the potential application as a reference or even gold standard assay, cf. Definitions.

The reaction in a first-order virus neutralization test is slowly progressing and enduring with increasing reaction times and the reacting antibodies are exclusively neutralizing. The test sensitivity is temperature-dependent and directly proportional to the reaction time. The very sensitive 37oC/24h configuration (reaction at 37 oC for 24 hours) is concluded to be the ideal reference and gold standard assay measuring neutralizing antibodies to viruses.

In the virus aggregation neutralization test, the inactivation of the virus by aggregation is prompt and short-lasting. All antibodies to the various antigenic determinants on the virus, neutralizing or non-neutralizing, react synergistically. The reaction depends strongly on the antibody concentrations, i.e., largely on the number of antigenic determinants on the virion. The test sensitivity is low and practically not adjustable, because of which the test is unsuited for demonstrating antibodies to viruses. This test is almost similar to a conventional 37oC/1h neutralization test, which by many has been considered a gold standard assay for demonstrating neutralizing antibodies.

In the complement-enriched neutralization test, the reacting antibodies of the highest concentration are non-neutralizing The reaction is almost instantaneous immediately after the addition of complement due to a prompt reaction with antigen-antibody complexes formed, but otherwise of first order with extended increasing reaction times following the first-order binding of non-neutralizing antibodies to the virus. The sensitivity of a 37oC/24h modification is high and for IgG antibodies equal to that of a conventional antibody ELISA with identical reaction conditions. However, it is laborious, because of which the latter will be a better alternative.

The conventional antibody ELISA is basically a first-order assay, and the reacting antibodies of the highest concentration determining the antibody titer and test sensitivity are non-neutralizing. Aggregation of virions is impossible, and the sensitivity is directly proportional to the length of the reaction time. A 37oC/24h configuration will be the ideal reference and gold standard antibody assay for measuring titers of non-neutralizing antibodies in the highest concentration and test sensitivities.

The sensitivity of the blocking antibody ELISA depends on the reaction temperature and time, but the reaction is not of first order. Increasing the reaction time from 1 to 24 hours in a herpesvirus test raises the sensitivity by approx. a factor of 4. The reaction rate is decelerating with increasing reaction time but still the sensitivity is relatively high with extended reaction. With herpesviruses, the sensitivity of a 37oC/24h test is twice that of the 37oC/24h first-order neutralization test, although 4 times lower than that of the conventional antibody ELISA. In its basic configuration, it is the test best suited for large-scale antibody examinations in diagnosing and controlling viral infections. The reactants can be varied to serve different objectives.

The sensitivity and specificity of 37oC/24h modifications of the three antibody tests found of special value are regularly over 99 percent when undiluted serum/plasma samples are examined.

SARS-CoV-2 antibody testing is referred to in relevant sections.