Single radial hemolysis test for quantitation of complement-fixing antibodies to non-hemagglutinating viruses

Abstract

A single radial hemolysis test, which overcomes many of the problems of conventional complement fixation tests, was developed for the quantitation of virus complement-fixing antibodies. The test procedure utilized staphylococcal protein A-coated sheep erythrocytes immobilized in agarose into which antigen was incorporated. Undiluted heat-inactivated serum samples were allowed to diffuse radially from wells punched in the agarose. Protein A served to concentrate the subsequent antigen-antibody reaction on the surface of the erythrocytes. Zones of hemolysis were developed by flooding with complement. With adenovirus as a model, basic test parameters were defined, and optimum reagent concentrations and diffusion conditions were determined. A positive linear relationship was found to exist between zone diameter and increasing log concentration of specific antiserum. No correlation was found between zone diameter and total concentration of immunoglobulin G in test sera. Sera rendered anticomplementary by the addition of carrageenan produced hemolytic zones equal to diameter to those observed with untreated sera. Seventy-seven human sera with known complement fixation titers were tested by this method. Good correlation (r = 0.74) between the complement fixation test and single radial hemolysis was observed. This test was highly reproducible and more sensitive than the conventional complement fixation test.

Overview of Serological Techniques for Influenza Vaccine Evaluation: Past, Present and Future

“…The haemolysis, mediated by complement and induced by the antibody-antigen complex, produces easily identifiable “zones of haemolysis”, whose size is proportional to the concentration of anti-influenza antibodies present in the sera [ 88 ]. SRH has been used to detect antibodies not only against the influenza viral hemagglutinin but also against numerous other viruses such as coronaviruses, parainfluenza virus, Dengue virus and Japanese Encephalitis virus [ 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 ].…”

“…This aspect is particularly advantageous in the case of H5N1 because the serological tests can be safely carried out under BSL-2 containment [ 97 ]. SRH is inexpensive, rapid, reliable, reproducible and the quantitative, unbiased results are available after an overnight incubation [ 89 , 91 , 98 , 99 , 100 , 101 ]. Other significant advantages to the SRH assay include the ability to simultaneously and rapidly test a large number of samples without pre-treatment (excluding complement inactivation), and the requirement for only a small volume of sera [ 93 ].…”