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The aim of this study was to evaluate a novel third-generation enzyme-linked immunosorbent assay (ELISA) for the high-sensitivity detection of autoantibodies to proteinase-3 (PR3) in patients with Wegener's granulomatosis (WG). First- and second-generation ELISA for the detection of antineutrophil cytoplasmic antibodies (ANCA) frequently demonstrate insufficient sensitivity due to inadequate presentation of autoantigenic epitopes. Human PR3 was immobilized on the solid phase of ELISA plates by anchoring technique. Anti-PR3 reactivity was measured in 34 C-ANCA positive patients with WG, 11 MPO-ANCA-positive patients with other autoimmune vasculitides, 65 patients with systemic lupus erythematosus (SLE), and 137 healthy blood donors. Thirty-three of 34 patients with WG (97.1%) showed positive anti-PR3 IgG antibody reactivity. None of 11 MPO-ANCA positive vasculitis patients, none of 137 blood donors, and 3 of 65 SLE patients expressed elevated IgG reactivity to PR3 (specificity: 98.4%). Comparison with another third-generation ELISA did not reveal different qualitative results. However, there was no significant correlation between quantitative results of both assays. Receiver operating characteristic (ROC) curve analysis revealed a significantly better assay performance compared with first (direct)- and second (capture)-generation assays (P = 0.011 and P = 0.001, respectively). Third-generation (anchor) anti-PR3 ELISA exhibit significantly higher sensitivity than previous generation assays. Anchoring of PR3 renders the granulocyte protein more autoantigenic compared with direct or capture immobilization.

Objective: In order to shorten the diagnostic procedures simple serological tests with high diagnostic value for Crohn's disease and ulcerative colitis are required. The aim of this study was therefore to evaluate the diagnostic value of a newly developed assay for the determination of antibodies to mannan of Saccharomyces cerevisiae in combination with antibodies to antigens of exocrine pancreas, goblet cells, human neutrophils and tissue transglutaminase., Methods: Sera from 112 patients with Crohn's disease (82 confirmed and 30 suspected cases), 65 patients with ulcerative colitis, 13 patients with indeterminate colitis, 212 patients with other diseases and 250 healthy blood donors were tested for IgA and IgG antibodies to mannan of S. cerevisiae and IgA to tissue transglutaminase by enzyme-linked immunoassays. Endomysial IgA antibodies and antibodies to antigens of exocrine pancreas, goblet cells and human neutrophils were determined by indirect immunofluorescence. Sensitivity, specificity, positive and negative predictive values of antibodies to mannan of S. cerevisiae and different antibody combinations were determined regarding the diagnosis of Crohn's disease and ulcerative colitis., Results: Antibodies to mannan of S. cerevisiae were detected in 50% of confirmed and 27% of patients suspected of having Crohn's disease. Among patients who did not have Crohn's disease only those suffering from gluten-sensitive enteropathy and primary biliary cirrhosis exhibited antibodies to mannan of S. cerevisiae (14%, 6%). With respect to antibody positivity, antibodies to mannan of S. cerevisiae and/or antibodies to antigens of exocrine pancreas combined with tissue transglutaminase antibody negativity yields a sensitivity, specificity, positive and negative predictive value of 60%, 100%, 100% and 90%, respectively, regarding diagnosis of Crohn's disease., Conclusions: Determination of antibodies to mannan of S. cerevisiae as a new marker for Crohn's disease is helpful for the differential diagnosis of inflammatory bowel disease. In combination with other established serological markers, antibodies to mannan of S. cerevisiae improve their diagnostic and predictive values considerably.

3,3' 5,5'-tetramethylbenzidine (TMB) is a high sensitive chromogenic substrate for horseradish-peroxidase as a marker enzyme. In an enzyme immunoassay (EIA) for alpha-1-fetoprotein and in an rapid EIA for myoglobin it reveals higher sensitivity compared to o-phenylenediamine, the increase depends on reaction time. The optimal peroxide concentration depends on reaction time of enzyme chosen in different assays. TMB used for histochemistry is also suitable for EIA if hydrochloric acid is used as stopping reagent. TMB lacks in mutagenic properties and it should preferred for peroxidase rather than all other chromogenic substrates applied up to now.

alpha 1-Microglobulin was purified from urine to a purity of 97.7% in a yield of 25.8%, and was used to produce antibodies in sheep. These antibodies, purified by affinity chromatography, were used to develop a rapid one-step and a two-step immunoenzymometric assay (IEMA). The equilibrium in the reaction between solid phase-adsorbed antibodies and antigen and between the antigen and enzyme-labelled antibodies was attained within 30 and 100 min, respectively. The one-step IEMA permits a good differentiation of low alpha 1-microglobulin concentrations after 30 min reaction time. Its detection limit is 0.35 micrograms/l, and its measurement range is between 0.5 and 100 micrograms/l. The IEMA correlates well with radial immunodiffusion (r = 0.973). The mean alpha 1-microglobulin serum concentration in women is insignificantly lower (33.2 mg/l) than in men (36.1 mg/l). In both sexes the alpha 1-microglobulin concentration increases with age. HIV-infected symptomless men have a significantly lower (15.9 mg/l) alpha 1-microglobulin concentration in serum than normal persons, whereas in AIDS patients it is significantly higher (45.5 mg/l).

Murine monoclonal IgG1 antibodies directed against four different epitopes of human Cu/Zn superoxide dismutase (SOD) were produced by immunization with recombinant Cu/Zn SOD. The antibodies reacted well with the recombinant protein and Cu/Zn SOD purified from human erythrocytes, with binding constants ranging from 8.8 X 10(9) to 2.2 X 10(10) l/mol. When mixed, these antibodies completely prevented the binding of rabbit and sheep polyclonal antibodies raised against erythrocyte Cu/Zn SOD. Whereas one antibody was directed against a common homology region of bovine and human Cu/Zn SOD, all the other antibodies reacted exclusively or preferentially with human Cu/Zn SOD. Only one epitope on the human Cu/Zn SOD molecule was accessible at two different sites as demonstrated in a homologous two-site assay with one and the same antibody used as both capture and indicator antibody. In the indirect two-site assay with unlabelled monoclonal antibodies, and additive effect with a steeper dose-response curve was obtained by mixing antibodies against different epitopes. A super-rapid one-step two-site enzyme immunoassay (overall duration 20 min) was established with antibodies against two different epitopes. Its detection limit was 0.5 micrograms SOD/l.

A direct two-site binding assay on the basis of antibodies from sheep for the quantification of human gamma-gamma enolase is described. The antibody was produced by immunization with human NSE coupled to horse spleen ferritin. The assay shows two feature: a decreased reactivity with NSE from rat and NSE from human serum in spite of 100% recovery of purified human brain NSE. The sheep antibody seems to react with epitopes less accessible on the rat NSE and on the NSE of human serum. The assay is characterized by gamma-gamma enolase specificity, a high sensitivity (2 pg) and a precision of CV = 3-7%.

Covalent linkage of antibodies directed against marker substances and antigen specific antibodies resulted in antibody chimera. Their usefulness was proved in an enzyme and erythro immunoassay for alpha-1-fetoprotein. Whereas the enzyme immunoassay showed the same sensitivity, precision and practicability when compared with assays using covalently linked enzyme antibody conjugates, the antibody chimera technique enables the application of crude enzyme preparations. The erythro immunoassay is well suited as screening procedure and allows also the quantitation of the antigen determining the pseudo peroxidase activity of bound erythrocytes.

The absorption-elution method allows the determination of the blood group properties of the ABO-System from human teeth. The results of own investigations carried out on a larger number of specimens are reported. The significance of the determination of the blood group antigens in teeth for the forensic stomatology is emphasized.

Fractions of retroplacental sera containing pregnancy-associated alpha 2-glycoprotein (PAAG) inhibited the lymphocyte transformation. The inhibitory effect did not correlate to the amount of this protein. It remained even after a specific depletion of the PAAG without altering the composition of the other proteins in the sera fractions. These results rule out a suppressive activity of the PAAG in the lymphocyte transformation.

The suitability of different carrier materials for absorption of serum was investigated with respect to a centralised screening for AFP serum levels in pregnant women, which needs a transmission of the samples. AFP was quantified in an indirect two-site binding enzyme immunoassay. AFP concentrations of native serum and of the eluate from the samples dried on filter paper were in a good agreement with a coefficient of correlation of 0.902. But storage of dried serum samples should not exceed one week because a two week storage diminished the coefficient of correlation to 0.766 and AFP concentrations of paper dried samples were overestimated.

Frequency of detectable Y-bodies in interphase nuclei after mepacrin staining of paraffin embedded formalin-fixed tissue was compared with those of cryostat sections of the native material and organ smears with and without formalin fixing. In formalin fixed smears from males 42--70% of investigated cells showed the Y-body. Sections of formalin-fixed and paraffin embedded male organs demonstrated in 12--24% of their cells the typical fluorescence for the Y-chromatin. High significantly less fluorescent Y-like bodies were found in smears and sections of all proofed organs from women prepared in the same manner. Mepacrin staining in combination with investigation of Barr-bodies permits a fast determination of sex of both organ smears and sections, too.

Labelling of IgG with various HRP isoenzymes purified by preparative isoelectric focussing influences the yield and the specific activity of the conjugates. Alkaline isoenzymes were preferably coupled by glutaraldehyde whereas application of the periodate method additionally formed relatively large quantities of conjugates with acidic isoenzymes of a high purity number, but a low specific activity. In an enzyme immunoassay for alpha fetoprotein the detection limit can be varied by a factor of 6 and even by a factor of 20 by use of the conjugates with different isoenzymes coupled by the glutaraldehyde method and the periodate method, respectively. In order to achieve enzyme immunoassays of the highest sensitivity, antibodies should be coupled to horseradish peroxidase after removing acidic isoenzymes from the enzyme preparations.

Separation of X- and Y-chromosome-bearing spermatozoa has been attempted using Sephadex gel filtration. Fractions rich in X-chromosome-bearing spermatozoa were eluted in dependence on length of separation distance and on speed of elution. Spermatozoa remaining at the gel showed a higher percentage of Y-bodies compared with the untreated control. Sedimentation pattern and amount of eluted cells is influenced by eluation conditions.

An indirect two-site binding enzyme immunoassay has been developed to quantify the pregnancy associated globulin (PAG) in rats. Anti-PAG from guinea pig is adsorbed to solid phase and binds serum-PAG, which then reacts with its remaining free determinants with anti-PAG from rabbit. Sheep anti-rabbit IgG, labeled with horse radish peroxidase (HRP), binds to the rabbit anti-PAG antibody and the enzymic activity of the solid phase is measured by incubation with the appropriate chromogenic substrate. The optical density is directly related to the quantity of PAG to be measured. Reproducible results are obtained in the range from 5 to 200 micrograms/l. The test detects 12 fmoles of PAG. Female rats show a 1000-fold higher serum level of PAG than male rats. Strain differences of the distribution of the PAG serum concentration were only found between male rats.

Three variants of the immunoenzymometric assay of human lysozyme with HRP-labeled antibodies were compared. The highest sensitivity (with a detection limit of 0.2 micrograms lysozyme/L) was achieved by a one-step assay lasting 2 h. Between-batch precision for the techniques was 6-11%. Lysozyme reference values were determined in serum, cerebrospinal fluid and urine. In serum they are age-dependent and in urine sex-dependent when related to creatinine excretion. Serum lysozyme is increased in only 57% of the patients with active rheumatoid arthritis and is also unreliable for indicating remission. In Crohn's disease the serum lysozyme reflects activity better, but it does not exceed the diagnostic value of alpha-1-acidic glycoprotein (orosomucoid). The lysozyme quantification in cerebrospinal fluid is useful in distinguishing between viral or bacterial meningitis.

Isolation of a glycoprotein from serum of pregnant rats has been accomplished by ammonium sulphate precipitation, gel filtration, ion exchange and affinity chromatography. Low concentrations of the protein were detectable in the serum of some of the male rats tested, while somewhat higher concentrations were detected also in the serum of non-pregnant female rats. No relationship could be established between this protein and the well known pregnancy-specific proteins or the alpha 2-acute phase-macroglobulin of the rat, whereas evidence was obtained for a strong cross-reaction with a serum protein of pregnant mice.

The use of a newly developed immunoassay for alpha-1-protein in testicular tumours is described. With 2 ng AFP/ml serum the lower detection group of the test is in the physiological range. Of 39 non-seminomatous testicular tumours, 59% had a high AFP-serum level post-operatively, of 51 on initial diagnosis 54%; 12 seminomas remained AFP-negative. Checking the AFP level ist valuable for diagnosis, staging and prognosis of all testicular tumours.

The use of a two-side-binding-enzyme-immunoassay for pregnancy-specific beta 1-glycoprotein (SP-1) in tumours of the testicles is described. The lower limit of evidence is with 4 ng/ml near to the physiological region. In 17 of 41 non-seminomatous germinal tumours of the testicles (41%) initially increased SP-1-serum concentrations were present, the other measuring values correlated with the course of the disease. In all tumours of the testicles with initially increased SP-1-titres the use of the SP-1-test gives a further possibility of the regulation of therapy and control of the course.

Antibodies coupled to horseradish peroxidase (HRP) were purified by gel filtration and by combination of affinity chromatography and gel filtration. Binding kinetics to solid phase bound antigens were studied of the antibody-enzyme conjugates in the presence and in the absence of unlabelled IgG. The detection limit in enzyme immunoassay was found to be lowered by factor 16 after elimination of unlabelled IgG from the conjugate.