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A quantitative micro-complement fixation test capable of detecting nanogram quantities of antigen is described. The test is simple to perform and is highly reproducible. Typical results from three antigen-antibody systems are given.
Complement fixation (CF) by bovine IgG1 or IgM antibodies to Brucella abortus was inhibited by specific non-complement-fixing antibodies of the IgG2 subclass. This inhibition may account for the appearance of prozones in CF titrations of some antiserums, and for the occurrence of serums which are positive to the Rose Bengal test, but negative to the CF test.
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The conglutinating complement fixation test was compared with the haemolytic complement fixation test for the detection of brucella antibodies in human sera. Conglutinating-complement-fixation antibodies were detectable during the early stages of acute brucellosis and persisted for approximately 1 year after the infection.
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We describe here a microneutralization procedure for conveniently testing large numbers of specimens for antibodies to enterovirus 70. The test utilized human rhabdomyosarcoma cells and was read by staining with crystal violet after 4 days of incubation. The test compares well with other serological assays, being more sensitive than the standard tube neutralization test and the complement fixation test, but less sensitive than the hemagglutination inhibition test. However, the hemagglutination inhibition test required concentrated, partially purified virus as antigen, as did the complement fixation test, and was difficult to read, so that its greater sensitivity may not be of practical significance. By all four test procedures, a recent isolate of enterovirus 70 was a more sensitive antigen than the prototype strain, as shown by greater geometric mean titers in sera of patients from various epidemics.
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Control of paratuberculosis in dairy herds is based on preventing the transmission of Mycobacterium avium subsp. paratuberculosis (Mptb) from cows to calves by management measures, supported by removal of cows excreting these bacteria by the fecal route (Mptb shedders). Fecal culture is the most accurate test for identifying Mptb shedders, but this technique is expensive and takes up to 16 weeks for results to be available. Serologic tests are inexpensive, rapid, and easy to perform. Of serologic tests, the complement fixation test (CFT) and absorbed enzyme-linked immunosorbent assay (ELISA) are the serologic tests used most frequently; the CFT is considered less accurate than the ELISA with respect to sensitivity and specificity. The commonly accepted absorbed ELISA is from the Australian Central Serum Laboratory. However, a European supplier has marketed a second ELISA that is supposed to be more sensitive in detecting Mptb shedders. These 2 absorbed ELISAs, designated ELISA-A and ELISA-B, and an in-house CFT were compared with data from 2 serum panels. The Mptb shedding panel consisted of sera from 198 culture-positive cows from 53 infected herds. The method used for culture of fecal samples was a modified Jørgensen method on individual samples. The Mptb shedder detection rate by the 3 serologic tests ranged from 29.8% to 39.4%. Detection rate for ELISA-A was lower than that for ELISA-B and CFT. For all 3 tests, detection rate was dependent on the level of Mptb shedding and the age of the animals. Detection rates increased as cattle age increased to 4 years. The specificity panel was initially composed of sera from 811 cows randomly selected from 41 herds without clinical paratuberculosis that were negative for Mptb based on whole-herd fecal culture. The modified Jørgensen method for culture was used on pooled fecal samples. Serologic test specificity ranged from 93.4% to 99.8%. The specificity of ELISA-A was higher than that of ELISA-B and CFT. Specificity of ELISA-B between herds was 75-100%. Specificity of CFT between herds was 62-100%. The low specificity of ELISA-B and CFT could not be explained by a higher sensitivity for Mptb-infected cows before onset of shedding, because in the 19 herds with 8 more subsequent negative whole-herd fecal cultures in the 4 years after sampling, specificity was not improved. The insufficient specificity of ELISA-B was not corrected sufficiently by heightening the cutoff value because Mptb shedder detection rate was lowered to 28.9%, equal to that of ELISA-A, and specificity only rose to 97%, much lower than that of ELISA-A. Taking into account the different test characteristics, serologic tests are a cost-effective alternative to fecal culture in high-prevalence herds. For certification programs, only ELISA-A is recommended because in a large number of nonsuspect herds specificity remained almost 100%.
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The complement fixation test was evaluated in a study of Salmonella dublin infection in cattle. In calves vaccinated with a live S dublin vaccine at three days of age, complement fixing (CF) antibodies were not detected in colostrum-fed calves although CF antibodies were detected at low titre in colostrum-deprived calves. In calves vaccinated at three months of age and in experimentally infected cows, CF antibodies persisted for three to six months but serum agglutinin (SA) antibodies persisted at diagnostic titres for longer periods. When serum samples from outbreaks of S dublin infection were examined, the CF results were in agreement with the SAT in the majority of samples obtained from adults and calves suffering from enteric disease. In samples from cases of S dublin abortion, a number of samples had negative CF titres although the SA titres were indicative of infection. Non-specific CF reactions occurred in serum samples from cattle infected with salmonella serotypes other than S dublin.
The preparation of complement-fixing antigens for the Coxsackie group of viruses (C virus) is described. This includes the manufacture of crude antigens, their subsequent treatment with protamine sulfate to remove non-specific interfering substances, and their concentration by ultracentrifugation. The plate complement fixation technique of Fulton and Dumbell is described in detail as it has been used for the Coxsackie viruses. Seven strains of C virus have been cross-tested in the plate complement fixation test and have been found to belong to six immunologically distinct types. The temporal pattern of complement-fixing antibodies in human beings infected with two types, Ohio-1 and Easton-2, respectively, has been studied. In the former the antibodies rise to a peak rather late in convalescence (3rd month) and in the latter, complement-fixing antibodies are already present at high levels in the acute phase serum. The problems of serodiagnosis are briefly discussed.
The immunodiffusion and complement fixation tests for histoplasmosis were compared on 2,090 initial sera from 70 proven cases of histoplasmosis, 177 routine chest hospital patients, and 1,843 coal miners from outside the major endemic area for histoplasmosis. The complement fixation test using histoplasmin as antigen detected antibodies in the sera of 72.8% of the 70 proven cases, while the complement fixation test using yeast antigen detected antibodies in 94.3% of the case sera, and the immunodiffusion test using histoplasmin antigen detected antibodies in 90% of the sera. The tests were also compared for sensitivity, specificity, and ease of performance.
Sheep and guinea pigs were immunized with cellular and extracellular antigen from Legionella pneumophila bacteria. After immunization, the animals developed immunoglobulin G titers against the immunizing agent. The same sera were also tested in a Mycoplasma pneumoniae complement fixation test. All the preimmunization sera from sheep showed positive M. pneumoniae complement fixation tests of varying titers, with significant antibody rises in two of five sheep as a result of the Legionella immunizations. In contrast to the sheep, all the guinea pigs were negative in the M. pneumoniae complement fixation test, both in their preimmunization sera and after completion of the Legionella immunizations. The results obtained with the sheep sera may be explained as a nonspecific booster effect of Legionella bacteria upon previously elicited immune responses.
Eight ponies infected with Babesia equi were investigated for their serological response to B. equi schizont and piroplasm antigen with the indirect fluorescent antibody test (IFAT) and complement fixation test (CFT). Piroplasm antigen was prepared from an infected splenectomized pony, while schizont antigen was produced from cultured lymphoid cells which contained B. equi macroschizonts. The IFAT detected a rise in antibody titres to schizont antigen as well as to piroplasm antigen, but differences were obtained in the duration of antibody detection. Significant antibody titres to piroplasm antigen were detectable for a longer period post infection than to schizont antigen. The complement fixation test was not effective in detecting specific antibodies to schizont antigen in contrast to piroplasm antigen. The schizont antibody titres were in general extremely low and not detectable in 3 horses. Neither test showed any serological cross-reaction with B. caballi and B. bigemina antiserum using schizont antigen.