Complement Fixation Procedures Research Articles

Review of human and animal cases of coccidioidomycosis diagnosed in Canada

The first Canadian case of coccidioidomycosis in a human was reported in 1952 and 11 more cases since then. This study provides details of other cases of coccidioidomycosis that have been diagnosed in Canada. Based on clinical details, isolation of Coccidioides immitis, detection of a specific antibody (F band) for coccidioidomycosis by macro- or microimmunodiffusion tests, concurrently used with the complement fixation procedure, and histopathological findings, 116 more cases of this disease were verified. The great majority (94%) of these cases were diagnosed in the western Canadian provinces of British Columbia, Alberta, Saskatchewan and Manitoba, and the others in Quebec, Ontario and Nova Scotia (5, 1, and 1 cases, respectively). Available information indicates that the C. immitis infections were contracted during visits to endemic areas in the United States (Arizona, California and New Mexico), Mexico, and Bolivia. Pulmonary infections were the most common type of coccidioidomycosis (93%) followed by the disseminated or meningeal types C. immitis infections occurred in individuals with or without predisposing factor(s) and were more common in males than in females. The exoantigen procedure was very useful and reliable in the accurate and rapid identification of suspected C. immitis isolates. Two cases of coccidioidomycosis were reported in animals in Ontario, Canada.

Detection of foot-and-mouth disease virus by competitive ELISA using a monoclonal antibody specific for the 12S protein subunit from six of the seven serotypes

Foot-and-mouth disease (FMD) prevention and control programs are dependent upon rapid, reliable diagnostic procedures. The widely used FMD diagnostic complement fixation (CF) procedures require a specific antiserum for each of the seven FMDV serotypes making the tests both cumbersome and difficult to standardize. An FMD diagnostic, monoclonal antibody based inhibition-ELISA procedure was developed. The test uses a single monoclonal antibody (MAb) that reacts with all European and South American FMDV isolates examined. The procedure detects a highly conserved epitope on the 12S protein subunit of FMDV which appears to be common to all FMDV's with the exception of the South African Territories 2 serotype. The results indicate that the sensitivity of this test is greater than CF and approaches that of virus isolation.

A competitive enzyme immunoassay for the detection of bovine antibodies to Brucella abortus using monoclonal antibodies

A competitive enzyme immunoassay (EIA) for the detection of circulating bovine antibodies to Brucella abortus has been developed using horseradish peroxidase conjugated monoclonal antibodies (MAb) raised against B. abortus cell surface antigens. Antibodies present in the serum of either vaccinated or infected cattle can apparently displace the conjugated MAb from the lipopolysaccharide antigen (LPS) in a quantitatively different manner allowing an assessment of immune status of the animal. The results from a panel of sera from animals with a known status of vaccination or infection indicated that the test was more selective in the detection and discrimination of infected from uninfected or immunized animals, than conventional complement fixation, agglutination or indirect enzyme immunoassay procedures.

Cross-reactivity of Haemophilus somnus antibody in agglutination and complement fixation tests and in the enzyme-linked immunosorbent assay.

The specificity and sensitivity of agglutination, complement fixation, and enzyme-linked immunosorbent assay (ELISA) procedures in the detection of antibodies to Haemophilus somnus was investigated. H. somnus rabbit immune sera were found to agglutinate Pasteurella multocida, Staphylococcus aureus, and Haemophilus agni and, in some instances, also Pasteurella haemolytica, Salmonella dublin, Streptococcus agalactiae, and Corynebacterium pyogenes. In complement fixation tests with saline extracts as antigens, only H. agni reacted with H. somnus antisera to any significant degree. In ELISA tests with sonicated or heat-extracted antigens, cross-reactions were seen with the two Pasteurella spp. and with H. agni. When whole cells and saline extracts were used as antigens in ELISAs, only H. agni showed any cross-reactivity. The greatest specificity in distinguishing homologous from heterologous reactions was achieved by ELISA with saline extracts as antigens. Escherichia coli and Brucella abortus antigens failed to react with H. somnus antibody in any of the tests. A rabbit serum containing antibody to bovine type isolates of P. multocida, P. haemolytica, S. aureus, S. agalactiae, S. dublin, C. pyogenes, and E. coli gave no positive reaction in ELISA tests with saline extract of H. somnus as antigen. It is concluded that such saline extract, which appears to consist largely of H. somnus common antigen, has the potential of being a useful diagnostic reagent in the study by ELISA of antibody response to H. somnus.

A large-scale radiometric micro-quantitative complement fixation test for serum antibody titration

A micro-quantitative complement fixation (CF) procedure based on 51Cr release is described. The method employs 50% hemolysis as end point and the alternation equation to calculate the amount of complement involved in the hemolytic reaction. Compared to the conventional CF tests, the radiometric procedure described here is very precise and consistently reproducible. Also, since only 3 4-fold dilutions of sera are used for the titration of antibodies over a wide range of concentrations, the test is very concise and is economical to perform. Its format is amenable to automation and computerization. This radiometric CF procedure is thus most useful for large-scale immunological research and epidemiological surveillance studies.

Antigenic relatedness of equine herpes virus types 1 and 3.

Antiserums prepared in specific pathogen free (SPF) ponies were used in direct and indirect immunofluorescence, immunodiffusion, complement fixation and serum neutralization procedures to study the interrelationships of the three types of equine herpes viruses (EHV-1, EHV-2, and EHV-3). Equine cell cultures infected with each type virus fluoresced when stained with homologous conjugated antiserum. In reciprocal tests EHV-1 and EHV-3 cross-fluoresced, but EHV-2 did not cross-fluoresce. Non-infected cell cultures did not fluoresce when stained with the 3 conjugates. EHV-1 and EHV-3 cross-fluoresced in reciprocal indirect fluorescent antibody tests, but no cross-fluorescence was shown with EHV-2. Antigens representing each type of equine herpes virus reacted with their homologous antiserum in the immunodiffusion test. In reciprocal tests, a common line(s) of identity formed with EHV-1 and EHV-3; however, the precipitin line(s) was not common with EHV-2. Antigen prepared from noninfected embryonic mule skin (EMS) cell cultures did not react with any of the antiserums. Specific complement-fixing antibodies were present in antiserums when tested against their homologous antigens. In reciprocal complement fixation tests EHV-1 and EHV-3 crossreacted, but no cross-reactivity was shown with EHV-2. Significant levels of neutralizing antibody were in an antiserum when tested against homologous virus, whereas cross-neutralization was not detectable in reciprocal tests. These studies indicate that each type of equine herpes virus contains specific antigenic components, and EHV-1 and EHV-3 share a common antigen(s) that is not shared with EHV-2.

Purification and serological characterization of a type-specific antigen of Streptococcus equisimilis

A microtiter complement fixation (CF) procedure was developed for use in detection of antibodies in sera of swine and rabbits vaccinated with Streptococcus equisimilis. Crude ultrasonic as well as acid-extracted preparations contained CF antigen, but the ultrasonic procedure resulted in a higher yield of active antigen. Evidence of serotype specificity with varying degrees of cross reactivity was detected with the CF procedure when representative strains of four different serotypes of S. equisimilis were compared by using their respective unadsorbed antisera. Fractionation of crude sonic extract or acid extract by centrifugation, precipitation with ammonium sulfate, and chromatography on carboxymethyl cellulose yielded a purified, type-specific antigen that reacted only with the homologous antiserum in the CF test and formed a single band by immunodiffusion. Complement-fixing antibodies in immune swine sera were predominately immunoglobulin G.

Hepatitis B antigenemia in remote tribes of Northern Kenya, Northern Liberia, and Northern Rhodesia.

Hepatitis B antigen (HB Ag) was detected by complement fixation procedures in 32 of 343 persons tested (9.3 %) from the Turkanas of Lokori and Kalokol (northern Kenya), the Korekore people living on the Urungwe Reserve (northern Rhodesia), and the Mano tribe residing in Nyao (northern Liberia). The prevalence of HB Ag was 12.2% in Turkana, 8.7% in Korekore, and 6.4% in Mano tribesmen. Turkana males exhibited the highest prevalence of HB Ag. By comparison, not a single male from the Mano people had detectable HB Ag. Individuals of all ages were positive for HB Ag, but no age-related trends were evident.

Indirect fluorescent-antibody and quantitative agar-gel immunodiffusion tests for the serological diagnosis of paracoccidioidomycosis.

The value of various serological tests in the diagnosis of paracoccidioidomycosis was studied. Quantitative agar-gel immunodiffusion and indirect immunofluorescent tests were performed, and the results were compared with those of complement fixation and qualitative agar-gel procedures. The quantitative immunodiffusion procedure was found to serve as the simplest and safest quantitative test that could be performed for evaluation purposes, whereas the indirect fluorescent-antibody test gave nonspecific reactions and, as such, proved unsuitable.

Current state of serological tests used to detect blood parasite infections

Various serologic tests used for immunodiagnosis of blood parasite infections are discussed, and the advantages and limitations of each are noted. The procedures include complement fixation, indirect fluorescent antibody, indirect hemagglutination, agglutination, and soluble antigen fluorescent antibody tests. The influence of the quality of antigen on the reliability of the tests is stressed, and the fact that no single procedure is suitable for all applications is emphasized. Problems peculiar to preparation of antigens from intraerythrocytic parasites are noted. In addition, an adaptation of the quantitatively standardized complement fixation procedure for critical, objective evaluation of antigens is described.

Indirect Fluorescent-Antibody and Quantitative Agar-Gel Immunodiffusion Tests for the Serological Diagnosis of Paracoccidioidomycosis

The value of various serological tests in the diagnosis of paracoccidioidomycosis was studied. Quantitative agar-gel immunodiffusion and indirect immunofluorescent tests were performed, and the results were compared with those of complement fixation and qualitative agar-gel procedures. The quantitative immunodiffusion procedure was found to serve as the simplest and safest quantitative test that could be performed for evaluation purposes, whereas the indirect fluorescent-antibody test gave nonspecific reactions and, as such, proved unsuitable.

Micro complement fixation assay for type-specific group A streptococcal antibody.

A micro complement fixation assay was devised to measure the type-specificity of anti-M antibody. Unabsorbed sera were from rabbits hyperimmunized with heat-killed streptococci and from humans with naturally occurring antibody or immunized with purified M protein vaccines. These sera fixed complement only in the presence of homologous M proteins (serotypes 1, 3, 6, 12, and 14). The complement fixation reaction paralleled the results obtained with the Lancefield bactericidal (opsonic) assay and did not exhibit the cross-reactions frequently seen with type-specific anti-M sera assayed by passive hemagglutination. All serological activity of the antisera resided in the fraction corresponding to 7S globulin eluted from a Sephadex G-200 gel filtration column. Because of the close correlation between micro complement fixation and the bactericidal assay for type-specific antibody, it is proposed that the micro complement fixation procedure, as we have described it, merits further evaluation as a substitute for the bactericidal test of immunity to group A streptococcal infection.

Preparation and standardization of an Australia antigen antibody of equine origin.

A horse has been immunized with Australia antigen (Au/SH) purified 20-fold by a procedure employing gel filtration of Cohn fraction IV derived from an Au/SH-positive human plasma pool. Hyperimmunization was initiated by the intramuscular injection of 20 ml of a mixture of equal parts of purified Au/SH and complete Freund's adjuvant. The 20-ml volume was divided into four 5-ml doses, two of which were administered on each side of the horse's neck. Booster doses of antigen alone were given as follows: 10 ml intravenously 30 days later and 5 ml intramuscularly on each of days 77 and 205. Au/SH antibody formed readily, beginning on day 17, and was demonstrated by the agar gel double-diffusion technique and the complement fixation test during the subsequent 6 months. Antihuman plasma protein antibodies were effectively removed from the horse serum by one absorption with 1 to 3 volumes of normal human plasma. Abrupt rises in anticomplementary activity observed shortly after the third and fourth antigen injections, when the horse had developed elevated and steady levels of Au/SH antibody, could possibly be due to formation of antigen-antibody complexes. After optimal conditions were determined, an Au/SH antibody reagent pool which met official requirements was prepared. It was found equally suitable for the agar gel double-diffusion, complement fixation, and counterimmunoelectrophoresis test procedures.

Measurement of human chorionic gonadotropin by means of isotope complement fixation.

ABSTRACT A complement fixation procedure is described, employing 51Cr-tagged sheep erythrocytes. With the HCG-anti-HCG system as a model, a considerable increase in sensitivity is demonstrated in comparison with established complement fixation procedures, approaching the range that may be obtained by means of radioimmunoassay.

Preparation and Standardization of an Australia Antigen Antibody of Equine Origin

A horse has been immunized with Australia antigen (Au/SH) purified 20-fold by a procedure employing gel filtration of Cohn fraction IV derived from an Au/SH-positive human plasma pool. Hyperimmunization was initiated by the intramuscular injection of 20 ml of a mixture of equal parts of purified Au/SH and complete Freund's adjuvant. The 20-ml volume was divided into four 5-ml doses, two of which were administered on each side of the horse's neck. Booster doses of antigen alone were given as follows: 10 ml intravenously 30 days later and 5 ml intramuscularly on each of days 77 and 205. Au/SH antibody formed readily, beginning on day 17, and was demonstrated by the agar gel double-diffusion technique and the complement fixation test during the subsequent 6 months. Antihuman plasma protein antibodies were effectively removed from the horse serum by one absorption with 1 to 3 volumes of normal human plasma. Abrupt rises in anticomplementary activity observed shortly after the third and fourth antigen injections, when the horse had developed elevated and steady levels of Au/SH antibody, could possibly be due to formation of antigen-antibody complexes. After optimal conditions were determined, an Au/SH antibody reagent pool which met official requirements was prepared. It was found equally suitable for the agar gel double-diffusion, complement fixation, and counterimmunoelectrophoresis test procedures.

An Automated Complement Fixation Procedure for Detecting Antibody to N. gonorrhoeae

An Automated Complement Fixation Procedure for Detecting Antibody to N. gonorrhoeae

鳥類血清の補体結合反応に関する研究

Various test conditions were examined to establish the complement fixation (CF) test procedures of chicken sera for practical application. From the experiment in which fresh duck, goose, and pigeon sera were tested for their cofactor (avian C′1) activity that is essential for CF reaction of avian antibody, duck serum was proved to be most suitable as cofactor. Thus, standard CF procedure was established by employing a given amount of fresh duck serum in the reaction system, and was shown to have higher reproducibility and to be more practical than the other methods previously reported. The procedure was successfully applied for detection of antibody to Newcastle disease virus in convalescent and hyperimmune chicken sera, and antibody to Mycoplasama galisepticum in convalescent chicken sera.

Comparison of methods for coccidioidomycosis complement fixation.

A Laboratory Branch Task Force of the National Communicable Disease Center has proposed a standardized complement fixation procedure (LBCF) and an adaptation of this to microtitration techniques (MT) as uniform methods for performing complement fixation (CF) tests. A common procedure should make CF results from one laboratory more comparable to another. In addition, it would be preferable if the common procedure reproduced the titer levels of a testing procedure which is to be replaced, particularly when valid clinical interpretations have been derived from the latter. Replicated sets of sera were tested by the LBCF, MT, and the standard Smith CF procedure for coccidioidomycosis. Results with all three procedures were highly reproducible within an acceptable one-tube variation of a twofold dilution series, but the frequency of one-tube variations was greater with the MT method than with the other two. There was no statistical difference in the titers obtained with the Smith and LBCF procedures, but there was a significant difference when the MT results were compared to those with the Smith method. The LBCF method should be acceptable as a standardized and uniform CF procedure for coccidioidomycosis, subject to comparative testing between different laboratories.

Australia antigen in a closed adult population monitored for serum glutamic oxalacetic transaminase.

A study of the presence of Australia antigen (Au/SH) was conducted over a period of 21 weeks among volunteer plasma donors living in a prison and being monitored for serum glutamic oxalacetic transaminase (SGOT). A good correlation was observed between the level of SGOT and presence of Au/SH, the latter being present in 33% of donors with SGOT values higher than 101 Karmen units and in 12% of those with SGOT values of 41 to 100 units. Furthermore, none of the 87 donors with all SGOT values below 40 was found positive for Au/SH. It should be noted, however, that single specimens only were tested from 72 of the 87 individuals. Au/SH was detected with equivalent efficiency by both agar gel precipitation and complement fixation procedures. Implications of these findings in the prognostication of hepatitis carrier state are discussed.

Australia Antigen in a Closed Adult Population Monitored for Serum Glutamic Oxalacetic Transaminase

A study of the presence of Australia antigen (Au/SH) was conducted over a period of 21 weeks among volunteer plasma donors living in a prison and being monitored for serum glutamic oxalacetic transaminase (SGOT). A good correlation was observed between the level of SGOT and presence of Au/SH, the latter being present in 33% of donors with SGOT values higher than 101 Karmen units and in 12% of those with SGOT values of 41 to 100 units. Furthermore, none of the 87 donors with all SGOT values below 40 was found positive for Au/SH. It should be noted, however, that single specimens only were tested from 72 of the 87 individuals. Au/SH was detected with equivalent efficiency by both agar gel precipitation and complement fixation procedures. Implications of these findings in the prognostication of hepatitis carrier state are discussed.