N. Schmeer

Enzymimmuntest (ELISA) zum nachweis von IgG1-, IgG2-und IgM-antikörpern bei der Q-fieber-infektion des rindes

By application of IgG1-, IgG2-, and IgM-specific conjugates in an enzyme-linked immunosorbent assay (ELISA), dominance of IgG1 in natural Q-fever infections of cattle could be demonstrated. In contrast, vaccination with an inactivated Q-fever vaccine predominantly induced IgG2 antibodies. Complement fixing activity was detected in positive sera (inactivated at 56 degrees C) in the IgG1 fraction only. Therefore, with serum samples containing exclusively IgM (10%), or IgG2 (4%), a serodiagnosis could be achieved only by ELISA. Furthermore, it could be shown that IgG2 and IgM may suppress fixation of complement by IgG1 antibodies, thus resulting in incomplete inhibition of hemolysis and even reduction of CF-titers. So, sera with low CF-titers may give incorrect negative results in the CF-test. Applying the ELISA with L-chain-specific conjugates, such problems could be avoided. For evaluation of the early and later stages of infection or the status of vaccination, IgM, IgG1-, and IgG2-specific conjugates were used. In comparison to sera, only 73% of corresponding milk samples were positive in the IgG1-ELISA. However, for seroepidemiological purposes testing of bulk milk samples by ELISA may be feasible.

Epidemiology and significance of Q fever in the Federal Republic of Germany

In the Federal Republic of Germany no large Q fever epidemics (more than 200 cases) have been encountered within the last 20 years; however, Q fever was prevalent throughout that period on a constant level (between 27 and 100 officially reported cases per year). Besides classical pneumonic Q fever, chronic forms associated with endocarditis, myocarditis and hepatitis were recently diagnosed for the first time in the Federal Republic of Germany. The disease Q fever in humans is often misdiagnosed as common cold or influenza, and more attention should be paid to this entity by the medical profession. Within ten years there has been a sharp increase of Q fever infections in livestock and pets as proved by seroepidemiologic investigations. Preliminary results of a seroepidemiological study indicate a parallel increase of seropositives in the human population, but further investigations on larger numbers of sera are required for statistic confirmation. There are reasons to believe that, in contrast to general opinion, in the Federal Republic of Germany C. burnetii is involved now in infertility in cattle, and besides being a zoonosis Q fever must be considered as a potentially important infectious disease of cattle causing economic losses in this country. Further investigations on this matter are required.

Isolation of a protein antigen from Coxiella burnetii

Antigens prepared from Coxiella burnetti, strain Frankfurt, phase II, propagated in persistently infected Buffalo Green Monkey (BGM) cell cultures were purified by guanidinium hydrochloride treatment and chloroform/methanol extraction. By ELISA analysis, chloroform/methanol residues (CMR) proved to be free of host cell antigens. The CMR were sensitive to trypsin, pronase E and proteinase K, as determined by absorption-kinetics of CMR suspensions at 600 nm and release of protein. Coomassie blue stained SDS polyacrylamide gels of proteinase hydrolysates from CMR revealed only a single component of apparently 27,000 D. Silver stained gels, however, showed a second component of apparently 12,000 D. In contrast, from untreated native C. burnetii a large variety of proteins, most of them protease-sensitive, were released by detergents at low temperatures, but the 27,000 D component was only solubilized at 60-100 degrees C. The 27,000 D component was obviously the major protein of CMR as well as of whole cells. Antigenicity of this 27,000 D protein could be demonstrated by agargel precipitation test, ELISA and immunoperoxidase techniques applying antisera raised against whole cells and against the extracted component. The component was also recognized in a dot immunobinding assay by sera from guinea pigs infected with cloned C. burnetii stain Nine Mile, phase I, thus indicating an important role of this antigen in C. burnetii specific immune response.

Enzyme-linked immunosorbent assay (ELISA) zum Nachweis von IgG- und IgM-Antikörpern bei Chlamydien-Infektionen des Menschen

Abstract The development and evaluation of an indirect ELISA for IgG and IgM antibodies in human sera against the genus-specific chlamydial antigen is described. Application of commercial anti-immunoglobulins for production of conjugates usually resulted in direct conjugate/antigen reactions. Therefore, all immunoreagents had to be prepared by the authors. Using anti-immunoglobulins purified by affinity chromatography for the production of anti-human IgG(H + L)-peroxidase conjugates, these conjugates showed high specific activity and could be diluted 1/10,000 for the test, whereby direct reactions with antigens were negligible (12 ± 12 ELISA units). Results with the μ-chain-specific IgM ELISA could be verified additionally by rheumatoid factor analysis and gel-chromatographic investigations. Reactions with sodium deoxycholate extracted antigen from cells not infected with chlamydiae (“negative antigen”) were observed with sera from persons working with cell cultures. In comparison with microimmunofluorescence (MIF) test and radioimmunoassay (RIA), the ELISA was the most sensitive method. However, for differentiation of urogenital infections associated with chlamydiae, the type-specific MIF test was superior to the genusspecific ELISA. In comparison to persons with chlamydia-associated prostatitis, the high percentage of IgM-positive reactions in patients with nongonococcal urethritis (10% and 56%, respectively) pointed to a predominant participation of genus-specific IgM antibodies in recent infections. As compared to ELISA values of patients with infections of the urogenital tract (up to 875 ELISA units), in some apparently healtly blood donors significantly higher ELISA values (up to 1400 ELISA units) were recorded. Such reactions may be attributed to systemic infections, e. g. chlamydial pneumonias. The importance to differentiate between IgG and IgM antibodies in such infections is discussed.

Detection of Coxiella burnetii by the immunoperoxidase technique

An immunoperoxidase method using Coxiella (C.) burnetii-specific hyperimmune serum raised in rabbits, and swine anti-rabbit horseradish peroxidase conjugate was applied to visualize C. burnetii in BGM cell cultures. The technique proved to be highly specific and did not result in any unspecific background staining. The technique was applied successfully to study the organism during multiplication in vacuoles of epithelial cells and in the phagolysosomes of macrophages. Preliminary results further indicate that the technique may be useful for the detection of the pathogen in tissues and milk samples.