Microcomplement Fixation Tests Research Articles

Seroprevalence of antibodies to Chlamydophila abortus in ovine in the State of Alagoas, Brazil

The goal of this study was to perform a seroepidemiological investigation and to identify risk factors associated with infection of Chlamydophila abortus of sheep herds in the Brazilian state of Alagoas. The study was conducted with samples of 274 ewes with ages equal to or higher than 24 months in 25 herds and in 23 towns located in three regions of the state (Sertão, Agreste and Eastern Alagoas). Anti-C. abortus antibodies were detected using the microcomplement fixation test. The risk factors, were determined based on questionnaires consisting of objective questions, about the farmer and general characteristics of the herd like size, sanitary situation and reproductive management. Among 274 sera samples analyzed for C. abortus, 59 (21.5%) were positive with titers ≥32, 187 (68.3%) negative and 28 (10.2%) suspect with titers ≥16. In the 23 towns studied, 20 had positive animals. Among herds 21 (77.7%) of had positive animals. The only variable which appeared to be significant in the multivariate analysis was the region, and Sertão was the most significant (p<0.001; OR=3.48; T.I. 1.79 - 6.76). Results indicate that infection by Chlamydophila abortus is widespread on sheep farms in the State of Alagoas. Others studies, however, have to be conducted to isolate the agent in order to confirm the role of the bacteria is reproductive disturbances in sheeps. In addition to that, control and prophylactic measures along with health promoting programs have to be encouraged on the studied farms so that infection reates are reduced.

Seroprevalence of antibodies to Chlamydophila abortus in Ovine in the State of Alagoas, Brazil

The goal of this study was to perform a seroepidemiological investigation and to identify risk factors associated with infection of Chlamydophila abortus of sheep herds in the Brazilian state of Alagoas. The study was conducted with samples of 274 ewes with ages equal to or higher than 24 months in 25 herds and in 23 towns located in three regions of the state (Sertão, Agreste and Eastern Alagoas). Anti-C. abortus antibodies were detected using the microcomplement fixation test. The risk factors, were determined based on questionnaires consisting of objective questions, about the farmer and general characteristics of the herd like size, sanitary situation and reproductive management. Among 274 sera samples analyzed for C. abortus, 59 (21.5%) were positive with titers ≥32, 187 (68.3%) negative and 28 (10.2%) suspect with titers ≥16. In the 23 towns studied, 20 had positive animals. Among herds 21 (77.7%) of had positive animals. The only variable which appeared to be significant in the multivariate analysis was the region, and Sertão was the most significant (p<0.001; OR=3.48; T.I. 1.79 – 6.76). Results indicate that infection by Chlamydophila abortus is widespread on sheep farms in the State of Alagoas. Others studies, however, have to be conducted to isolate the agent in order to confirm the role of the bacteria is reproductive disturbances in sheeps. In addition to that, control and prophylactic measures along with health promoting programs have to be encouraged on the studied farms so that infection reates are reduced.

Sensibilidade toxicológica e especificidade do teste de microfixação de complemento na detecção de toxinas botulínicas C e D em meio de cultura e fígado de camundongos

No presente estudo pretendeu-se verificar a sensibilidade toxicológica e especificidade do Teste de Microfixação de Complemento (MCF) na detecção de toxinas botulínicas C e D no sobrenadante de cultivos bacterianos e em fígados de camundongos inoculados com doses letais e subletais. As toxinas foram produzidas em meio de cultura Hemoline, tituladas através da determinação da DL50 pelo Bioensaio em Camundongo e diluídas nas concentrações de 10, 1, 0,1, 0,01 e 0,001 DL50. Desta forma, foram utilizadas em dois modelos experimentais, onde foi determinada a sensibilidade toxicológica do MCF no sobrenadante do meio de cultura com as diluições descritas acima e ainda em extratos hepáticos de camundongos com peso corporal de 20g, inoculados com as mesmas diluições. A tentativa de evidenciação das toxinas botulínicas nos extratos hepáticos de camundongos foi realizada através da sua extração após a morte pela administração das doses letais e ainda pelo sacrifício dos animais inoculados com doses subletais, em intervalos de 5 dias. Os resultados evidenciaram uma sensibilidade toxicológica para o MCF de 100% para os dois tipos de toxinas ao nível de 0,01 DL50, quando testados os sobrenadantes de meio de cultura, portanto 100 vezes superior ao Bioensaio em Camundongo. A sensibilidade toxicológica do MCF, quando examinados extratos hepáticos de camundongos inoculados com 1 e 10 DL50 de toxinas botulínicas C e D, foi inferior, com valores de 100, 80, 89 e 72%, respectivamente. Pelo teste foi possível detectar toxinas botulínicas tipos C e D nos extratos hepáticos de camundongos inoculados com doses subletais até 15 dias após a sua inoculação. A especificidade do MCF foi de 88 e 92%, quando testados extratos hepáticos de camundongos sadios, e confrontados com as antitoxinas C e D; e 100% no sobrenadante do meio de cultura. Os resultados apontam para uma possível utilização do teste como importante instrumento de pesquisa e ainda na eventual substituição dos testes in vivo pelas suas implicações éticas e limitações práticas.

Cell specific nuclear antigens of boar spermatozoa.

Demembranated boar sperm heads were differentially extracted at conditions involving high salt-urea, proteolysis and DNase I cleavage that mimic the conditions promoting the in vivo decondensation of the fertilizing sperm nucleus in the egg ooplasm. The sperm-unique subset of proteins was studied which remained bound in the residual salt-resistant nuclear structure operationally defined as sperm nuclear matrix. By means of polyvalent antisera the immune specificity of the sperm nucleoprotein complex was estimated using ELISA and microcomplement fixation test as compared to somatic type dehistonized chromatin of boar liver. To define immunologically specific sperm DNA-associated proteins hybridomas were generated by fusing lymphocytes immunized with boar sperm protein/DNA complex. Monoclonal antibodies were selected (Mab 1A8, 1B3, 2B5, 2H5 and 3A4) which identified protein moieties in the sperm DNA-tight binding proteins complex resistant to cleavage with DNase I and sensitive upon digestion with high concentration of proteases. No appreciable reactivity was recorded of the antibodies to somatic chromatin and no significant binding to ssDNA. A polypeptide in the residual sperm nuclear structure of apparent Mr 27 kDa was recognized by Mab 3A4 as detected by Western blotting. The enhanced reactivity to the DNase I digested sperm nuclear fraction (except for Mab 2H5) suggests that DNA protected from nuclease digestion by a protein might be essential for immune reactivity and full antigenic integrity as well as the dependence of the cognate proteins on the binding to DNA for antigenicity and immune specificity. The functioning of the identified putative sperm specific proteins is anticipated in the structural rearrangement of chromatin in the zygote.

The sequence-immunology correlation revisited: Data for cetacean myoglobins and mammalian lysozymes

Quantitative microcomplement fixation tests employing rabbit antisera were done to compare immunologically 13 cetacean myoglobins and 15 mammalian lysozymes c of known amino acid sequence. In both cases there was a strong correlation between immunological distance (y) and percent sequence difference (x), as had been found for several other globular proteins. For myoglobin the relationship could be described by y = 10.5x and for lysozyme by y = 8.5x. The coefficients in both of these equations are appreciably higher than the values of 5.1-6.9 reported for three other vertebrate globular proteins (bird lysozyme c, mammalian ribonuclease, and mammalian serum albumin), and they imply that rabbit antisera to mammalian myoglobins and lysozymes are more sensitive to evolutionary substitutions. A strong inverse correlation (r = -0.95) was found when the slope of the line relating y to x for these five data sets was plotted against the percent sequence difference between the rabbit's own protein and the proteins immunized with. Specifically, the cetacean myoglobins on average differ in amino acid sequence from rabbit myoglobin by less than 13% and exhibit the steepest slope (10.5), while bird lysozyme sequences differ by nearly 40% from rabbit lysozyme and exhibit the shallowest slope (5.1).

Immunogenicity of free histones and of histones complexed with RNA

Histone antibodies have been obtained by immunizing rabbits with histones H1, H2A, H2B, H3, H4 and triacetylated H4, uncomplexed to RNA. The reactivity of these antibodies was investigated by ELISA using as antigen isolated histones and chromatin as well as thirty-five different synthetic peptides covering the entire sequence of the four core histones, two peptides of H1 and two acetylated peptides of H4. The binding of these antibodies to histones was also measured in immunoblotting and in microcomplement fixation (MCF) tests. In parallel experiments using the same assays the various antigens were tested with antisera raised against histones complexed with RNA. Antibodies induced in the absence of RNA did not react with histones in MCF tests nor with chromatin in ELISA but reacted with the histones in ELISA, although the antibody titers were somewhat lower than in the case of antisera to histone-RNA complexes. Antibodies to RNA-histone complexes reacted with histones in both ELISA and MCF tests. When they were tested with peptide-coated microtiter plates in a direct binding ELISA format, antibodies induced with uncomplexed histones recognized very few fragments which were mainly located in the N- and C-terminal ends of the histones.

Immune status of young adults to mumps virus infection in northern Jordan.

Eight hundred and seventy-five sera, collected randomly from university students aged 18-24 years, were tested for mumps virus antibodies. The prevalence of seropositivity was 85.3% for males and 87.1% for females using the enzyme linked immunosorbent assay method (ELISA) and 83.4% for males and 77.6% for females using the micro-complement fixation test (CFT). The remaining 14.7%-16.7% of males and 12.9%-22.3% of females, who had non-reactive sera in ELISA and CFT tests respectively indicated the presence of a sizeable population with a high risk of contracting the infection and this recommends the implementation of routine vaccination against mumps in Jordan. The two techniques correlated well, though ELISA exhibited a higher sensitivity than CFT.

Diversity of non-histone protein fraction NHCP2 from hamster Kirkman-Robbins hepatoma and liver.

Non-histone protein fraction NHCP2 eluted from hydroxyapatite with 100 mM phosphate buffer (pH 6.8) of undigested, nuclease-sensitive and nuclease-resistant nuclei of hamster Kirkman-Robbins hepatoma and liver was studied by two-dimensional gel electrophoresis and microcomplement fixation test in the presence of antibodies elicited against NHCP2 of examined tissues. The NHCP2 of undigested nuclei as well as from two chromatin fractions with different susceptibility to nuclease of both tissues, besides many common components, showed some differences in their non-histone patterns especially within molecular weights of 17,000-24,000, 36,000-44,000 and 60,000-90,000. Immunological analysis confirmed the high specificity of hepatoma non-histone components of the NHCP2 fraction. However, these components appeared not to be exclusively localized either in nuclease-sensitive or nuclease-resistant part of chromatin of neoplastic tissue.

Quantitative microcomplement fixation tests using chicken anti-viral antibody extracted from egg yolk

Yolk immunoglobulins obtained from hens immunized with human rotavirus and two plant virus antigens were used in quantitative microcomplement fixation tests. Difficulties inherent in the use of avian complement were overcome by utilizing a mixture of the C1 component of chicken complement and guinea-pig complement devoid of its own C1. The method is suitable for detecting small quantities of viral antigen and antibody and for detecting antigenic activity present on peptide fragments of viral proteins.

Comparative studies on glucose phosphorylating isoenzymes from vertebrates—VIII. Immunochemical studies on mammalian hexokinases A

1. 1. An immune serum elicited in a rabbit by injection of homogeneous brain hexokinase A was shown to be specific for the antigen. Other rat hexokinase isoenzymes (hexokinases B, C or D) did not present cross-reaction when tested by immunoinhibition of enzyme activity, double immunodiffusion and immunoadsorbent columns. 2. 2. The enzyme activity of hexokinase A from several mammals (rodents, lagomorphs, artiodactyls) was partially inhibited by the immune serum. In the case of the mouse enzyme, the amount of serum required to inhibit 50% of the activity was five-fold higher than in the case of the rat enzyme. Enzymes from cow or sheep brain were only marginally affected. 3. 3. Hexokinases A isolated from various mammals, tested against the rat enzyme, showed faint lines of precipitation and marked spurs in double immunodiffusion plates even when enzymes from closely related rodents were analyzed. Immunoadsorbent columns, on the other hand, were able to retain most of the activity of hexokinases A from the mammals studied. 4. 4. Micro-complement fixation tests showed that hexokinases A from mammals outside the Order Rodentia were only partially recognized by the anti-hexokinase A rat serum. 5. 5. The results suggest that amino acid substitutions on the hexokinase A molecule have occurred at a rather fast rate.

Comparison of leucine aminopeptidase from human lens, beef lens and kidney, and hog lens and kidney

Antisera specific for leucine aminopeptidase (LAP) were prepared. Using these antisera in immunodiffusion tests, the identity of LAP isolated from beef lens and kidney is demonstrated. The same pertains to hog lens and kidney LAP. As indicated by only partial fusion of the immunoprecipitates in immunodiffusion plates, leucine aminopeptidases isolated from hog and beef are antigenically similar but not identical. These tests also indicate substantial similarity between a component in human lens homogenates and bovine or hog LAP. Microcomplement fixation tests corroborate these observations and indicate that, under these conditions, LAP from beef lens and kidney, or hog lens and kidney, are indistinguishable. However, there is an approximate 8·5% amino acid sequence difference between beef and hog LAP. Microcomplement fixation tests with human lens homogenate also corroborate immunodiffusion results and indicate an approximate 19% amino acid sequence difference between beef and human LAP. These data establish that LAP is a species-specific enzyme and they indicate that it is not organ specific. Maximal complement fixation occurs at approximately 0·1 μg antigen per tube in those assays in which pure aminopeptidases were tested. This permits standardization of the microcomplement fixation assay for LAP under these conditions. Maximal complement fixation occurs at 160–200 μg human lens homogenate per tube. Assuming that in this quantity of homogenate there is 0·1 μg LAP, then it can be calculated that LAP comprises about 0·05% of the lens protein. This agrees closely with the percentage of LAP in hog and beef lenses. Thus, the reduced LAP activity reported for human lens tissue appears not to result from an absence of the enzyme but rather, may be due to diminished catalytic competency of the enzyme in aged human lens tissue (see Taylor and Juhngen, 1984). The unit evolutionary period, 4·7–5·8 × 10 6 years, indicates that LAP has been highly conserved during evolution.

A modified microcomplement fixation test for antigen or antibody determination in soluble immune complexes, using gel filtration.

A Modified Microcomplement Fixation Test (MMFT), useful for detecting and quantifying soluble immune complexes (ICs) and their components in chromatographic separations, is described. This method is based on the addition of excess specific antibody to the ICs against any of their components in order to increase the ICs' anticomplementarity. The concentration of ICs is expressed as the sample dilution which fixes 50% of the added Complement. The MMFT was applied to profiles of ICs obtained in vivo and in vitro. MMFT allows great sensitivity, good reproductibility and the detection of noncomplement fixing ICs without any interference of free antigen (Ag) or free antibody (Ab).

Humoral response of melanoma patients to two different tumor-associated antigens.

Sera of 8 stage II melanoma patients undergoing surgical adjunctive immunotherapy with bacille Calmette Guérin (BCG) plus melanoma cell vaccine (MCV) were assayed for humoral response by the complement-dependent antibody cytotoxicity (CDAC) assay and by the microcomplement fixation (MCF) test. The patients developed high levels of cytotoxic (CTX) and complement-fixing (CF) antibodies to the UCLA-SO-M14 (M14) cells, one of the three melanoma cell lines in the MCV. Significant rises in CTX and CF antibodies occurred one month post-immunotherapy. While the level of CTX antibodies was maintained for 11 months thereafter, the titer of CF antibodies was sustained for seven months, then gradually declined. When the sera were absorbed with lymphoblastoid (ML14) cells which are autologous to the M14 cells, two residual peaks of CTX antibodies, one and four months postimmunotherapy and two peaks of CF antibodies, one and seven months postimmunotherapy, emerged. Two sera that exhibited high levels of CTX and CF antibodies one month postimmunotherapy were absorbed with ML14 cells and human fetal brain tissue. The reactivity of one serum in both the CDAC and MCF assays were abolished, whereas the reactivity of the other serum was not significantly diminished in either assay. These data indicate that the stage II post-surgical melanoma patients developed a humoral immune response to at least two distinct tumor antigens on the membrane of the M14 cells. One of these antigens appeared to be of fetal origin (OFA), the other M14-associated (TAA). Both antigens, OFA and TAA, were involved in complement-dependent antibody cytotoxic and complement fixation reactions in vitro.

Tissue and cell-specific antigens in chromatin from cultured rat Sertoli cells

Cell-specific chromatin antigens have been detected in rat Sertoli cells. Antisera were raised in rabbits to dehistonized chromatin prepared from 5- to 6-day cultures of rat Sertoli cells and immunoreactivity was assessed with microcomplement fixation tests or immunoidentification of antigens separated electrophoretically and transferred to nitrocellulose sheets. Tissue specificity was confirmed further by immunoabsorption. These antisera recognized only components of Sertoli cell chromatin; chromatins prepared from rat liver, kidney, thymus, Novikoff hepatoma, a testes germinal cell fraction, or purified rat DNA showed little or no immunoreactivity. Nitrocellulose transfers of total chromosomal proteins revealed the presence of two high-molecular-weight antigens (greater than 200,000) and a broad range of weaker immunologic species ( M r about 50,000–200,000) in Sertoli cell chromatin but not liver or thymus chromatin. Deproteinization of Sertoli cell chromatin with concentrated salt and urea at pH 6 or 8 produced an increase in complement-fixing activity of the fraction sedimenting with DNA and micrococcal nuclease digestion studies showed these fractions to depend in part on DNA for antigenicity. Individual antigens in the protein-DNA pellets of pH 8 salt and urea fractionated chromatin could not be identified with antigens on nitro-cellulose sheets. Collectively, these observations suggest that at least two groups of cell-specific antigens exist in Sertoli cell chromatin; one group is detected after electrophoretic separation and transfer to nitrocellulose and the other group, reactive in complement fixation, cannot be detected through this method, apparently becoming antigenically inactive once the complex of protein and DNA is dissociated.

Species specificities of l-glutamic acid decarboxylase: Immunochemical comparisons

The relationship of catfish brain l-glutamic acid decarboxylase (GAD) with those from other species were examined by comparing the extent of their interactions with antibodies against catfish brain GAD. Several immunochemical methods, such as immunodiffusion, inhibition of enzyme activity and microcomplement fixation tests were employed for these studies. Antibodies against GAD from catfish brain were observed to cross-react with the enzymes from goldfish, frog, chick and turtle. Rabbit and bovine GAD did not cross-react with these antibodies. In the presence of the antibodies, enzymes from goldfish, frog. Drosophila, chick and crayfish were inhibited 27, 46, 32, 38 and 57% respectively. Enzymes from all the other species examined were not affected at all. Microcomplement fixation tests showed that goldfish, frog and chick GAD were quite similar in immunoreactivities. These immunochemical studies demonstrated that GAD from lower vertebrates such as chick, goldfish and frog and some invertebrates such as Drosophila and crayfish are closely related to catfish GAD. In contrast, mammalian GAD shows little or no cross-reactivity with antibodies against catfish GAD.

Enzyme-linked immunosorbent assay for use with nuclear protein-DNA complex antigens.

An enzyme-linked immunosorbent assay (ELISA) was developed for identification and quantification of nuclear antigens (nonhistone protein-DNA complexes from chromatin). Until now, the complement fixation assay has been the only immunoassay routinely applied to nonhistone protein-DNA complexes. The ELISA is considerably more sensitive than the micro-complement fixation test for assaying the immunospecificity of nuclear protein-DNA complexes. Dilutions of rabbit antisera as great as 1:6400 could be used to detect nanogram quantities of antigen, chicken reticulocyte chromatin or dehistonized rat liver chromatin.

Identification and quantification of leucine aminopeptidase in aged normal and cataractous human lenses and ability of bovine lens LAP to cleave bovine crystallins.

Rabbit antisera to bovine lens leucine aminopeptidase (LAP) have been prepared. These LAP-specific antisera cross-react with a component, LAP, in human lens homogenates. Bovine and human lens LAP are similar but not identical. Immunodiffusion tests show that LAP is present in a vast majority if not all cataractous and normal human lens homogenates. Results from immunoelectrophoresis indicate that LAP is found in these homogenates as several metazymes as well as in an albuminoid--and possibly membrane-associated form. In contrast to many activity-based studies which imply that very little LAP is present in human lenses, micro-complement fixation tests indicate that the concentration of LAP in aged human lenses is similar to that found in calf lenses. Taken together, these data indicate that LAP undergoes age-related changes. These alterations of enzyme (or environment) result in an enzyme of markedly reduced activity, hence, the discrepancy between amount of LAP and the amount of enzymatic activity in aged human lenses. Active LAP is shown to enhance the rate of hydrolysis of alpha-2 and alpha-B crystallins but not alpha-A2, beta H, beta L or gamma crystallins. The ramifications of LAP inactivation with respect to cataractogenesis are discussed.

Enzyme-linked immunosorbent assay in the study of histone antigens and nucleosome structure

The application of an enzyme-linked immunosorbent assay to the study of histone antigens is described. This method is much more sensitive than microcomplement fixation tests and allows the interaction between histone antibodies and nucleosomes to be measured directly on the solid phase under a variety of ionic conditions. The results indicate that the enzyme-linked immunosorbent assay is a sensitive assay for investigating different conformational states of chromatin and for detecting low levels of histone antibodies in antisera.

Monoclonal antibodies against mouse nerve growth factor produced by somatic cell hybrids.

Spleen leukocytes from rats and mice immunized against mouse 2.5S nerve growth factor (NGF) and peripheral blood leukocytes from rabbits hyper-immunized against the same antigen were fused with the mouse plasmacytoma P3X63Ag8. Hybridomas were screened by immunological assays (micro-complement fixation test and solid phase radioimmunoassay) for production of antibodies that reacted with NGF. Significant variations were seen between culture fluids from different hybrid cells. In addition, most but not all hybridoma antibodies that reacted immunologically with NGF prevented neurite outgrowth from 8-day chick embryo sensory ganglia explants after binding to NGF. These results suggest that the hybridoma antibodies produced by the different clones react with different antigenic sites on the NGF molecule.

Quantitative immunological studies of the albumins of North American squirrels, family Sciuridae

1. 1. Rabbit antisera against purified serum albumin of Spermophilus tridecemlineatus, Spermophilus lateralis, Ammospermophilus nelsoni, Eutamias amoenus, and Sciurus niger were used to probe relationships within the Sciuridae. 2. 2. Quantitative micro-complement fixation tests indicated that ground squirrel ( Spermophilus and Ammospermophilus) and chipmunk ( Eutamias) albumins were more similar to one another than any were to tree squirrel ( Sciurus) albumins. 3. 3. Spermophilus tridecemlineatus appears more distantly related to other Spermophilus than are Ammospermophilus, Marmota, and Eutamias.