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The indirect complement-fixation test was used in experiments for the subtype differentiation, and with the agar gel diffusion and the serum neutralization test was studied the immunity in cattle that had already recovered from foot-and-mouth disease or were immunized against the disease. It was found that the indirect complement-fixation test is instrumental in the demonstration of the antigenic differences between the foot-and-mouth viruses. Comparative experiments have shown that the indirect test in this case is more expedient and of higher economic value than the direct CF reactions after Traub and Möhlmann and after Marucci. The assessment of the results of comparative investigations of sera carried out by the indirect complement-fixation test, the agar gel diffusion test, and the serum neutralization reaction revealed that the indirect reaction is a convenient, quick, and prompt method of work. Discussed is the sensitivity of the reaction and its advantages and its advantages in studies on a large scale on the immune status of animals immunized against F.M.D. or animals that have recovered from the disease.
Paired specimens of serum from patients from whom Bacteroides fragilis had been isolated were tested by complement fixation against a crude B. fragilis antigen. A high titre or a rise in titre to B. fragilis was obtained in each of five patients with infection after abdominal surgery but in none of 11 patients with postpartum pyrexia nor in nine with vaginitis.
Sera from patients suspected of having rickettsial infections were tested in the complement fixation test with antigens prepared from the rickettsiae of Rocky Mountain spotted fever (SF), rickettsial pox (RP), murine typhus, epidemic typhus, and from Rickettsia canada (RC). Eight units of antigen were used in all cases and two units in man. Only those patients with antibody titers of 1:16 or higher were included in the study. Largely on the basis of comparative titers, the patients were divided into two groups: 102 with SF and 35 with infections by one of the members of the typhus group. The antibody titers were higher with SF antigen than RP antigen in 72% of the SF patients, and in only two SF patients was the RP titer higher, and then by only one tube (twofold dilution). There seemed little advantage in including the RP antigen in the battery of rickettsial antigens. Cross-reaction with at least one of the typhus antigens was observed in the sera from 64% of the SF patients. It was extensive enough to be confusing (within one tube) in 17% with eight units of antigen, but the differentiation was more distinct with two units of antigen. The cross-reaction with typhus antigens was as frequent in children with SF as it was in adults; thus, it is unlikely that these cross-reactions resulted from previous typhus vaccination. The serological differentiation between murine typhus and epidemic typhus was frequently difficult, but the epidemiological background was distinct. Five patients had higher titers to RC antigen, and four of these may possibly have had RC infections.
The practical value of the indirect fluorescent antibody technique (I FAT), the micro complement fixation test (M CFT) and the agar gel immunodiffusion test (AGIDT) for the detection of antibodies to bovine leucosis virus (BLV) was investigated. For this purpose 1495 serum samples were examined. There was a remarkably good agreement between the three tests in the demonstration of antibodies to BLV especially if sera with a sufficient high concentration of antibodies ("late serum") were under investigation. These positive sera were derived only from farms which exploited one or more imported animals. A disagreement of results of the three tests was observed in 38 (2.5%) cases. This was due to: (1) difficulties in reading of the test; (2) presumably by the demonstration of different classes of antibody and (3) the fact that in the AGIDT sera could be used undiluted. This discrepancy was especially evident with sera with a low concentration of antibodies ("early or incubation sera"). A drawback for the M CFT is the anticomplementary activity found in 17% of the bovine serum samples.
Tween-ether toxoplasma antigen for the complement fixation test was verified on a more extensive clinical material. From a series of 949 patient's sera, positive reaction was obtained in 44% of samples with the tween-ether antigen and in only 33.5% of samples with the FT antigen. All sera, giving positive results only with the tween-ether antigen, were also positive in the Sabin-Feldman test. The authors believe that by using this type of more sensitive antigen, containing also the cell-wall components of Toxoplasma gondii, it would be possible to standardize serological examination by the complement fixation test on the basis of an international standard serum.
A complement fixation test (CFT) based on that used for brucellosis (Brinley Morgan and others 1971) has been developed for use on the sera of horses exposed to the contagious equine metritis (CEM) organism. None of 50 single samples from horses thought to be unexposed to the CEM organism was positive to the test, although five showed inconclusive reactions. Samples were examined from 41 mares either proved to be infected or from an infected stud. Of these 21 were positive, 11 were inconclusive and nine were negative. The relationship of the CFT to reactions in the other tests used in this condition is discussed--the serum agglutination test (SAT) and antiglobulin test (AGT) (Benson and others 1978). Complement fixing antibodies appear to remain for a longer period in the infected animal, and are therefore more likely to be of use in the diagnosis of the chronic carrier state.
The gonococcal complement fixation test (GCFT) was investigated with regard to its sensitivity and specificity by testing serum specimens from (a) female patients attending a VD out-patient clinic because of suspected gonorrhoea, (b) patients with a proven, uncomplicated, urogenital gonococcal, infection, (c) patients with disseminated gonococcal infection (DGI). Three different pools of gonococcal (GC) antigens were used which were comprised of GC strains from two different geographical areas. It was found that 39% of the females with culture-proven uncomplicated gonorrhoea had a positive GCFT whereas 10% of the females with negative GC cultures had a positive GCFT. The latter were found to have either a history of gonorrhoea or strong clinical suspicion of recent GC infection. One of the GC antigen pools gave a much lower diagnostic yield than the other two pools in the GCFTs with serum specimens from patients with uncomplicated gonorrhoea. However, no differences were found between the antigen pools in the tests with serum specimens from patients with DGI. These findings indicate the presence of various strain antigens participating in the immune response to complicated as well as uncomplicated GC infections. The results are presented in detail and discussed.
Sera from 452 patients and blood donors were tested for antibodies to Tryp-anosoma cruzi. The complement fixation test and a counterelectrophoresis test were used. The results showed that both tests agreed in 92% of the sera. However, the counterelectrophoresis test was not as sensitive as the complement fixation test.
Eight reference California group viruses of North America were typed using sera of immune hamsters bled 21 days after a single inoculation. The complement-fixation test reactions were relatively specific, although only 2-fold differences were observed reciprocally with the closely-related La Crosse and snowshoe hare viruses. Hamster serum taken 10 days post inoculation was more specific than a 21-day serum. Sepcificity after second inoculation was lost with some antigens. Jamestown Canyon and South River viruses were identical by complement-fixation test and showed minor differences in the plaque reduction neutralization test.
The utility of complement-fixation test with an antigen from commercially lyophilized BCG was investigated in the diagnosis of leishmaniasis. The following results were obtained: Visceral leishmaniasis: 4 positivity on 5 cases; Oriental sore: 3 positivity on 6 cases; Canine leishmaniasis: 6 positivity, at high level, and 2 negativity; Other diseases: 5 positivity, on 17 sera of tbc; No positivity in sera from patients with various diseases, in control and in normal dogs. The results are compared with those quoted in medical literature and discussed. The meaning of the reaction is also briefly discussed, and the hypothesis of the presence of partigens between BCG and leishmania, or presence of autoantibodies is prospected.
Complement-fixation test based in 50% hemolytic end point was applied to investigate the immune status to rabies of dogs vaccinated with heigh egg-passage Flury vaccine. The complement-fixation titer was compared with serum neutralization results. Twenty-five sera was employed and the complement fixation titer varied of 0 to 256. Three sera was anticomplementary. The results indicated a lack of quantitative correlation, but was found a qualitative correlation between the two methods.
For the circumoval precipitin test (COPT) blood was taken on quantitative blood sampling filter paper by finger prick from outpatients at the Schistosomiasis Control Pilot Project, Palo, Leyte, Philippines. The volume of serum available per strip of filter paper was 0.04 ml and this was extracted at 1:3, 1:5 and 1:8 dilutions. Lyophilized eggs of Schistosoma japonicum were mixed with the diluted serum on a microscope glass slide and incubated at 37 degrees C for 2 days. The reaction was read following the criterion made by Yokogawa et al. [11]. The serum at 1:8 was too dilute to make correct diagnosis; serum at 1:3 dilution contained too much hemoglobin which made microscopic observation difficult and the extract at 1:5 was found to be appropriate. There was no remarkable difference in antigenicity among 3 preparations of lyophilized eggs from Kofu strain, Japan, and those of new and old preparations from Philippine strain. Under the best condition, false negative results appeared in 15.3% of 152 outpatients in Leyte and false positives in 2% of 50 human sera collected in Tokyo. This method of COPT is not satisfactory for the diagnosis of individual cases but is useful in the epidemiological assessment of Schistosoma infections because of the simplicity of blood sampling from dwellers of infested areas and also because it shows nearly the same sensitivity as that of a single fecal examination by the MIFC method. A microtiter technique of complement fixation test (CFT) was also studied. This method, however, was less sensitive than the COPT or a single fecal examination as to give 23.7% false negatives. Frequency distributions of CF and COP titers were analysed among egg positive, egg negative and treated groups. The results showed that treatment with stibophen had little influence in lowering the serum response, especially in COPT.
The radial complement fixation test has to be made in forms in 1.4% layer of agar in physiological saline in a Petri dish. These forms have to be of the same thickness as the reaction itself, usually about 1 mm. In the first layer, it is recommended to mix antigen with complement and sheep erythrocytes in agarose and this mixture is to be poured over the tissue fragments examined. Following the primary incubation (at room temperature overnight), another mixture of haemolysin in agarose is poured over the first layer in form of optical convex lenses. The haemolytic phase is performed at room temperature, too. The complement is titrated together with the optimal amount of antigen and a drop of positive serum is placed in the well just in the centre of the first layer. In this way an optimal dilution of complement is detected, which is capable of producing a full hemolysis outside the zone of the positive serum diffused during the first, primary incubation period, whereas the zone itself remains untouched with the haemolytic activity of complement.
Coccidioidin, an extract from the saprophytic mycelial form of Coccidioides immitis, has been a very useful antigen preparation in serological tests for coccidioidomycosis. Its sensitivity has been very good for detecting most types of clinical disease, but tests with coccidioidin have been negative for 40% or more of patients with chronic pulmonary disease, the clinical entity which must be differentiated from other cavitary, nodular, or fibrotic pulmonary disease, e.g., tuberculosis and cancer. The specificity of coccidioidin has also been good although it results in positive tests for an average of 16% among patients with noncoccidioidal mycoses. Recently spherulin, an extract from the parasitic endosporulating spherule form of C. immitis, was reported to be more sensitive than coccidioidin in concurrent complement fixation tests with sera from selected cases. We have compared coccidioidin and spherulin in concurrent complement fixation tests with 614 sera submitted routinely for coccidioidal serology and with 159 selected sera from patients with noncoccidioidal mycoses. Among the former, spherulin was positive with 25% and coccidioidin with 23%, and correlation of titer scores was highly significant. Statistical analysis revealed no significant differences with respect to frequency of positive specimens, titer scores, or diagnosis for current coccidioidomycosis. The results with sera from noncoccidioidal mycoses revealed marked differences. Coccidioidin was positive with 20%, and spherulin was positive with 48%. The titer scores with spherulin were consistently and significantly higher, and there was no correlation for results with the two antigens. Thus, coccidioidin and spherulin were equally sensitive, but spherulin was considerably less specific.
Several methods for increasing yield and specificity of the contagious bovine pleuropneumonia complement fixation test antigen which is derived from Mycoplasma mycoides subsp mycoides, strain V5, were examined. Changes in culture conditions that increased the cell mass per unit volume of culture did not result in comparable increase in antigen yield. Sixteen to 60-day-old cultures yielded more boiled cell antigen than younger cultures. Some antigen in young cultures appeared to be masked, probably by galactan. The yield of antigen extracted from boiled cells with ethonol was as much for two to eight-day-old cultures as for older cultures. The ethanol extract antigen was less reactive with false positive bovine sera than standard boiled antigen while reactivity with anti Mycoplasma mycoides sera was similar to that of standard antigen. Adsorbed gamma globulin was not detected in either boiled or ethanol extract antigen. The data suggest that several complement fixing antigens were present in antigens derived from older cultures.
An enzyme immunoassay (EIA) for the detection of rotaviral antibodies was developed, using a purified, cell culture-grown SA 11 viral antigen and alkaline phosphatase as an enzyme label. This technique was evaluated by comparative testing with tube neutralization and complement-fixation assays on a collection of simian sera. There was close correlation between positive and negative results obtained by EIA and by neutralization. The EIA was as easy to perform as complement fixation testing, but showed greater sensitivity and fewer nonspecific reactions. Thus, EIA was shown to be a very suitable test for routine detection of rotaviral antibodies in serum. Results of neutralization tests suggested that the monkeys (mostly rhesus macaques) in the present study were infected with viruses varying in their antigenic relatedness to SA 11 virus and to a British isolate of calf rotavirus.
Type-specific antigens for herpes simplex virus type 1 and 2 were prepared by rigorous absorption of cell extracts with heterotypic immune sera. Type-specificity was demonstrated by immunodiffusion and complement-fixation tests against immune sera prepared in rabbits. Specific type 1 complement-fixing reactivity was detected in eleven of fifteen sera from Roman Catholic nuns and in two convalescent sera from patients with recurrent herpes labialis; these sera had been previously shown to contain neutralising and complement-fixing antibody to herpes simplex virus. Three of the non-reacting sera contained low or absent levels of type-common complement-fixing reactivity and other contained no type-specific neutralising antibody. With the exception of three "acute" sera, specific type 2 complement-fixing reactivity was detected in every convalescent or interim serum obtained from patients with a virologically-proven history of type 2 herpes virus infection. It is suggested that complement-fixation testing using these absorbed type-specific antigens preparations may provide a convenient and rapid method for the identification of type-specific antibody in human sera.