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Rapid detection of IgG and IgM antibodies for cytomegalovirus by the enzyme linked immunosorbent assay (ELISA).

A simple solid phase enzyme immunoassay for the detection of immunoglobulin G and M to cytomegalovirus (CMV) is described. Using this test IgM antibodies to CMV were detected in 0.7 per cent of newborns and regularly after CMV infection in transplant patients, furthermore in these latter patients IgM production was prolonged for several months. For the determination of IgG the enzyme immunoassay was more sensitive than the complement fixation test (CF) and the antibody titres were 4 to 8 fold higher. Since the ELISA test is rapid, specific and unexpensive it can become an acceptable routine diagnostic procedure.

Adult↗

Reliability of results of the Rose Bengal test performed for export control in northern Somalia.

Sera from sheep and goats in northern Somalia which are exported to countries of the Persian Gulf are systematically checked for brucellosis by local veterinary teams. The standard test used is rapid seroagglutination using the Rose Bengal test (RBT) and seropositive animals are not exported. In order to assess the reliability of the serological results, three randomised batches of samples (653 sera), corresponding to an equivalent number of shipments (October and December 1994 and March 1995) were counterchecked. Control RBTs were carried out by expatriate veterinarians working on behalf of international non-governmental organisations and by the Istituto Zooprofilattico Sperimentale of Padua, Italy, which also performed the complement fixation test (CFT). A fourth batch (n = 100), including a group of sera found positive by the local veterinary teams, was also checked. Agreement ranged from 96.3% to 98.5%.

Agglutination Tests↗

Detection of adenovirus antibody by counterimmunoelectrophoresis.

Counterimmunoelectrophoresis was adapted as a rapid, sensitive assay for detection of adenovirus group-specific antibody. This assay was six times more sensitive than the complement fixation test and was successfully adapted for use with microliter quantities of serum or whole blood.

Adenoviruses, Human↗

Membrane filtration enzyme immunoassay, a novel, rapid method for measurement of virus-specific immunoglobulins G and M and detection of viral antigens.

A novel membrane filtration enzyme immunoassay (MF EIA) is described in which virus-antibody complexes formed are trapped onto the surface of membranes with low protein-binding affinity by vacuum filtration. Class-specific immunoglobulin G (IgG) or IgM antibody was measured by adding enzyme-conjugated antiimmunoglobulin and incubating prior to the final wash and addition of enzyme substrate. Influenza A virus-specific IgG antibodies measured by MF EIA showed similar sensitivity for detecting seroconversion in volunteers administered influenza virus subunit vaccines and subtype specificity comparable to that observed by the hemagglutination inhibition technique. Cytomegalovirus-specific IgG antibodies measured by MF EIA with commercially available complement fixation antigens gave results similar to those of conventional enzyme-linked immunosorbent assay and complement fixation tests. The MF EIA method is also suitable for detection of rotavirus antigen in feces.

Antibodies, Viral↗

An evaluation of the enzyme-linked immunoabsorbent assay (ELISA) for quantitation of antibodies to Paracoccidioides brasiliensis.

The ELISA procedure was adapted for quantitation of antibodies against Paracoccidioides brasiliensis. Using a yeast cytoplasmic antigen and sera from patients with proven paracoccidioidomycosis, we found that 66% of sera reacted at titers greater than or equal to 1:128. Titers of this magnitude were obtained only for 4-5% of sera from healthy blood donors, tuberculosis patients and patients with other systemic mycoses. The exception was sera from patients with histoplasmosis (36% had titers greater than or equal to 1:128). Follow-up of 10 paracoccidioidomycosis patients during the course of therapy indicated a gradual decrease in antibody titers. Because of the technical advantages of the ELISA procedure in comparison with the standard complement fixation test, the ELISA test has potential utility for the quantitative determination of antibodies in patients with paracoccidioidomycosis.

Antibodies, Fungal↗

Studies on bluetongue. 3. Comparison of two complement-fixation methods.

The complement-fixation test used at Onderstepoort was compared with the method used at A.D.R.I. on infected calf and sheep sera. In the first method, the tests are incubated at 37 degrees C for 90 minutes and the test sera are inactivated at 53 degrees C; whereas in the A.D.R.I. method, the test sera are inactivated at 60 degrees C for 30 minutes, incubation is at 9 degrees C for 18 hours, and guinea-pig complement is supplemented with 5 per cent fresh, non-inactivated, normal calf serum. Serial serum samples from one of six experimentally infected calves were negative in the Onderstepoort test, three calves gave only trace reactions and two showed maximum titres of 1:10 whereas all six had maximum serum titres of 1:10 to 1:80 in the A.D.R.I. test. A good correlation was obtained, however, between the results of the two methods with the sera of experimentally inoculated sheep although titres 3 to 8 times higher were obtained with the A.D.R.I.'s test. Post inoculation bleedings from each sheep reacted in both tests.

Animals↗

A single dilution enzyme-linked immunosorbent assay for the quantitative detection of antibodies to African horsesickness virus.

A single dilution enzyme-linked immunosorbent assay (ELISA), utilizing a reference serum with a known, predetermined end-titre was used to compute antibody titres of serum samples against African horsesickness virus. Compared to the complement fixation test it was found to be highly reproducible and sensitive. The assay also reduced the effect of between-test variations on test results and proved superior to an existing single-dilution ELISA method.

African Horse Sickness Virus↗

Radioimmunoassay for antibodies against Brucella abortus: a new serological test for bovine brucellosis.

A radioimmunoassay (RIA) has been developed to measure antibodies against Brucella abortus in bovine serum and can be used in the diagnosis of bovine brucellosis. The RIA measures the amount of specific antibody of the IgG1 and IgG2 subclasses but is insensitive to IgM, a characteristic which may make it more suitable than the complement fixation test (CFT) or the serum agglutination test for distinguishing infected animals from those which have been vaccinated with Br. abortus strain 19. The RIA is not subject to prozoning or ambiguous reactions, both of which interfere with the interpretation of the CFT.

Agglutination Tests↗

Serologic diagnosis of human infections with lymphocytic choriomeningitis virus: comparative evaluation of seven methods.

The aim of this work was to compare different procedures for the serodiagnosis of human infections with the lymphocytic choriomeningitis (LCM) virus and to single out those which are both reliable and practicable. By the use of 46 sera from as many persons who had undergone infection with this virus either some time in the past or very recently and of 26 control sera, seven methods were evaluated. For making a rapid diagnosis soon after infection, determination of antibody by the immunofluorescence procedure appears to be the method of choice. Of equal reliability, though less easy to perform, is demonstration of sensitizing antibody by a plaque reduction assay, this procedure having the additional advantage of detecting antibody many years after infection, probably for life. For the demonstration of neutralizing antibody, three of four methods which were analyzed in this study gave the expected information, two employing mice, one employing cell cultures for the determination of residual infectivity. Neutralizing antibody was found to appear relatively late after infection and cannot, therefore, be recommended for the demonstration of seroconversion early in covalescence. This antibody, however, persists for many years, presumably lifelong, and is well suited to detect infections with this virus if they have occurred in the more distant past. Based on previous observations by ourselves and others, as well as on the work reported here, the complement fixation test appears to be of little value for the serological diagnosis of infection with LCM virus.

Animals↗

[Evaluation of usefulness of OWD and OIEP in diagnosis of epidemically occurring infections caused by Mycoplasma pneumoniae].

Blood serum samples of 3593 persons clinically suspected of infection with Mycoplasma pneumoniae were tested. Of these, patients with pneumoniae constituted 66.5%, upper respiratory tract infection--24.0% and with symptoms localized outside the respiratory system--9.5%. These studies were performed by application of complement fixation test (OWD) and immunoelectroprecipitation (OIEP) methods, accepting as a diagnostically significant--titer 1:60 or higher and/or occurrence in OIEP reaction with serum diluted 1:2 or more. Among patients studied prevailed children in the age of 3 to 16 years (61.6%). Mycoplasmosis was detected in these patients in 1071 out of 2236 cases (47.9%). Compatible results in both tests were obtained in 90.6% patients, whereas OWD only in 3.0% and OIEP only in 6.4% cases. Simultaneous application of both tests increased detectability of infections caused by M. pneumoniae by 3% in relation to OIEP and by 6.4% in relation to OWD.

Adolescent↗

Serologic responses, biosafety and clearance of four dosages of Brucella abortus strain RB51 in 6-10 months old water buffalo (Bubalus bubalis).

Thirty water buffalo were obtained from a brucellosis-free farm in order to evaluate antibody responses, bacterial clearance and safety to Brucella abortus strain RB51 vaccine in a dose response study. The animals were randomly divided into five treatment groups. Groups I-V received the recommended dose of RB51 vaccine (RD) once, RD twice 4 weeks apart, double RD once, double RD twice 4 weeks apart and saline once, respectively. Antibody responses to RB51 were monitored at 2, 4, 6, 8, 10, 12, 16 18, 22, 24 and 27 post-initial-inoculation weeks (PIW). Clearance of RB51 from the prescapular lymph node was evaluated at 2, 4, 6, 12, 18 and 24 PIW for groups 1, III and V and at 6, 8, 10, 16, 22 and 27 PIW for groups II and IV. To evaluate shedding of the RB51 strain, nasal, conjunctival, vaginal or preputial swabs were taken from all experimental animals at 1, 2, 3, 4, 6, 8 and 12 PIW. Sera taken at all PIW were negative for field strain B. abortus by both the buffered plate agglutination test (BPAT) and competitive enzyme-linked immunosorbent assay (c-ELISA). Antibody responses to RB51 were demonstrated in all vaccinates but not in the controls, up to 12 PIW, by complement fixation test (CFT) and the dot-blot assay with an 83.7% agreement for both tests. Clearance of RB51 occurred between 6 and 12 PIW in group I but less than 2 weeks after booster vaccinations in groups II and IV and between 4 and 6 PIW in group III. RB51 was not recovered at any time from swabs obtained from either RB51-vaccinates or non-vaccinates. The results of this study indicate that serologic responses to RB51 vaccination can be monitored by both CFT and dot-blot assay in water buffalo. Our data also indicates that RB51 vaccination does not interfere with brucellosis sero-surveillance and is safe (no serological and bacteriological evidence of spread to non-vaccinates, no adverse clinical signs or detectable abnormalities on haematology and serum biochemistry) for use in water buffalo.

Agglutination Tests↗

Detection and serotype-specific differentiation of vesicular stomatitis virus using a multiplex, real-time, reverse transcription-polymerase chain reaction assay.

A multiplex, real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed that allowed simultaneous detection and rapid differentiation of vesicular stomatitis virus strains--New Jersey (VSV-NJ) and Indiana 1, 2, and 3 (VSV-IN1-3). This assay involves use of a set of VSV universal primers located in the L gene that amplify VSV-IN1-3 and VSV-NJ using probes that allow differentiation of the major serotypes Indiana and New Jersey. The assay was evaluated using reference VSV, foot-and-mouth disease virus, swine vesicular disease virus, and vesicular exanthema of swine virus. To estimate diagnostic sensitivity, 159 epithelial samples collected between 1996 and 2002 from naturally infected cattle in Colombia were used. The assay cut off was calculated by testing RNA extracted from 150 virus-negative bovine tissues consisting of tongue, soft palate, muzzle, coronary band, and lymph node. All infected cattle were test positive for VS by results of real-time RT-PCR analysis; results for 156 of 159 (98.1%) agreed with the serotype determination from the complement-fixation test. Amplification did not occur in any of the negative bovine epithelial samples, allowing the cut-off values for the assay to be set. The real-time RT-PCR assay was documented to be sensitive and specific for the detection of VSV-NJ and VSV-IN (1-3) strains from field samples in a single reaction, thereby supporting use of this assay in the differential diagnosis of vesicular virus diseases in cattle.

Animals↗

Estimation of sensitivity, specificity and predictive values of two serologic tests for the detection of antibodies against Actinobacillus pleuropneumoniae serotype 2 in the absence of a reference test (gold standard).

Latent-class models were used to determine the sensitivity, specificity and predictive values of a polyclonal blocking enzyme-linked immunosorbent assay (ELISA) and a modified complement-fixation test (CFT) when there was no reference test. The tests were used for detection of antibodies against Actinobacillus pleuropneumoniae serotype 2 in a survey of respiratory diseases in Danish finishing pigs. The estimates were obtained by maximum-likelihood and also by a Bayesian method (implemented with Gibbs sampling). Possible dependence of diagnostic errors was investigated by comparing models where independence was assumed to models allowing for conditional dependence, given the true disease status. No strong evidence of conditional dependence in either test sensitivity or specificity was found. Assuming independence, maximum-likelihood estimates and 95% confidence intervals of the sensitivity and specificity of the ELISA were 100% and 92.8% (90.1-95.5%) and the corresponding values of the CFT were 90.6% (85.8-95.4%) and 98.6% (98.0-99.3%), respectively. Bayesian estimates and posterior 95% credible intervals of the sensitivity and specificity of the ELISA were 99.7% (98.7-100%) and 92.7% (89.9-95.3%) and of the CFT were 90.6% (86.0-95.3%) and 98.7% (98.0-99.3%). The sensitivity and specificity of a combined test, where the CFT is subsequently applied to the pig sera that test positive in the ELISA, were estimated at 90.2% (85.6-95.0%) and 99.9% (99.8-100%), respectively. The cost of the combined test was less than the cost of the use of the CFT alone, at prevalences <54%. Prevalences and predictive values and their 95% limits were estimated in six sub-samples of data. The estimates of sensitivity and specificity obtained in the present investigation generally validate those reported from other sources.

Actinobacillus Infections↗

SPECIFIC FRACTIONATION OF HUMAN ANTIDEXTRAN ANTIBODIES. II. ASSAY OF HUMAN ANTIDEXTRAN SERA AND SPECIFICALLY FRACTIONATED PURIFIED ANTIBODIES BY MICROCOMPLEMENT FIXATION AND COMPLEMENT FIXATION INHIBITION TECHNIQUES.

Human antidextran of one individual, absorbed specifically on sephadex, was fractionated into two populations of antibody molecules by successive elution with oligosaccharides of the isomaltose series of increasing size. The purified antibody fractions and some whole antidextran sera were found to fix complement with dextrans of molecular weight of 195,000 and above. It could be demonstrated by quantitative microcomplement fixation inhibition assays that the antibody eluted with isomaltotriose had a higher affinity for smaller oligosaccharides relative to isomaltohexaose, indicating a high content of antibody molecules with smaller combining sites, while with the second fraction, eluted with isomaltohexaose, the small haptens were very poor inhibitors and the larger oligosaccharides inhibited readily, presumably due to a higher proportion of molecules with larger combining site size. Assays of similarly prepared fractions, obtained from earlier bleedings of the same individual (1), with inhibition of complement fixation were in good agreement with those obtained by inhibition of precipitation. The two purified antidextran fractions were shown to differ with respect to their complement-fixing capacity. The fraction with molecules with smaller size-combining sites fixed only about half as much complement per unit antibody N as did the fraction containing largely molecules with larger combining sites suggesting that the strength of complement fixation is affected by the strength of the antigen-antibody interaction.

Antibodies↗

Detection and identification of diagnostic Histoplasma capsulatum precipitates by counterelectrophoresis.

Studies were carried out to develop and evaluate a counterelectrophoresis (CEP) technique for the rapid and specific identification of the diagnostically important histoplasmosis H and M precipitin bands. Well-defined and centrally located precipitin bands were produced by using a discontinuous buffer system and a gel matrix composed of agarose and ionagar no. 2. A template was devised which allowed the selective identification of the H and M precipitins. Comparative evaluations were performed with the microimmunodiffusion (ID) and complement fixation tests. In 52 sera from persons with histoplasmosis, either the H or M precipitin, or both, were identified in 42 (81%) of the cases with the CEP technique and in 43 (83%) with the ID test. With sera from 28 persons with heterologous diseases, the CEP technique, like the ID test, failed to react. The specificity of the CEP technique was dependent upon the use of the identity template. The CEP technique is recommended for routine use in laboratories testing moderate numbers of sera. It provides accurate and reproducible results within 90 min, in contrast to the ID test, which requires 18 to 24 h.

Antibodies, Fungal↗

Gonococcal serology. A comparison of three different tests.

Three serological tests for the detection of gonococcal antibodies were compared: an enzyme-linked immunosorbent assay (ELISA), an indirect haemagglutination reaction (IHA), and a gonococcal complement-fixation test (GCFT). The ELISA was performed with gonococcal pili of a Rotterdam strain (1443) as antigen, the IHA with pilus antigen of an American strain (2686, Buchanan), and the GCFT with whole gonococci of a single strain (46695, Oliver) as antigen. The tests were performed on sera from the same groups of Dutch patients; samples of sera were taken at the first examination and generally 11-22 days later. The ELISA and the IHA were more sensitive than the GCFT. The specificity of the tests was equal in low-risk groups, but the GCFT was slightly more specific in high-risk groups. The ELISA and the IHA did not differ in sensitivity and specificity. The agreement between the ELISA and IHA for patients with uncomplicated gonorrhoea was low (chi = 0.44), but the agreement between the GCFT and the two pilus assays was less (chi = 0.26 and 0.20). The sensitivities were highest for sera from patients with oropharyngeal gonorrhoea or with gonococcal complications; again the ELISA and the IHA were more sensitive than the GCFT.

Adult↗

Localization of antigenic determinants of a viral protein by inhibition of enzyme-linked immunosorbent assay (ELISA) with tryptic peptides.

A new method is described for localizing antigenic determinants in globular proteins by means of inhibition of enzyme-linked immunosorbent assay (ELISA) with tryptic peptides. The inhibitory activity of 10 tryptic peptides of the coat protein of 2 strains of tobacco mosaic virus (TMV) was demonstrated by inhibition of ELISA and verified by inhibition of complement fixation tests. These results bring to 7 the number of antigenic determinants of TMV protein that have been defined.

Amino Acid Sequence↗

Evaluation of anti-complement immunofluorescence test in cytomegalovirus infection.

The anti-complement immunofluorescence (ACIF) technique was evaluated for the diagnosis of human cytomegalovirus (CMV) infection in a group of sera derived from renal transplant recipients and donors by comparing it with the indirect immunofluorescence (FA) and complement fixation (CF) TESTS. The ACIF and FA tests yielded similar results. However, the ACIF test had a distinct advantage over the indirect FA test, since it eliminated the nonspecific cytoplasmic staining that may result in false positive readings in inexperienced hands. Both the indirect FA and ACIF tests were more sensitive than the CF test. In primary CMV infection, the FA and ACIF antibodies appeared earlier and had significantly higher titer than corresponding CF titers. This difference in titers was not seen in seropositive individuals who lacked overt infection. Our previously reported correlation between the seropositivity of the donor and CMV infection in seronegative recipients has been confirmed.

Antibodies, Viral↗