Characteristics of the Reactions in Tests for Antibodies to Viruses and Their Significance for Standard Assays and Adequate Routine Tests
Authors: Bitsch Viggo 
Published: June 2023

Viggo Bitsch: Characteristics of the reactions in tests for antibodies to viruses and their significance for standard assays and adequate routine tests.
Interactions between antibodies and viruses are diverse and are used in different ways in tests for the demonstration of either virus or antibody. Three tests, each based on one of three different neutralization reactions by antibodies, and another two ELISA modifications of which one demonstrates a binding reaction and the other one a blocking reaction by antibodies, represent the five basic types of antibody tests.
These five tests are 1) the first-order virus neutralization test (f-ord-VNT), 2) the virus aggregation neutralization test (aggr-VNT), 3) the complement-enriched virus neutralization test (C-enr-VNT), 4) the conventional antibody ELISA (conv-ab-ELISA), and 5) the blocking antibody ELISA (bl-ab-ELISA).
Basic versions of these five tests are evaluated. The reaction in each test is described. The reacting antibodies in the highest concentration determining the antibody titer and the test sensitivity are defined as either neutralizing or non-neutralizing, and modifications with a sensitivity found optimal are evaluated for routine practical use and for the potential use as a reference or even gold standard assay (cf. Definitions).
In the f-ord-VNT with appropriately extended reaction, the reacting antibodies are exclusively neutralizing. The reaction is of first order, implying that the sensitivity is depending on the temperature and is proportional to the reaction time. A 37oC/24h configuration (reaction at 37 oC for 24 hours), being approx. 16 times more sensitive than a 37oC/1h version, is judged to be the ideal reference and gold standard assay for measuring truly neutralizing antibodies to viruses.
In the aggr-VNT, the inactivation by aggregation is explosive and short-lasting. All antibodies to the various antigenic determinants react synergistically. The reaction is highly dependent on the antibody concentration and can readily be diluted away, but the sensitivity is low and not variable, and the test is of no significance for the demonstration of antibodies to viruses.
The reaction in the C-enr-VNT is explosive immediately after the addition of complement, but otherwise of first order with appropriately extended reaction times. The sensitivity of a 37oC/24h modification is equal to that of a conv-ab-ELISA with identical reaction conditions, but the test is more laborious and therefore not relevant for practical use.
The conv-ab-ELISA is a first-order assay, and the reacting antibodies in the highest concentration determining the test sensitivity are non-neutralizing. Aggregation of virions is impossible, and the sensitivity is directly proportional to the length of the reaction time. The 37oC/24h conv-ab-ELISA is an ideal reference and gold standard antibody assay for comparative measurements of the test sensitivity. Because of its high sensitivity, it is excellent for controlling questionable results by other tests, and it is well-suited for routine large-scale examinations.
The sensitivity of the bl-ab-ELISA, is dependent on both the reaction temperature and time, but the reaction is not immediately of first order. An increase of the reaction time from 1 to 24 hours at 37 oC will raise the sensitivity by approx. a factor of 4, but still the sensitivity is relatively high. The reactants can easily be varied in different ways to serve different objectives. In its basic configuration, it is the test best suited for large-scale antibody examinations in connection with the diagnosis and control of viral infections.
The sensitivity and specificity of 37oC/24h modifications of the three antibody tests found of special value are regularly over 99 percent when undiluted samples are examined.
SARS-CoV-2 antibody tests are referred to in relevant sections.