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Isolation of specific peptides from Mycobacterium paratuberculosis protoplasm and their use in an enzyme-linked immunosorbent assay for the detection of paratuberculosis (Johne's disease) in cattle.

An antigen was isolated from the protoplasm of Mycobacterium paratuberculosis by a combination of gel filtration, ion exchange, and affinity chromatography. The purified antigen constituted 7.8% of the total protein in the protoplasm. The specificity and sensitivity of the enzyme-linked immunosorbent assay (ELISA) for paratuberculosis, using the purified antigen, were evaluated with sera from 104 cattle which were examined (surveyed) for M paratuberculosis infection by fecal cultural technique. The ELISA was positive in 50 of 60 infected animals. Five of 44 noninfected animals were also test-positive. When a crude protoplasmic extract was used as antigen in the ELISA, sera from 37 infected and from 18 noninfected animals were test-positive. Cross-reactions were encountered in both complement-fixation test and the ELISA between crude or partially purified M paratuberculosis antigens and antisera to Nocardia asteroides, M avium, M phlei, and M fortuitum. The purified antigen gave no complement-fixation reaction with any of these antisera. In the ELISA, cross-reaction was not found when purified antigen was used and the sera were screened at 1:40 dilution.

Animals↗

Impaired humoral responses to subgenus D adenovirusenovirus infections in HIV-positive patients.

HIV-positive patients are at increased risk of developing adenovirus infection, particularly of the gastrointestinal tract and with unusual subgenus D strains. To investigate humoral immunity to these strains of adenoviruses, the humoral immune response was examined in longitudinal samples of serum against isolates collected from a prospective study of HIV-positive patients with subgenus D adenovirus infection. Of 10 HIV-positive patients developing adenovirus infection, 3 had chronic infection (8->27 months) with one serotype, 3 had chronic infection (>/=10 months) with changing serotypes and 4 had acute and self-limiting adenovirus infection (<1 month). Fifty-one sera were tested, and samples collected before adenovirus infection were available in 8 patients. Neutralising assays were performed against the patient's own isolate (adenoviruses 9, 17, 19, 19/23, 19/37, 23, 26, 23/26, 43 and 46) and common circulating strains of adenovirus 1-5. Indirect immunofluorescence tests were carried out against the autologous isolate and complement-fixation tests were undertaken using a standard assay. Immunofluorescence test antibodies were detected (titre >/=160) in all patients, and present to high titre (>/=320) in 8/10 patients. Complement-fixing antibodies were not detected in significant titre. Of particular note, there was no significant neutralising antibody response to the patient's own isolate after acute infection. Neutralising antibody to adenovirus 3 (titre 20) was transiently detected in two patients. In the remaining patients neutralising antibody directed against adenoviruses 1-5 was not detected. Persistent carriage of subgenus D adenoviruses in HIV-positive patients is probably the result of failure of cell-mediated immune responses to clear primary infection. Nevertheless, there is marked impairment of B cell responses resulting in poor neutralising and complement-fixing antibody production even though immunofluorescence test determined antibodies are produced in high titre. These possibly reflect impairment of effective B cell priming mechanisms within the germinal centres of lymph nodes, or the polyclonal activation of B cells driven by HIV infection.

AIDS-Related Opportunistic Infections↗

RELATIONSHIP OF HISTOPLASMIN AGAR-GEL BANDS AND COMPLEMENT-FIXATION TITERS IN HISTOPLASMOSIS.

Wiggins, Geraldine L. (Communicable Disease Center, Atlanta, Ga.), and Joseph H. Schubert. Relationship of histoplasmin agar-gel bands and complement-fixation titers in histoplasmosis. J. Bacteriol. 89:589-596. 1965.-Culture filtrates of various strains of Histoplasma capsulatum were studied with sera from patients with histoplasmosis, by use of the agar-gel and complement-fixation tests. It was found that the filtrates represented three types: those containing H and M components, those with H only, and those with neither H nor M. The same strain of H. capsulatum often produced culture filtrates of more than one type. Complement fixation showed that only the filtrates having both the H and M components were sufficiently sensitive as diagnostic antigens to detect the antibodies in histoplasmosis. H and M fractions prepared by ion-exchange chromatography were found to produce more than one band; therefore, it was concluded that the available fractions were not pure. The formation and identification of bands were affected by the concentration of the antigen and the arrangement of the wells containing the antigen and serum. The optimal concentration of the antigen varied, depending on the position of these reagents. The method found most practical for the identification of bands in unknown sera consisted of selecting the optimal concentration of the components with a box titration, by use of antigen having both the H and M components, placing known positive sera in wells adjacent to the unknown sera, and studying the sera for lines of identity. The M band was usually closest to the antigen well, and the H was nearest the serum well. These bands could be reversed by dilution, adjusting the H antibodies so that they equalled or exceeded the M antibodies. One human serum in which the bands were reversed showed the H bands at a higher serum dilution than that obtained with the M band. Patients with culturally proven histoplasmosis had the M band in their serum regardless of the stage of disease or length of duration; only 4 of 10 patients in the active stage of histoplasmosis also had the H band. The low prevalence of the H band would eliminate it as a diagnostic tool for detecting the active stage of the disease. Other bands obtained with histoplasmin antigen in the sera from proven cases were of no assistance in determining the clinical status of the patient.

Agar↗

[The diagnostic significance of the indirect hemagglutination test for the surveillance of toxoplasmosis during pregnancy (author's transl)].

In order to clarify the relevance of the indirect hemagglutination test (IHAT) for the surveillance of toxoplasmosis during pregnancy 2417 sera of pregnant women were tested for antibodies against Toxoplasma gondii in this test and in the fluorescent-antibody test (FAT). In addition 1319 of these sera were tested in the Sabin-Feldman test (SFT) adn 681 samples also in the complement fixation test (CFT). Significant correlations were found between the results obtained in the IHAT on one hand and in the FAT and SFT on the other hand: 95% of the sera which were negative in the FAT or SFT did not react in the IHAT as well, while most (but not all!) sera which were positive in the FAT or SFT gave positive results in the IHAT also. No correlations could, however, be found between titres obtained in the IHAT on one hand and in the CFT on the other hand. The suggestion recently published by several authors that it might be possible to decide from one single serum sample only whether or not a fresh infection had occurred by correlating FAT- (SFT-) and CFT-titres with IHAT-titres could not be confirmed. For a definite interpretation of serological results with respect to the risk of an infection of the fetus with Toxoplasma gondii it will still be necessary to test a second serum sample. As a screening method for detection of antibodies against Toxoplasma gondii -- without determination of the duration of infection -- the indirect hemagglutination test appears to be, however, a highly effective and economic method.

Antibodies↗

The antigenicity of myelin encephalitogenic protein: production of antibodies to encephalitogenic protein with deoxyribonucleic acid--encephalitogenic protein complexes.

Rabbits immunized with monkey encephalitogenic protein (EP) complexed to deoxyribonucleic acid (DNA) formed complement-fixing antibodies that reacted well with EP from monkey, bovine, and human brain and the large EP of rat brain. Reactivity with the small EP of rat brain was much less. The antibodies, analyzed by the quantitative micro-complement fixation test, were of the 7S class, were produced in high titer, and failed to react with histone, lysozyme, or acidic liver extracts. Immunohistochemical studies revealed that the antibodies bound to central nervous system white matter from human, rat, guinea pig, and mouse as well as human peripheral nerve myelin. These findings demonstrate that the immunogenicity of EP is greatly enhanced by attachment to DNA and perhaps other negatively charged molecules. This method makes available anti-EP with which to investigate immunochemically the antigenic and conformational features of EP.

Animals↗

Characteristics of the major internal protein and RNA-dependent DNA polymerase of bovine leukaemia virus.

A virus designated bovine leukaemia virus (BLV), associated with leukaemia in cattle and previously demonstrated to induce the disease in sheep, was purified from chronically infected sheep cell cultures. Electrophoretic analysis showed a major protein of mol. wt. about 24,000 (p24) which reacted in gel diffusion and complement-fixation tests with sera from naturally infected cattle, experimentally infected sheep, and guinea pigs immunized with p24. BLV p24 has an isoelectric point of 8-6. Interspecies antigenic reactivities characteristic of mammalian Type C virus p30s were not detected in disrupted BLV or on p24. Sheep and guinea pig antisera to BLV, reactive with p24, also did not precipitate several Type C virus p30s in radioimmunoassays. BLV is also distinguished from Type C viruses and resembles mouse mammary tumour virus and Mason-Pfezer virus in having an RNA-dependent DNA polymerase which is preferentially active in the presence of Mg++ when synthetic templates are used. Along with previously published morphological data, the above indicates that BLV is not a Type C virus as classically defined. Four hundred and forty one human sera from cancer patients and matched controls were non-reactive with disruped BLV, BLV infected cells, and BLV p24 in complement-fixation tests.

Animals↗

The presence of liver auto-antibodies induced by Entamoeba histolytica in the sera from both naturally infected humans and immunized rabbits.

Auto-antibodies against normal human liver have been detected in the sera of humans with highly positive indirect hemagglutination (IHA) amebiasis titers and with clinically-proven amebic liver abscess. Sera of amebiasis patients and rabbits immunized with killed Entamoeba histolytica were tested for anti-amebic antibodies by the IHA test and for auto-antibodies by the complement fixation test, using the antigens prepared from extracts of human liver and rabbit liver. A direct correlation was found to exist between high anti-Entamoeba antibody titers and the presence of anti-liver antibody in the serum. It is proposed that, in addition to direct parasite damage to host tissue, immunological damage could result from the attachment of circulating antigen to the cell surfaces of host tissues such as the liver.

Adolescent↗

An epidemic of histoplasmosis on the Isthmus of Panama.

Forty-seven men on the Isthmus of Panama were exposed to histoplasmosis in an old bunker inhabited by bats. The resulting epidemic was studied with serial clinical, serological, and radiological examinations. Thirty-seven (78.7%) of the men showed serological evidence of infection and 26 (70.3%) had symptoms. Incubation periods ranged from 4 to 30 days. A general relationship between severity of illness and degree of exposure was noted. The agar gel diffusion test for precipitin antibodies was more sensitive than the complement-fixation test or slide test in detecting infection with Histoplasma capsulatum. Decontamination procedures and environmental studies are described.

Amphotericin B↗

Diagnosis of acute respiratory tract infections: serology and new methods.

No test is available that can identify all potential pathogens in acute respiratory tract infections. Each diagnostic test is associated with limitations with respect to sensitivity and/or specificity and/or speed, and thus a combination of tests has to be used. Even so, no etiologic agent is found in 30--60% of cases. The following methods are recommended for use in the routine laboratory for the diagnosis of infections: (1) Legionella --- direct immunofluorescence test, culture, serology and antigen detection (available only in specialized laboratories); (2) Chlamydia pneumoniae --- micro-immunofluorescence test, complement fixation, but not direct antigen detection by immunofluorescence; (3) Mycoplasma pneumoniae - complement fixation and/or particle agglutination or other evaluated methods; (4) Coxiella burnetii --- immunofluorescence test (IgG and IgM) and complement fixation; (5) viruses --- complement fixation or other similar test (ELISAs often lack adequate evaluation). Culture for Chlamydia, Mycoplasma pneumoniae, Coxiella burnetii and viruses should only be performed by very experienced laboratories. Most procedures deliver results only retrospectively or too late. The most promising diagnostic tools for the future are nucleic acid amplification techniques (NAT) or PCR, which could solve many of the problems connected with conventional techniques, but there are enormous contamination problems. Further research and worldwide aid in the development of standardized NAT, especially by industrial companies, is urgently encouraged to improve the laboratory diagnosis of these pneumonias.

Journal Article↗

Evidence of chronic persistent infections with polyomaviruses (BK type) in renal transplant recipients.

Ten renal transplant recipients showing a significant increase in human polyomavirus antibodies, indicative of an acute infection, were followed up serologically over periods ranging from two months to more than two years. Fifty-four serum specimens were available for the study and they were tested by both haemagglutination-inhibition and complement-fixation. Polyomavirus antigens were prepared from the BK and SV40-like strains of polyomaviruses, and from the SV40 virus. One strain of polyomavirus, related to the BK strain was isolated from the urine of one of these patients. Two other BK strains were recovered from the urine and kidney, respectively, of transplant recipients not included in this study. Sera of these two patients were not obtained until the transplantation was made; they were already highly positive for polyomavirus antibodies, precluding the demonstration of an increase in antibody titer. Serologic results have shown that HAI antibodies persist at high titers throughout the observation period. This persistence ranged from two to four months (four cases), seven to eleven months (three cases) and thirteen to twenty months (three cases). In none of the cases could a decrease of high titer be demonstrated. Moreover, density gradient studies have shown that specific IgM antibodies also tend to persist over many months. Similar serologic results were obtained in complement-fixation tests with a BK antigen. Titers were at least 1 in 30 in the study group, but were not observed among healthy blood donors. All sera were uniformly negative for SV40 and SV40-like antigens. One polyomavirus isolation was successful from urine obtained six months after initial serologic evidence for a polyomavirus infection. The other two viruses were isolated from materials taken four and seven months after first detection of polyomavirus antibodies at high titer. Both serologic evidence and viral isolations seem to indicate that polyomaviruses (BK type) might cause a chronic infection in humans.

Animals↗

Studies of the alternate pathway in chelated serum.

Activation of the alternate pathway in chelated serum by cobra venom factor (Co F), endotoxin of Escherichia coli, inulin, and zymosan was studied by immunoelectroprhoresis, radial immunodiffusion, and complement fixation tests. In serum chelated by 10 mM EGTA and 10 mM MgCl-2 (10 mM MgEGTA) activation by the classical pathway was blocked, but activation of C3PA and C3 occurred normally. By radial immunodiffusion assay, consumption of C3 and properdin was demonstrated to be similar to consumption in nonchelated sera. By immunoelectrophoresis, C3PA and C3 were shown to be activated by CoF in serum chelated with 6 mM but not with 10 mM EGTA without added Mg++. Inulin and endotoxin did not result in activation of either C3PA or C3, while C3PA but not C3 was activated by zymosan in the presence of 10 mM EGTA without Mg++ added. Consumption of properdin and C3, measured by radial immunodiffusion, was inhibited by 6 to 10 mM EGTA following activation by inulin, but not following activation by zymosan. The usefulness of 10 mM MgEGTA-chelated serum for the selective investigation of the alternate pathway in chemotaxis was demonstrated.

Blood Protein Electrophoresis↗

The use of skin delayed-type hypersensitivity as an adjunct test to diagnose brucellosis in cattle: a review.

Brucellosis, caused by bacteria of the genus Brucella, is a contagious disease that causes economic loss to owners of domestic animals due to loss of progeny and milk yield. Because cattle, sheep, goats, and to a lesser extent pigs are considered to be the source of human brucellosis, serological tests have been used to screen domestic animals for antibodies against Brucella. Although the serological tests helped to eradicate brucellosis in many countries, serological tests are not always adequate to detect latent carriers of Brucella. Therefore, the use of the skin delayed-type hypersensitivity (SDTH) test, which is independent of circulating antibodies, might improve the diagnosis of brucellosis. In the literature, however, there are conflicting reports as to the value of the SDTH test for the diagnosis of brucellosis. Some studies consider the test unreliable, whereas others advocate its use because it detects brucellosis earlier than serological tests. The objectives of this study were therefore to assess the characteristics of the SDTH test, to select a Brucella strain that will yield a suitable brucellin for use in the field, and to determine whether the use of serological tests in combination with the SDTH test improves the detection of brucellosis. The results of this study clearly show that the SDTH test detects latent carriers of Brucella and confirms brucellosis in cattle with ambiguous serological test results. Brucellins prepared from smooth or mucoid strains of Brucella are better suited for use in the field than brucellins prepared from rough strains because they detect brucellosis in cattle with acute as well as chronic infection. The SDTH test is highly specific (99.3% specificity), and repeated testing of naive cattle or cattle infected with microorganisms that serologically cross-react with Brucella does not sensitize cattle to subsequent SDTH tests. However, it is possible that some naive cattle may serologically react to the injection of brucellin. The effect of these serological reactions on the sero-diagnosis of brucellosis is limited, because cattle may only now and then react serologically either with the serum agglutination test (SAT) or the complement fixation test (CFT). Nevertheless, cattle infected with microorganisms that serologically cross-react with Brucella may test seropositive for brucellosis 4 to 7 weeks after injection of brucellin, depending on the cross-reacting microorganism. The value of the SDTH test for the diagnosis of brucellosis was demonstrated after an outbreak of brucellosis. When the SDTH test was used in combination with SAT and CFT at diagnostic threshold > or =2 mm or > or =1 mm (increase in skinfold thickness), respectively, 39/44 (88%) or 42/44 (95%) of the infected cattle were detected compared with only 27/44 (61%) when SAT and CFT were used. When cattle in areas of low prevalence or in areas free from brucellosis are tested with the SDTH test an increase > or =2 mm in skinfold thickness should be considered indicative of infection. When the control and eradication of brucellosis is based on test-and-slaughter, an increase of > or =1 mm in skinfold thickness should be considered indicative of infection. Repeated serological testing complemented with the SDTH test in this programme will shorten the quarantine (movement control) period of a suspect herd, limiting the financial loss incurred during outbreaks of the disease. Consequently, since the SDTH test usually does not interfere with the serological diagnosis and can safely be used to establish the infection status of cattle in a suspect herd, it is opportune to consider adding the SDTH test to the procedure currently used to diagnose brucellosis in individual animals.

Animals↗

Micro indirect hemagglutination test for Cytomegalovirus.

In an effort to obtain the flexibility and ease of performance of a rapid, serological test for detection of cytomegalovirus antibody, the indirect hemagglutination (IHA) technique was investigated by using a microserological system. Antigens were prepared from tissue cultures of infected human fibroblasts. The specificity of the cytomegalovirus antibody response detected by the IHA test correlated well with the standard neutralization test. The IHA method was more sensitive than the complement fixation test in detecting antibody in congenitally infected newborns. There appeared to be some heterologous antibody response with Herpesvirus hominis or varicella virus infections. The IHA test pattern was found to be very stable with excellent persistence of agglutination.

Antigen-Antibody Reactions↗

A preliminary study of the development of 19S and 7S immunoglobulins in sera of two sheep vaccinated with Brucella abortus, strain 19.

The pattern of antibody response to vaccination with Brucella abortus, strain 19, was studied in two sheep. Agglutinative activity was detected by the third and fifth days and complement - fixing activity by the fifth and seventh days post-vaccination. Density gradient centrifugation and DEAE cellulose column chromatography showed the 19S antibody developed first, followed soon after by 7S antibody. The former had disappeared by the 25th day but the latter persisted longer in both sheep. A small amount of 19S antibody was detected in sheep 1 following a booster dose of vaccine but 7S antibody constituted the major secondary response. The standard tube agglutination test was found to be more efficient than the complement-fixation test for titration of 19S antibody. An increase in the salt concentration to 10% in tube agglutination test rendered it more sensitive in demonstrating 7S antibody.

Agglutination Tests↗

Seroepidemiology of Anaplasma marginale in white-tailed deer (Odocoileus virginianus) from Louisiana.

Antibodies to Anaplasma marginale were detected by the indirect fluorescent antibody test (IFA) in six of 331 (2%) serum samples of white-tailed deer (Odocoileus virginianus) from Louisiana. None of the serum samples were positive using the A. marginale modified rapid card agglutination test. Of the six IFA positive sera retested by the complement fixation test four sera gave anticomplementary and two gave seropositive reactions. The low A. marginale reactor rate in this white-tailed deer population was probably a reflection of the lack of cohabitation between cattle and deer and the fact that the primary arthropod vectors in Louisiana are tabanids. The validity of the indirect fluorescent antibody test for A. marginale antibodies in white-tailed deer should be evaluated.

Agglutination Tests↗

A complement fixation method for quantitative differentiation of reactions to 45/20 vaccine and Brucella infection.

Brucella complement fixing antibodies may be titrated independently in adult vaccinated (strain 45/20) and in naturally infected cattle by serological tests utilizing a specially prepared antigen. Serum samples are subjected to the standard complement fixation test for the diagnosis of brucellosis (MacKinnon 1963) and subsequently retested by the same method but with a saline extract antigen prepared from strain 45/20 Brucella abortus. The results obtained in the two tests are compared in five specific categories of brucella reactors. An evaluation and discussion of the test method, on the basis of the results obtained, indicates that informed diagnoses can be made in parallel with a continuous adult vaccination programme.

Animals↗

Complement fixation by antibodies to the alpha toxin of Staphylococcus aureus.

When rabbits were injected with the heat-denatured alpha toxin (toxoid) of Staphylococcus aureus, the immune response was demonstrated by an increase in antitoxin that fixed complement. Such antitoxin was detected in 72% of normal human sera. After fractionation of the antitoxin into two types (the antibinding antibodies and the indirect hemagglutinating antibodies), both types of antibodies were found to fix complement in the standard serological complement fixation test. In addition, the indirect hemagglutinating antibodies were capable of fixing complement when the antigen (alpha toxin or toxoid) was covalently or noncovalently bound to erythrocyte membranes. The fixation of complement by membrane-bound immune complexes did not result in lysis of the carrier erythrocytes. The prevalence of complement-fixing antitoxin in normal humans and animals raised the concern that the outcome of in vivo experiments involving alpha toxin could be influenced by the immune status of the host.

Animals↗